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Poster Presentations.

Luminescence. 2014 Aug;29(S1):55-106


P0001 Theoretical study of oxalic peracid derivatives; insights on the high-energy intermediate of the peroxyoxalate system Felipe A. Augusto, Noriberto A. Pradie, Antônio C. Borin, Erick L. Bastos, Wilhelm J. Baader Instituto de Química, São Paulo, SP, Brazil The peroxyoxalate reaction is used as an analytical tool for the detection of several analytes due to its low cost and high sensibility.(1,2) Our group has proposed a simplified mechanism for the reaction of bis(2,4,6-trichlorophenyl) oxalate (oxalic ester) with hydrogen peroxide in the presence of imidazole (base) and 9,10-diphenylanthracene (activator).(3,4) More recently it has been possible to directly observe the chemiexcitation step, obtaining for the first time kinetic data related to this step.(5) Although being known for more than half a century and having been extensively studied, the peroxyoxalate system still has particularities that are a matter of discussion.(1,2) Among them are the reason for its high efficiency when compared to similar systems and the identity of the high-energy intermediate (HEI).(1,2) Several HEI have been proposed along the years, but only a few of them based on experimental evidence and even less were effectively studied or discarded.(6) In the present work, several oxalic peracid derivatives have their geometries optimized and their energy calculated by theoretical means (B3LYP/6-31 + G(d)), then, for each peracid, a proposed reaction path is studied. This path is selected based on several structures proposed as HEI, like the 1,2-dioxetanedione and other cyclic peroxidic derivatives. Based on the results obtained for each step and for each peracid, it is possible to draw the energetic profile for these reactions. Using data from the third and fifth step Hammett plots were made which indicate the charge distribution during the course of the reaction (Fig. ). [Figure: see text] The results obtained in the study of specific reaction steps of the peroxyoxalate reaction for several oxalic peracid derivatives indicate that the proposed mechanism for this reaction is energetically viable.(3,4) The Hammett plots obtained with the calculated data indicate the involvement of a negative charge in the transition state, as also indicated by kinetic results from our group (unpublished work). References 1. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;2012:391. 2. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APE, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader WJ. Quim. Nova 2011;34:544. 3. Stevani CV, Lima DF, Toscano VG, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;989. 4. Da Silva SM, Casallanovo F., Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 5. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. 6. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. P0002 Investigation into the phosphate buffer role on the peroxyoxalate system in aqueous medium Glalci A. Souza, Monica M. M. Peixoto, Wilhelm J. Baader Instituto de Química da Universidade de São Paulo, São Paulo-SP, Brazil The peroxyoxalate reaction is known as the only chemiluminescence system which involves the intermolecular Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism that possesses proven high quantum yields (up to 60%).(1,2) The mechanism of this reaction has been intensively studied in non-aqueous medium, however, is still not yet completely understood.(3-6) Contrarily, no detailed mechanistic studies on this reaction were performed in aqueous medium, important for many analytical applications.(2) In this work we report the results of a kinetic study of the reaction of oxalic esters such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis(4-methoxyphenyl) oxalate (BMPO) with hydrogen peroxide and phosphate buffer as catalyst, using 2,5-diphenyloxazole as activator. The reaction was performed in a binary system using 1,2-dimethoxyethane as the organic phase and phosphate buffer as the aqueous phase in a proportion of 1;1 (v/v). The influence of the reagent concentration as well as the pH of the medium on the kinetic parameters and the chemiluminescence quantum yields has also been investigated. Kinetics studies with TCPO using different H2 O2 concentrations allow the determination of the rate constant for oxalic ester perhydrolysis (kper ), from the linear correlation between the hydrogen peroxide concentration and the observed rate constants (kobs ). The results show that TCPO is more reactive at pH 8 (kper  = 14.7 ± 0.7 L mol(-1) s(-1) ), followed by pH 7 (kper  = 6.2 ± 0.3 L mol(-1) s(-1) ) and less reactive at pH 6 (kper  = 0.82 ± 0.02 L mol(-1) s(-1) ). Additionally, kinetics studies performed with BMePO at different H2 O2 concentrations showed the same reactivity tendency over the pH range from 6 to 8 as with TCPO. However the kper values are slightly higher for BMePO (pH 8; kper  = 61 ± 3 L mol(-1) s(-1) ; pH 7; kper  = 20 ± 1 L mol(-1) s(-1) and pH 6; kper  = 9.9 ± 0.1 L mol(-1) s(-1) ). Finally, BMPO presented a very interesting reactivity pattern in aqueous medium. Contrarily to TCPO and BMePO, BMPO is considerably more reactive at pH 6 than at pH 8 and its reactivity at pH 7 (kper  = 34 ± 1 L mol(-1) s(-1) ) is comparable to BMePO. At pH 6 and 8, the reaction is so fast that is not possible to measure the rate constants at high hydrogen peroxide concentrations (> 15 mmol L(-1) ) in order to establish the linear correlation between the hydrogen peroxide concentration and kobs values. The highest rate constants possible to measured were kobs  = 1.23 ± 0.5 s(-1) at pH 6 for [H2 O2 ] = 10 mmol L(-1) and kobs  = 1.18 ± 0.06 s(-1) at pH 8 for [H2 O2 ] = 15 mmol L(-1) . These results indicate that for TCPO general base catalysis by phosphate is predominant, which is more efficient at pH 8. For BMePO at pH = 6 the general acid catalysis is also important as indicated by it much higher kper value as compared to TCPO. Finally, for BMPO acid catalysis is predominant as indicated by the higher kper values in more acid pH values for this oxalic ester. Financial support; FAPESP, Capes, CNPq. 1. Stevani CV, Silva SM, Baader WJ. Eur. J. Org. Chem. 2000;4037. 2. Augusto F. A., Souza G. A., Souza Jr. S. P., Khalid M., Baader W. J. Photochem. Photobiol. 2013;89:1299. 3. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. 4. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 5. Silva SM, Wagner K, Weiss D, Beckert R, Baader WJ. Luminescence 2002;17:362. 6. Ciscato LFML, Bartoloni F. H, Bastos E. L, Baader WJ. J. Org. Chem. 2009;74:8974. P0003 Lanthanide Complexes with N'-(2-hydroxybenzylidene)-3-methoxybenzohydrazide; Synthesis, Thermal Behaviour, Biological Activities and Luminescent Properties. Abdulaziz Ajlouni(a) , Ziyad Taha(a) , Waleed Al Momani(b) (a) Department of Applied Chemical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan, Jordan (b) Department of Allied Medical Sciences, Al Balqa' Applied University, Amman, Jordan Novel Ln(III) complexes with N'-(2-hydroxybenzylidene)-3-methoxybenzohydrazide (L) have been synthesized. The ligand and its complexes were characterized based on elemental analyses, molar conductance, IR, 1H and 13C-NMR, UV-vis., and TGA studies. The conductivity data show a 1;2 electrolytic nature with a general formula [LnL2(NO3)2]NO3. The IR spectra reveal the coordination of the ligand through the azomethine nitrogen and the hydroxyl O-atom in addition to the carbonyl oxygen to the lanthanide ion. The coordinated nitrate ions behave in a bidentate fashion. Under the excitation, the luminescence emission properties for Sm, Tb, Eu and Dy complexes are observed. These observations show that the ligand favor energy transfers to the emitting energy level of these lanthanide ions. Furthermo re, the antimicrobial activities of all complexes were studied against different types of bacteria. It was observed from the results that most of the synthesized complexes of the tested series possessed good antibacterial activity against bacteria and the microbial activities of the complexes in most cases are higher than that of the corresponding ligand. P0004 Comparative study of apoptosome formation and ATP oscillation in apoptosis and differentiation processes using luciferase Shiva Akbari-Birgani(a) , Saman Hosseinkhani(a) , Hossein Baharvand(b) (a) Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran (b) Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran A powerful and sensitive tool to monitor cytochrome c release and pursuing apoptosome formation based on split-luciferase complementary assay has been recently developed (1). This biosensor detects and report apoptosome formation based on Apaf-1 oligomerization (Fig. P0004;1). In this study the split luciferase biosensor is used to compare apoptosome formation in differentiation with apoptosis processes. ATP oscillation is also measured in both processes using luciferase based assay (2). The mESC line Royan B16, derived from the C57BL6 mouse strain (3) was cultured according to the standard protocol. Mouse Embryonic Stem Cells (mESCs) were differentiated to cardiac cell by static suspension culture and ascorbic acid as a cardiac differentiation inducer of mESCs. In parallel with differentiation, Doxorubicine, an apoptogenic chemotherapy drug, was used to induce apoptosis in another group of mECS. Sampling was performed in series time. To monitor the release of cytochrome c, western blotting was applied. To assess apoptosome formation during apoptosis and differentiation, mESCs were transfected with two vectors containing split luciferase genes connected to APAF1 gene; pcDNA-NLuc-APAF1 and pcDNA-CLuc-APAF1, and then apoptosis and differentiation were induced. Luciferase activity was measured at different times after induction. Cellular ATP content was also compared in these two processes using luciferase. Our evidences have revealed that cytochrome c accumulates in cytosol and apoptosome machinery forms during differentiation of mESCs as well as apoptosis. But they are delayed and reduced in differentiation relative to apoptosis. Cellular ATP measurements showed ATP oscillations go parallel with apoptosome formation during both processes. In total our results indicate that mitochondrial apoptotic pathway involves in cardiac differentiation of mESCs as well as apoptosis. However, timing and intensity of cytochrome c release, apoptosome formation and ATP increment is delayed in differentiation. [Figure: see text] 1. Torkzadeh-Mahani M, Ataei F, Nikkhah M, Hosseinkhani S. Design and development of a whole-cell luminescent biosensor for detection of early-stage of apoptosis. Biosens Bioelectron 2012;38:362-368 2. Mohammadi S, Nikkhah M, Nazari M, Hosseinkhani S. Design of a coupled bioluminescent assay for a recombinant pyruvate kinase from a thermophilic Geobacillus. Photochem Photobiol 2011;87:1338-1345 3. Hassani SN, Totonchi M, Farrokhi A, Taei A, Larijani MR, Gourabi H, Baharvand H. Simultaneous suppression of TGF-beta and ERK signaling contributes to the highly efficient and reproducible generation of mouse embryonic stem cells from previously considered refractory and non-permissive strains. Stem Cell Rev 2012;8:472-481 P0006 Effects of alcohols on the fluorescence of Ca(2+) -discharged photoprotein obelin Roza Alieva(a) , Nadezhda Belogurova(b) , Alena Petrova(a) , Nadezhda Kudryasheva(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Photoprotein obelin is stable enzyme-substrate complex of polypeptide and 2-hydroperoxycoelenterazine, which is responsible for bioluminescence of the marine hydroid Obelia longissima [1]. The bioluminescent is triggered by calcium ions. Obelin is not fluorescent, but the product of the bioluminescent reaction, enzyme-bound coelenteramide, is a fluorescent protein called "Ca(2+) -discharged" obelin. Discharged obelin is stable and nontoxic and its spectra are variable, it can be applied as fluorescent biomarker to visualize biochemical processes in biological and medical investigations. Variation of the color of biomarkers is important for these applications. As we showed previously [2-4], fluorescence spectra of discharged obelin are not completely stable; they depend on Ca(2+) concentration, exposure to higher temperature, and excitation wavelength. The effects of ethanol on bioluminescence of mutant obelins were found in [5]. Availability of the active center of the obelins to exogenous ethanol molecules was suggested as a reason for bioluminescence spectra changes. Influence of alcohol molecules on light-induced fluorescence of Ca(2+) -discharged obelin was not studied yet. Here we examined the intensity and color of light-induced fluorescence of Ca(2+) -discharged photoprotein obelin in the presence of alcohols (ethanol and glycerol) which are widely used as biomedical agents. Light-induced fluorescence spectra of Ca(2+) -discharged obelin were measured at different concentrations of the alcohols at 350- and 280-nm photoexcitation (corresponding to polypeptide-bound coelenteramide and tryptophan absorption regions). Emission spectra at 280 nm excitation (tryptophan absorption region) included three peaks with 348, 504, and 657 nm maxima, corresponding to fluorescence of tryptophan, enzyme-bound coelenteramide, and hypothetical indole-coelenteramide exciplex, respectively. The latter was found in photoprotein emission spectra recently [4]. The spectra were deconvolved into Gaussian components - ultraviolet (tryptophan emission), blue-green (coelenteramide emission), and red (hypothetical indole-coelenteramide exciplex emission). The addition of alcohols increases covariantly the fluorescence intensities and contributions of ultraviolet (346 nm, tryptophan), violet (420 nm, protonated form of coelenteramide), and red (655 nm, exciplex) components and decreases fluorescence of blue-green (503 and 565 nm, partly deprotonated forms of coelenteramide) components of Ca(2+) -discharged obelin fluorescence. The effects are related to changes of the proton transfer efficiency in the fluorescent state of coelenteramide. Therefore, two peculiarities should be taken into consideration when applying the discharged obelin as a fluorescent biomarker; (1) variation of fluorescence color and intensity in the presence of alcohols, and (2) dependence of emission spectra on the excitation wavelength. Details of these peculiarities are reported in [6]. Acknowledgements This work partly supported by; the Program 'Molecular and Cellular Biology' of the Russian Academy of Sciences; the Grant Ministry of Education and Science RF 11.G34.31.0058; the Grant B-14 of Ministry of Education and Science RF assigned to Siberian Federal University. References 1. Vysotski ES, Markova SV, Frank LA. Molecular Biology. 2006;40:355-367. 2. Belogurova NV, Kudryasheva NS. J Photochem Photobiol B 2010;101:103-108. 3. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. LUMINESCENCE 2012;27:96. 4. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem 2013;405:3351-3358. 5. Belogurova NV. Kudryasheva NS, Alieva RR. J Mol Struct. 2009;924-926:148-152. 6. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem DOI; 10.1007/s00216-014-7685-z. P0007 Mitochondrial genome Organization and Phylogenetic analysis of bioluminescent Elateroidea Danilo Trabuco Amaral(a,b) , Yasuo Mitani(c) , Yoshihiro Ohmiya(d) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar - Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil (c) Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan (d) Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan The bioluminescence in the superfamily Elateroidea is observed within Lampyridae (fireflies), Phengodidae/Rhagophthalmidae (railroad worm) and Elateridae (click-beetles) families. Bioluminescence may have evolved independently in these families, since most of the Elateroidea families such as Eucnemidae, Throscidae, Drilidae, Cantharidae, Lycidae do not display luminescence. In this study, molecular phylogenetic analyses with mitochondrial genome were performed to answer this question. We sequenced the mitochondrial genomes of (Elateridae) Hapsodrilus ignifer, Pyrearinus termitilluminans, a non-luminescent Elateridae (Lampyridae), Bicellonycha lividipennis, (Phengodidae) Brasilocerus sp.2 and Phrixothrix hirtus using Long-PCR reactions and the primer walking methodology. Elateridae and Lampyridae species showed a typical Colepotera mitochondrial genome, however, in Phengodidae genomes several rearrangements were observed (Fig. 1). In Brasilocerus sp.2 genome, we did not observe the tRNAs for Trp, Gln and Ile, probably deleted in this species. In P. hirtus genome, the tRNA-Tyr was rearranged before the tRNA-Trp and we observed a duplicated region of tRNA-Leu/COX2 between NADH2 gene and the control region. The phylogenetic analyses using the Bayesian methods showed four clades within Elateroidea, 1. Eucnemidae; 2. Elateridae (with Drilidae) 3. Lampyridae and Lycidae and 4. Phengodidae, Rhagophthalmidae and Cantharidae, (Cantharoidea). As discussed by Viviani et al. (2009) and Amaral et al. (2013), the bioluminescence within Elateroidea may have originated at three different times from the similar ancestor luciferase-like enzymes which was pH-insensitive. Within this scenario, the Elateridae and Phengodidae/ Rhagophthalmidae luciferases evolved directly from the ancestral enzyme and the pH sensitivity may have evolved later in lampyrids or within Lampyridae/Lycidae clade. Financial Support; FAPESP and CNPq Reference 1. Amaral DT, Arnoldi FGC, Rosa S.P., Viviani VR. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera; Elateroidea) in southern and central Brazil. Luminescence. Luminescence, DOI 10.1002/bio.2561. 2. Viviani VR, Prado RA, Arnoldi FCG, Abdalla FC. An ancestral luciferase in the Malpighian tubules of a non-bioluminescent beetle! Photochem Photobiol Sci 2009;8:57-61. P0008 Report of two new cases of luminous termite mounds inside Amazon forest; phylogenetic considerations Danilo Trabuco Amaral(a,b) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar - Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil The phenomenon of luminous termite mounds is known from Central Brazil cerrados (savannas) and is caused by infestation of larvae of the click beetle Pyrearinus termitilluminans. During warm spring nights, larvae expose their bright thorax, and attract flying insects that will serve as preys. The Pyrearinus genus comprises more than 40 species distributed in South America, most of them occurring in Brazil, including species inhabiting termite mounds. Although, there are also reports of luminous termite mounds inside the Amazon forest (Costa and Vanin, 2010), information about these cases are still missing. Here we report the observation of two new cases of luminous termite nests inside the Amazon forest; (I) one in transitional area between Cerrado and Amazon forest at the margins of Araguaia´s river in the northwestern region of Tocantins state, which is also caused by P. termitilluminans, and (II) the other occurring inside Amazon forest at the margins of Juruena river in the northwest region of Mato Grosso state, which is caused by a different species, Pyrearinus fragilis. Our molecular studies, using COI and NADH2 mitochondrial genes showed a close relationship between P. termittiluminans occurring in forest termite mounds in Caseara-TO and in Cerrado termite mounds of PNE-GO, despite the distance of 1,000 Km and the distinct habitats (cerrado and forest). Our data also show that P. termitilluminans and P. fragilis that inhabit termite mounds in different regions separated by 2 basin rivers, form a monophyletic group. These results indicate that niche selection for termite mounds may have occurred in a common ancestor, and this characteristic was maintained in the group. Financial support; FAPESP and CNPq Reference 1. Costa C, Vanin SA. Coleoptera Larval Fauna Associated with Termite Nests (Isoptera) with Emphasis on the Bioluminescent Termite Nests from Central Brazil. Psyche; A Journal of Entomology 2010;2010:1-13. P0009 Molecular Insights of luciferase evolution in Elateridae family Danilo Trabuco Amaral(a,b) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar - Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil Bioluminescence in beetles occurs in Elateroidea superfamily, within Lampyridae (fireflies), Phengodidae/Rhagophthalmidae (railroadworms/starworms) and Elateridae (click beetles) families. In Elateridae, more than 9,000 species were described, however, less than 300, display bioluminescence. Most of them are found in the Neotropical region with Brazil hosting the richest diversity, with species occurring in the Amazon, Atlantic rain forest and Cerrado (savanna). Adult click beetles usually display two lanterns emitting green bioluminescence in the dorsal region and a lantern emitting green to orange bioluminescence in ventral region. The luciferases from these lanterns were shown to be coded by paralogous genes. However, the ontogenic origin of these luciferases is not clear. Thus, to elucidate the evolution of the lanterns in Elateridae, we sequenced the partial cDNA luciferase sequences of Brazilian species and compared with the phylogeny of the group inferred from the mitochondrial genome. The reconstructed tree from luciferases sequences separated the species in two clades (I) South-american species, and (II) Central-american species. Biogeographic events, as discussed by Feder & Velez (2009), may have influenced the variation of bioluminescence colors in Elateridae. In our study, we did not observed the diversity of bioluminescent colors occasioned by vicariant events, as occur in Pyrophorus plagiophthalamus population, however the genetic signature found in our study with the luciferases of Pyrearinus, Fulgeochlzus and Ptesimopsia genera suggest the occurrence of some vicariant events resulting in two well-defined clades in the phylogeny. Our phylogenetic analyses of Elateridae grouped in the same clade the luciferases with similar bioluminescence spectra. The similarity among luciferase sequences displaying similar color, could be explained by intergenic recombination which increases the relationship among luciferases of different lanterns, as already reported luciferases of dorsal and ventral lanterns of Jamaican Pyrophorus plagiophtalamus (Feder & Velez, 2009). On the other hand, the luciferases of larval stage (Pyrearinus termitilluminans and Pyrearinus fragilis), were grouped at the same clade, between abdominal and dorsal luciferases of adult stage. The larval luciferases displayed a lower similarity with adult luciferases. These results showed a divergence between adult and larval luciferases, indicating that luminescence in adult and larvae are produced by two different isoenzymes. Financial Support; FAPESP and CNPq Reference 1. Feder JL, Velez S. Intergenic exchange, geographic isolation, and the evolution of bioluminescent color for Pyrophorus click beetles, Evolution, 2009, 5, 1203-1216. P0010 Development of a highly sensitive and rapid chemiluminescent assay for hydrogen sulfide Hidetoshi Arakawa, Chiaki Nishijima Showa university, Tokyo, Japan Hydrogen sulfide (H2 S) is attracting attention as one of three endogenously generated gaseous signaling compounds, the others being carbon monoxide and nitric oxide. The hydrogen sulfide in live cells is generated by the following three enzymes; cystathionine β-synthase (CBS); cystathionine γ-lyase (CSE); and 3-mercaptopyruvate sulfurtransferase (3MST). These enzymes are involved in neurotransmitter regulation and vasodilatation. However, hydrogen sulfide, the odorous component of waste and sewage, is a toxic gas; therefore, a highly sensitive and specific method for monitoring H2 S is desired in order to protect human health and the environment. Hydrogen sulfide is generally measured by gas chromatography, but this method requires special equipment. Fluorescent probes for hydrogen sulfide have also been recently developed as a simpler method. In order to analyze hydrogen sulfide rapidly and sensitively, we have developed a novel method using lucigenin chemiluminescence in the presence of copper ion (II). Materials and method (1) Assay method; Lucigenin chemiluminescent solution (0.2 mL; 5 µmol/l copper chloride (II), 0.04 mg/mL lucigenin, 0.1 mg/mL TritonX-100) was added to Na2 S solution (20 µl) diluted with phosphate buffer (pH 11.7). Chemiluminecence intensity was measured using an Aloka luminescence reader (Aloka Co. Japan) (waiting time, 10 s; integration time, 10 s). (2) Electron spin resonance method (ESR) for the analysis of radicals; DMPO (5 µl) and lucigenin luminescence reagent (250 µl) was added to Na2 S (25 µl), and the radicals generated were measured by ESR. Superoxide dismutase (SOD) and catalase were used as scavenging enzyme. Results and discussion This is a novel chemiluminescence method based on the principle that light is emitted by metal ions and hydrogen sulfide in the presence of lucigenin. The effects of several metal ions (copper (II), copper (I), zinc, magnesium and aluminum) were studied. Intense luminescence was generated with copper (II). Analysis of reactive oxygen species; Reactive oxygen involved in this chemiluminescent reaction was analyzed using ESR by the addition of a scavenging enzyme, SOD. The emission disappeared upon the addition of SOD. In addition, the generated radical species were analyzed by ESR using the spin trapping agent, DMPO. An ESR signal was observed in the presence of lucigenin. Lucigenin was essential for the generation of reactive oxygen. With the addition of both catalase and SOD, this signal essentially disappeared. This result indicates that the radical species is a superoxide anion. The concept is depicted in Fig. P0010;1. Next the effects of pH, lucigenin, copper (II) and phosphate salt concentration were examined to determine the optimal conditions. The results are shown in (1) of the Materials and Method. Time course; When the time course of chemiluminescence intensity was examined following the addition of Lucigenin chemiluminescent solution, the maximum light emission intensity was obtained within a few seconds, and then the emission gradually decreased. Consequently, this method is capable of rapid measurement (within 10 seconds). Validation of sensitivity, specificity and precision; Under the above conditions, a standard curve of Na2 S shows 1 µmol / l (20 pmol / assay) ~ 10 mmol / l (20 nmol / assay), and reproducibility was from 1.5 to 11.7% (n = 7), with 6.0% as the mean. The specificity of the method was examined using cysteine and glutathione as SH compounds. When compared to sodium sulfide standards at the same concentration, the emission intensity was 2.9% and 4.1 %, respectively, for cysteine and glutathione. Further, by adding maleic imide to the luminescent reagent, the specificity was able to be improved. Thus, this method was found to show high specificity for Na2 S. On-site assay for H2 S The development of an on-site analytical method for hydrogen sulfide is required for environmental and hot spring water analyses. Therefore, hydrogen sulfide was measured using a portable (6 × 16 cm) Lumitester emitting instrument (PD-20, Kikkoman, Japan). The sensitivity of the PD-20 instrument is low compared with more commonly used highly sensitive luminescence instruments. Therefore, for performing on-site measurements, it is necessary to further enhance the emission intensity. Screening of enhancer; The effects of 17 surfactants to enhance chemiluminescence intensity were examined. 3-((3-Cholamidopropyl) dimethylammonio) -1- propanesulfonate (CHAPS) was selected. This zwitterionic detergent increased the emission intensity about tenfold. In the presence of CHAPS, it was possible to obtain a detection limit of Na2 S of 1 × 10(-5) mol /l using the PD-20 instrument. Currently, we are investigating the application of this methodology to biological samples and environmental studies. [Figure: see text] P0011 Chemiluminescent investigation of the antioxidant properties of halogen derivatives of salicylaldehyde benzoyl hydrazone in ROS generating systems Vera Hadjimitova, Nadya Avakumova, Trayko Traykov Department of Medical Physics and Biophysics, Medical University of Sofia, Sofia, 1431, Bulgaria Hydrazones are considered as an important class of organic compounds possessing various biological activities viz antimicrobial, antiviral, anti-inflammatory, anticancer etc. [1]. The aim of the present investigation was to determine in vitro the antioxidant and radical scavenging properties of two derivatives of the iron chelator salicylaldehyde benzoyl hydrazone (SBH) (5-bromosalicylaldehyde-4-hydroxybenzoylhydrazone- B1 and 5-bromosalicylaldehyde-isocotinoilhydrazones - B2 ) and to examine the influence of the molecular structure on their interaction with reactive oxygen species (O2 (─•) , HO(•) ) [2]. ROS were registered by two types of luminol-dependent chemiluminescence assay - luminol-dependent CL in a system of potassium superoxide (KO2 ) - produced O2 (─•) and luminol-dependent CL in a system of iron-dependent hydroxyl radical formation. Detailed description of the methods was published by Hadjimitova et al [3]. The antioxidant and radical scavenging properties of the tested substances were investigated in the concentration range of 3-100 µmol/l. The chemiluminescent response was determined by calculating the area under the obtained chemiluminescent light curve (integral chemiluminescence) for samples containing the tested hydrazones and the control samples. The ratio of CL in the presence and in the absence of the drug was termed CL scavenging index (CL-SI). Over the whole studied concentration range all of the three hydrazones showed slight effect on the luminol-dependent chemiluminescence in the system of non-enzymatically generated O2 (─.) (Fig. ). The decrease of CL-SI was significant only for the highest concentration of 100 µmol/l and its value was about 75%. B2 showed moderate radical scavenging properties which remained unchanged through the tested concentration range. The obtained results demonstrate that the bromine atom doesn't possess electron-donating activity. In the system iron-dependent hydroxyl radical formation the tested components decreased the chemiluminescence response in a concentration dependent manner (Fig. P0011;2). In the studied concentration range B2 has the strongest antioxidant effect against the HO(•) . At concentration of 10μmol/l the observed effect of B2 is twofold bigger compared with the one of SBH. At the highest concentration B1 and B2 decreased equally the chemilumnescent response. CL-SI is 30% less than this of SBH measured in the same experimental condition. From the obtained results it can be concluded that the investigated hydrazones posses well expressed antioxidant properties against hydroxyl radicals, compared with the demonstrated versus the superoxide radicals. References; 1. Kumar P, Narasimhan B. Hydrazides/hydrazones as antimicrobial and anticancer agents in the new millennium. Mini Rev Med Chem 2013 Jun;13(7);971-87. 2. Nikolova-Mladenova B, Halachev N, Iankova R, Momekov G, Ivanov D. Characterization and cytotoxicity of new salicilaldehyde benzoydrazone derivatives as poten tial antiproliferative agent. Journal Arzeimittelforschung/drug research 2011;12-F:714-718. 3. Hadjimitova V, Traykov T, Mileva M, Ribarov St. Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2 (─•) , HO(•) , HOCL. Z Naturforsch C 2002;57:1066-1071. [Figure: see text] One ml sample of PBS, pH 7.4, is containing 0.1 mM luminol and the drug in shown concentrations (in control sample drug was omitted). The CL was measured immediately after addition of 20 µl KO2 solution. Therefore, CL was measured using "flash assay" option of the MultiUse program, every 50 miliseconds. [Figure: see text] One ml sample of PBS, pH 7.4, is containing 0.1 mM luminol, 0.1 mM Fe (3+) , 0.1 mM EDTA, 0.1 mM ascorbate, 0.1 mM H2O2 and either of tested drugs at concentration between 3 and 100 μM, or buffer for the controls. The CL was measured using "flash assay" option of the MultiUse program, every 50 milliseconds. P0012 Inhibition of oxygen free radicals induced luminol-dependent chemiluminescence by 4-methoxy derivatives of salicylaldehyde benzoyl hydrazone Nadya Avakumova, Vera Hadjimitova, Trayko Traykov Department of medical Physics and Biophysics, Medical University of Sofia, Sofia, 1431, Bulgaria The fact that reactive oxygen species are implicated in various pathological complications has initiated the development of design strategies for novel synthetic antioxidants possessing optimized antioxidant activity and solubility and reduced potential toxic effects [1]. It has been reported that among the vast spectrum of pharmacological activities viz. antimicrobial, antimalarial, analgesic, anti-inflammatory, antitumoral some hydrazone derivatives possess also antioxidant properties [2]. In this investigation we studied the antioxidant activities and free radical scavenging capacity of newly synthesized 4-methoxy derivatives of salicylaldehyde benzoyl hydrazone (SBH) - 4-methoxy-salicylaldehyde benzoyl hydrazone (M1), 4-methoxy-salicylaldehyde-4-hydroxybenzoyl hydrazone (M2), 4-methoxy-salicylaldehyde-isocotinoyl hydrazone (M3) [3]. The purpose was to compare the results measured using a luminol-dependent chemiluminescence in the presence of in vitro generated O2 (─•) and HO(•) in order to determine the influence of the molecular structure of the hydrazones on the studied properties. The ratio (in percentage) between the chemiluminescent response in the presence and the absence of the tested hydrazone was termed chemiluminescent scavenging index (CL-SI) and reflects the antioxidant properties of the investigated compound. A detailed overview of the method is given in [4]. In both assays the three derivatives present themselves as antioxidant with strong influence of the concentration on the CL-SI values.The ability of the SBH derivatives to scavenge O2 (─•) was tested in a system with potassium superoxide (Fig. 61). The obtained results suggest that the replacement of ─ CH group (M1) with ─ COH group or N atom (M2 and M3 respectively) in the hydrazide ring increases the antioxidant effect. The measurements of the luminol-dependent chemiluminescence in the system of iron-dependent hydroxyl radical formation showed more than threefold decrease in the CL-SI index, at concentration 100 µmol/l (Fig. 62). On the bases of the results it was proposed that the studied 4-methoxy derivatives of SBH are scavengers of HO(•) and the different tested substitution patterns on the benzene ring have slight but not significant influence on the investigated property at the highest tested concentration. The observed activity could be well explained with the electron-releasing inductive effect of the methyl group which redistributes the electron density in the molecule and improves the direct antioxidant electron-donating effect. References; 1. Schulman HM, Hermes-Lima M, Wang EM, Ponka P. In vitro antioxidant properties of the iron chelator pyridoxal isonicotinoyl hydrazone and some of its analogs. Redox Rep. 1995;1:373-378. 2. Rollas S, Küçükgüzel SG. Biological Activities of Hydrazone Derivatives Molecules 2007;12(8);1910-1939 3. Mladenova B, Momekov G, Ivanov D. Synthesis and physicochemical characteristics of new salicilaledehyde benzoylhydrazone derivatives with high cytotoxic activity. Pharmacia 2011;LVIII(1-4);41-44. 4. Hadjimitova V, Traykov T, Mileva M, Ribarov St. Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2 (─•) , HO(•) , HOCL. Z Naturforsch C 2002;57:1066-1071. [Figure: see text] The solution in the sample cuvette comprises 1 ml phosphate buffer solution pH 7.4, containing 0.1 mM luminol and the hydrazone derivatives at concentration as indicated. In control sample the studied hydrazones were omitted. The CL was measured immediately after addition of 20 µl KO2 solution [Figure: see text] The solution in the sample cuvette comprises 1 ml phosphate buffer solution pH 7.4, containing 0.1 mM luminol, 0.1 mM Fe(3+) , 0.1 mM EDTA, 0.1 mM ascorbate, 1mM H2 O2 and the hydrazone derivatives at concentrations as indicated, or a buffer for the controls. P0013 Peroxyoxalate reaction in aqueous carbonate buffer media Fernando H. Bartoloni(a,b) , Ana Paula E. Pagano(a) , Felipe A. Augusto(a) , Wilhelm J. Baader(a) (a) Departamento de Química Fundamental, Instituto de Química, Sao Paulo, SP, Brazil (b) Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brazil The peroxyoxalate system is commonly known as the reaction of an oxalic ester with hydrogen peroxide in the presence of a base and an activator.(1,2) In analytical chemistry it is very important as a detection tool, because of its low costs and high sensibility.(2) Although the behavior of the system is well known in organic media,(3-5) most of these applications are for the detection of analytes in aqueous media, in which competitive processes like ester hydrolysis can reduce the high chemiluminescence quantum yields normally obtained by this reaction.(2,6) Therefore, kinetic studies of this reaction in aqueous media are extremely important to understand mechanistic details like the relationship between ester hydrolysis and perhydrolysis and how this relation affects the chemiexcitation efficiency. In order to characterize the peroxyoxalate reaction in aqueous medium, the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) with hydrogen peroxide and fluorescein (FLU) as activator was studied in aqueous carbonate buffer. The observed rate constants (kobs ) of the chemiluminescence emission intensity decay showed to increase with increasing medium pH; typical experiments were performed in pH = 10.4, where carbonate shows maximum buffering capacity. The kobs values showed no dependence on the buffer concentration in a wide range of hydrogen peroxide concentrations (Fig. 63, for [H2 O2 ] = 10 mmol L(-1) ), indicating the occurrence of specific base catalysis. Thus, the observed rate constants showed linear dependence on the [HO2 (-) ] (Fig. 1), with a bimolecular perhydrolysis rate constant of kper  = 5.9 10(3) L mol(-1) s(-1) ; the pseudo-first order hydrolysis rate constant in these condition is k0  = 1.2 s(-1) ; leading to a bimolecular hydrolysis rate constant by hydroxide ion of khyd  = 4.8 10(3) L mol(-1) s(-1) (Fig. 63). [Figure: see text] The present study indicates that the peroxyoxalate reaction in aqueous carbonate buffer medium leads to reproducible kinetic curves and this system can therefore be utilized for analytical assays in aqueous environment. Although dark hydrolysis occurs in this system, the emission efficiency is high enough for possible applications. The rate constants are linearly dependent on the peroxide concentration, however, independent of the carbonate concentration indicating specific base catalysis. Financial support; FAPESP, Capes, CNPq. References 1. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APF, Santos CS, Oliveira S., Augusto FA, Pagano APE, Baader WJ. Quim. Nova 2011;34:544. 2. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;20 12:391. 3. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 4. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. 5. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. 6. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. P0014 Change of fluorescence spectra of discharged photoprotein obelin under variation of physico-chemical conditions Nadezhda Belogurova(a,b) , Nadezhda Kudryasheva(a,b) (a) Institute of Biophysics SB RAS, Krasnoyarsk, Russia (b) Siberian Federal University, Krasnoyarsk, Russia The assay of calcium-regulated photoproteins is highly sensitive, non- hazardous and their bioluminescence reactions triggered by calcium ions are rapid and simple. It makes photoproteins attractive for applications as a reporter protein [1]. Obelin isolated from hydroid Obelia longissima is one of the most studied among photoproteins. The product of the bioluminescent reaction of obelin (enzyme-bound chromophore, coelenteramide) is a fluorescent protein; It is called 'discharged' obelin. Because discharged obelin is stable and nontoxic it can be used as a fluorescent marker in biological and medical investigations, for example, in cytology, histology, and cryology. Fluorescence spectra of discharged obelin (and hence, emission color) are variable; they might depend on external physic-chemical conditions [2,3]. Therefore, it is important to study changes of fluorescent characteristics of the discharged obelin under variation of physico-chemical conditions - temperature and/or accompanying agents. Fluorescent spectra of discharged obelin were studied under exposure to 40°C (0 - 12.5 hours) [4] and addition of glycerin (С = 0.06 - 0.36 М), ethanol (С = 0.01 - 1.18 М) [5], dimethyl sulfoxide (С = 0.002 - 2.65 М), and polyethylene glycol (С = 1.7 10-5 - 1.7 10-2 М). We chose glycerin, ethanol, dimethyl sulfoxide and polyethylene glycol as accompanying agents extensively used in cytology, histology, and cryology. Increase of exposure time and concentration of the agents decreased intensity and changed color of discharged obelin fluorescence. The discharged obelin spectra obtained under different physico-chemical conditions were analyzed by deconvolving into spectral Gauss components. A red peak (λmax  = 660 nm) attributed to indole-coelenteramide exciplex was newly discovered in the discharged obelin emission induced by UV radiation. The expansion of the known group of colors (from violet to yellow) by adding the red color increases the potential of discharged obelin as a colored biomarker for monitoring of biochemical processes. Decay of the fluorescence intensity at exposure to 40°C was analyzed [6]. Permissible ranges of the accompanying agent concentrations were defined. Acknowledgements This work supported by the Pogramme "Molecular and Cellular Biology" of the Russian Academy of Sciences. References; 1. Markova SV, Vysotski ES, Lee J. In Bioluminescence and Chemiluminescence, Case JF, Harring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE, Eds. Singapore; World Scientific Printers (S) Pte Ltd 2000;11:115-118. 2. Belogurova NV, Kudryasheva NS. J Photochem Photobiol B 2010;101:103-108. 3. Belogurova NV, Kudryasheva NS. LUMINESCENCE 2012;27:100. 4. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. LUMINESCENCE 2012;27:96. 5. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem DOI; 10.1007/s00216-014-7685-z. 6. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem 2013;405:3351-3358. P0015 Purification of a Novel Luciferase from Luminous Fish, Parapriacanthus ransonneti Manabu Bessho, Yuichi Oba Nagoya University, Nagoya, Japan The shallow water fish Parapriacanthus ransonneti uses cypridinid luciferin for its bioluminescence. Haneda and Johnson (1958) demonstrated the luciferin-luciferase cross-reaction between P. ransonneti and luminous ostracod Vargula hilgendorfii. Then, Johnson et al. (1961) showed that the structure of luciferin in both species was identical, and concluded that P. ransonneti emits blue light by using ingested luciferin from luminous ostracods. However, the identity of the luciferase in this unique luminous system remains unresolved. This study seeks to identify the luciferase from P. ransonneti. We performed purification of the luciferase from the thoracic luminous organs of 200 P. ransonneti specimens collected in Japan using anion exchange chromatography, gel filtration chromatography and SDS-PAGE. We successfully obtained nearly pure protein, whose relative luminescence activity was 28 times higher than that of crude extracts from the thoracic luminous organs. Peptide sequencing of the active fractions is in progress. P0016 Chitosan- induced Au/Ag alloy nanoparticles dispersed in ion liquid and its application in developing an ultrasensitive glucose chemiluminescence biosensor Mohammad Javad Chaichi, Seyedeh Olia Alijanpour University of Mazandaran, Babolsar, Mazandaran, Iran Introduction Biosensors have attracted much attention during recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, pharmaceuticals, and food processing industries due to their fast response and ease of operation [1,2]. A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H2 O2 was constructed by one covalent immobilization of glucose oxidase in CL cell. The chemiluminescence-based biosensors are developing due to their high sensitivity and excellent performance. Reagents immobilization onto proper substrates plays an important role in the development of the high-quality CL-based biosensors [3]. In this regard, the immobilization of enzyme is a key step for constructing an enzyme-based CL biosensor. Emergence and recent advance of nanoscience and nanotechnology open new opportunities for the application of nanoparticles in bioanalysis [4]. Imidazolium ion based ion liquids (IL) have recently been shown as promising compounds for preparation and stabilization of nanomaterial, and have found application in transition metal catalyzed CL reactions [5]. This paper describes the synthesis and characterization of chitosan-induced Au/Ag alloy NPs dispersed in IL, and use of them in the construction of a novel biosensor for determination of glucose in pharmaceuticals sample by CL reaction. Materials and methods Chemiluminescence measurements were carried out by a Sirius tube luminometer (Berthold detection system, Germany) with a photomultiplier tube detector in a light-tight globular bottom glass cell of 10 mm diameter. The chitosan- induced Au/Ag alloy NPs dispersed in ion liquid were synthesised base on microwave method [6]. The CL biosensor based on covalent immobilization of glucose oxidase and gold-silver alloy NPs dispersed in ion liquid on glutaraldehyde-functionalized glass cell was fabricated. Results and discussion The analytical performance of the proposed biosensor was examined under the optimum condition. The calibration graph of emission intensity (I) versus glucose concentration was linear in the range of 1.8 × 10 (-6) to 7. 5 × 10 (-3) M (insert in Fig. 1), and the detection limit was 4.5 × 10(-7) M (S/N = 3). The regression equation was I = 1 × 10(+6) C + 14983 (where C is the glucose concentration, M) with a correlation coefficient of 0.9975 (n = 10). A complete analysis, including sampling and washing, could be performed in less than 2 min with a relative standard deviation of less than 2% for 5.0 × 10(-4) M glucose (n = 12). All those results indicated that the performance of the CL biosensor was compatible with those of the known and well assessed glucose CL sensors. The results with the present method for serum samples agreed with those obtained by the laboratory method. [Figure: see text] References; 1. Ionescu RE, Cosnier S, Marks RS. Anal. Chem. 2006;78:6327-6331. 2. Li B, Lan D, Zhang Z. Anal. Biochem. 2008;374:64-70. 3. Zhang Z, Zhang S, Zhang X . Rev. Anal. Chim. Acta 2005;541:37-47. 4. Haghighi B, Bozorgzadeh S.. Microchem. J. 2010;95:192-197. 5. Zhou Y. Curr. Nanosci. 2005;1:35-42. 6. Fan C, Li W, Zhao S, Chen J, Li X. Mater. Lett. 2008;62:3518-3520. P0017 The Study of Electrobioluminescence of Microscolex phosphoreus that found in Iran Mohammad javad Chaichi, Moslem Mansour Lakouraj, Shahram Ghasemi, Afsaneh Nemati University of Mazandaran, Babolsar, Mazandaran, Iran Introduction A method of the electrical pulse stimulation of an bioluminescence animal or electrobioluminescence(EBL) generates luminescence during a potential sweep or potential step programming [1-2] when the potential of electrode reaches to negative potential. This method has been previously demonstrated by Santhanam [1-4]. In EBL of the earthworm by potential sweep or electrolysis of an earthworm as electrode in an inert electrolyte solution produces luminescence [3]. This luminescence is due to the inflow of electron into the electrode, where oxygen is reduced to peroxide [3]. In continuation of these studies, we investigated the effect of addition salts solution to electrolyte solution on EBL process. It is a first report of existing of bioluminescent earthworm in Iran. Experimental In this method we used from connection Berthold detection systems, Sirius-tube luminometer and Dropsens, bipotentiostat/galvanostat (μStat 400) to each other. Each of instruments was connected to individual computer via different interface. Bioluminescent earthworms, that collect in the north of Iran, M. phosphoreus were washed well with distilled water and the outer surface were dried between the fold of filter paper. For producing earthworm electrode a gold wire (0.3mm × 5cm) was inserted into 15 segment of the post-clitelium region. Earthworm electrode as working electrode together with two graphite (2mm × 5cm) as the counter and reference electrode were placed into a cell which was made of pyrex. As shown in Fig. 65 the electrobioluminescence cell was placed instead of the common cell into luminometer instrument and it was connected to a μStat 400 by a function generator. Emission of light was investigated by luminometer instrument. Electrochemical behavior was investigated by cyclic voltammetry. [Figure: see text] Result and discussion The cyclic voltammetry of the earthworm electrode as base state recorded in 8mL of 0.1M phosphate buffer (pH = 7.5) as electrolyte solution with repetitive five cycles of the potential from 0.5 to -1.0 V and potential sweep rate of 50 mVs. Emission of the light was occurred continuously in the sweep towards negative potentials and the intensity of the emission was higher at more values of negative potential. First step is oxygen reduction. This step initiates luminescence in about E = -0.56 V. Generally it tends to move toward negative values. The second step in the reduction of O2 occurs at a more negative potential due to H2 O2 . This step would occur at about E = -0.90 V and in this potential, the intensity of the emission is maximum, after this potential at the return cycling toward positive potential, the light emission would return to the base value. For investigation the effect of addition salts solution to electrolyte solution on EBL process, 10μL of 10(-6) M salts added into electrobioluminescence cell and the effect of each other of the salts investigated individually. The curve of the cyclic voltammograms in presence of salts solution almost similar behavior during input potential programming. For example as seen in Fig. 66 for about Fe(3+) ion, the other ions such as Cu(2+) , Ca(2+) , Fe(2+) and Zn(2+) have enhance effect on the electrobioluminescence output, but Fe(2+) ion had more effective than the others. Therefore we can determine the ions and hydrogen peroxide by the EBL procedure. [Figure: see text] References; 1. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1990;24:249-256. 2. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1988;19:9-19. 3. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1986;15:341-351. 4. Ismail SA, Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1985;14:405-416. P0018 BRET(1) -assay using the FDSSμCell imaging plate reader; monitoring agonist-induced β-arrestin recruitment to a G protein-coupled receptor (GPCR) Frédéric Finana(a) , Jean Marc D'Angelo(b) , Luc De Vries(a) , Isabelle Rauly Lestienne(a) , Quynh-nhu Trinh-Xuan Kramer(b) , Romain Kramer(b) , Cyril Guerinot(a) (a) Centre de Recherche Pierre Fabre, Castres, France (b) Hamamatsu Photonics Europe, Massy, France The GPCRs represent the largest family of cell surface receptors and are the main target for drugs available on the pharmaceutical market. To prevent receptors from both acute and chronic overstimulation, GPCR activity is regulated by an intensively studied mechanism called desensitization or internalization. Following ligand exposure, arrestins interact with phosphorylated GPCRs, uncoupling them from their cognate G protein,blocking further activation and promoting endocytosis (2). Interaction between receptors and β-arrestins is a measurable functional event in the GPCR-mediated signaling cascade. The biophysical technique named Bioluminescence Resonance Energy Transfer (BRET) has been widely used to monitor and quantify agonist-promoted β-arrestin recruitment, including high throughput screenings. For the first time, Pierre Fabre Research Institute presents this BRET1 application on the FDSS/μCELL imaging plate reader (HAMAMATSU PHOTONICS) by monitoring the activity of the dopaminergic D2 receptor (short splice form), a prototypic and well characterized GPCR. P0019 Opsin-based extraocular photoreception in a luminous brittle star Jérôme Delroisse(a) , Esther Ullrich-Lüter(b) , Olga Ortega-Martinez(c) , Jérôme Mallefet(d) , Patrick Flammang(a) (a) Biology of Marine Organisms and Biomimetics, University of Mons, Mons, Belgium (b) Museum für Naturkunde, Berlin, Germany (c) Department of Biological and Environmental Science, University of Gothenburg, Kristineberg, Sweden (d) Laboratory of Marine Biology, Catholic University of Louvain, Louvain-La-Neuve, Belgium In the marine world, photoreception and bioluminescence constitute two opposite but intermingled phenomena related to light. Photoreception is a prerequisite for the bioluminescence perception and a luminous signal have to be perceived - by a prey, a predator or a conspecific - to be functionally efficient. Additionally photoreception could also be needed for the control of the bioluminescence process. In 2009, Tong et al. showed that the bioluminescent organ of a sepiolid squid possess light detection capability and use it to control the bacterial population of the photophore (and indirectly the light emission) [1]. More recently [2], molecular markers of photoreception - such as opsins - have also been identified in the photocyte of a ctenophore leading to the same conclusion of an intimate coupling between light perception and emission. Behavioral, morphological and molecular studies have shown that at least some echinoderms species have developed photoreception capabilities [3, 4]. Because of the lack of distinguishable eyes, echinoderm photoreception is usually considered as diffuse and tegumentary which makes that group particularly enigmatic! With the publication of the purple sea-urchin genome, a new window was opened on the understanding of the sensory capabilities in the phylum of echinoderms. The luminous brittle star Amphiura filiformis is known to use photoreception to synchronize its suspension feeding activity [5] but data are lacking concerning the molecular actors involved in the light perception. Because brittle star luminescence is considered to be highly controlled [6], could extraocular photoreception be linked to the light emission in these particular organisms? As previously shown in non-related marine species, extraocular photoreception could be used to control the photogenesis in brittle stars. Using transcriptomic and genomic data, we reported a decade of new putative opsin genes in the luminous brittle star A. filiformis. For nine A. filiformis o psin candidates, the Schiff base needed for the chromophore linkage was identified and the bona fide opsin status was confirmed. A. filiformis genome indeed codes for a large diversity of opsins including rhabdomeric and ciliary opsins but also minor groups of opsins such as Go opsins, neuropsins and peropsins. Transcriptome analysis and immunodetections revealed opsin expression in various organs of the brittle star. Interestingly, ciliary opsins and luciferase-like were co-immunodetected in the spines of the brittle star also described as the only light emitting areas of the brittle star. As light emission and perception actors seem to be expressed in a common organ, we hypothesize a dual role for the brittle star spine in both light emission and reception. A possible linkage between the two light-mediated processes is proposed. The optical implication of the spine skeleton in light perception and/or light emission is also suggested. Acknowledgements Contribution to the "Centre interuniversitaire de la Biologie Marine" (Belgium). Work supported in part by a FRFC Grant n° 2.4590.11. References 1. Tong D, Rozas NS, Oakley TH, Mitchell J, Colley NJ, McFall-Ngai MJ. Evidence for light perception in a bioluminescent organ. Proceedings of the National Academy of Sciences 2009;106(24);9836-9841. 2. Schnitzler CE, Pang K, Powers ML, Reitzel AM, Ryan JF, Simmons D, et al.. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi; a new view of ctenophore photocytes. BMC biology 2012;10(1);107. 3. Hendler G. An echinoderm's eye view of photoreception and vision. In Echinoderms; Munchen; Proceedings of the 11th International Echinoderm Conference, 6-10 October 2003, Munich, Germany. Taylor & Francis, 2006;339. 4. Ullrich-Lüter EM, D'Aniello S, Arnone MI. C-opsin Expressing Photoreceptors in Echinoderms. Integrative and comparative biology 2013;53(1);27-38. 5. Delroisse J, Flammang P, Mallefet J. Between emission and perception; do luminous brittlestars perceive their own light? In Proceedings of the 17th international symposium on Bioluminescence and Chemiluminescence, 2012. 6. Dewael Y, Mallefet J.. Luminescence in ophiuroids (Echinodermata) does not share a common nervous control in all species. Journal of experimental biology 2002;205(6);799-806. P0020 Luciferase-based microfluidic bioassays Ivan Densiov(a) , Anton Yakimov(a) , Kirill Lukyanenko(a) , Peter Belobrov(a,b) (a) Sibarian Federal University, Krasnoyarsk, Russia (b) Institute of biophysics SB RAS, Krasnoyarsk, Russia Bioluminescence inhibition assay based on components of bioluminescence system of luminous bacteria is the perspective express method for detection of organic and inorganic pollutants in liquids [1]. The method is based on interaction of pollutants with bacterial luciferase and NADH;FMN-oxidoreductase form the bacterial luminescence system, that leads to quenching of light emission and changing of a shape of measured kinetic curves. To make the portable device for environmental monitoring based on this method the automation is necessary. The purpose of the work was to test the possibility of automation of such bienzyme bioassay process through the integration of all reagents in one microfluidic chip. Channelized surface of the microfluidic chip was formed on the plate of polymethyl methacrylate (PMMA) by direct cutting [2] with Roland MDX-20. Luciferase from a recombinant strain of Escherichia coli, NADH-FMN-oxidoreductase from Vibrio fischeri, NADH and aldehyde were immobilized in starch gel [1] and placed in the reactor chamber (Fig. 67). FMN for reaction activation was deposited in a special chamber by the process of drop drying. Hermetic sealing was carried out by sticking a second plate of PMMA on 3M 467MR adhesive. [Figure: see text] Sample proceeded through the input channel into a chamber with FMN, where it dissolved FMN, and then stirred with it in the serpentine mixer [3]. When the sample with FMN entering the chamber with immobilized reagents and bioluminescence reaction starting. The kinetics of bioluminescence reactions was recorded during 200 seconds by the GloMax 20/20 single tube luminometer (Promega, USA). The test measurements of bioluminescence were conducted after 6 months of storage of chips with immobilized reagents in room conditions (23˚C, 1 atm, 30% relative humidity, direct sunlight). Second check was conducted after another 6 months. It was shown that the proposed microfluidic chip allows automating the process of bioassays; mixing of the sample with bienzyme systems and substrates - all in a single chip with pre-defined proportions of substrates without attracting qualified staff. The automation of bioassays in microfluidic chip allows softens requirements for the storage of immobilized reagents, as evidenced by the performance of chips stored under normal room conditions. This is true when stored for six months. After one year of storage the luciferase activity drops to 5% level. It was found that the adhesive used for hermetic sealing is hydrophobic and additional equipment (pump) must be used to fill the capillaries with the liquid sample. It was found that the luminescence intensity depends on the shape of the chamber with FMN (Fig. 68). [Figure: see text] The process of the luciferase-based bioassay based on quenching measuring can be automated by the usage of microfluidic technology and immobilization methods. The proposed topology, although in need to be further developed, but still demonstrates the ability to adapt the luciferase bioassay for use in more complex devices for personal ecology. 1. Esimbekova E, Kondik A, Kratasyuk V. Bioluminescent enzymatic rapid assay of water integral toxicity. Environmental Monitoring and Assessment 2013;185(7);5909-5916. 2. Howell Jr PB, Golden JP, Hilliard LR, Erickson JS, Mott DR, Ligler FS. Two simple and rugged designs for creating microfluidic sheath flow. Lab Chip 2008;8:1097-1103. 3. Capretto L, Cheng W, Hill M, Zhang X. Micromixing Within Microfluidic Devices. Top Curr Chem 2011;304:27-68. P0021 Quantum dynamics simulations of model chemiluminescence systems Ignacio Fernández Galván, Hans Karlsson, Michael Stenrup, Roland Lindh Uppsala Universitet, Uppsala, Sweden A common mechanistic feature of chemiluminescent and bioluminescent reactions is the existence of an "entropic trap", a region in the configuration space where the system dwells for a relatively long time and where the ground-state electronic surface is degenerate or quasi-degenerate with some of the excited states [1, 2]. It is generally assumed that this feature allows a transfer of population between the states, such that a significant proportion of the products is in an electronic excited states, capable of emitting light. The simulation of photochemical reactions, especially when electronic state surface crossings (or weakly avoided crossings) are involved, is generally performed with semi-classical methods, such as trajectory surface hopping (TSH) [3]. These methods have been used with success in cases where the electronic surfaces cross or approach at a localized point in the reaction coordinate, but there is some doubt on their appropriateness when the degeneracy exists for a finite length. The majority of quantum dynamics simulations of real or model systems have similarly dealt with single-point crossings as well. In this work, we perform quantum dynamics simulations of a model system, prototype for a chemiluminescent reaction, that includes a finite-length region of degeneracy between the states involved. We focus on how the reaction outcome, and in particular the relative population of the states, depends on the length of the degeneracy region, the energy difference and the coupling between the states. The model system was created by constructing diabatic potential energy curves based on accurate ab initio potentials for the 1,2-dioxetane chemiluminescence reaction [2]. The quantum dynamics simulations solve the time-dependent Schrödinger equation for the nuclei, subject to the model potentials. To render the problem tractable with current techniques, the model system was created with reduced dimensionality. 1. Yue L, Liu Y-J, Fang W-H. J. Am. Chem. Soc. 2012;134:11632-11639 2. Farahani P, Roca-Sanjuán D., Zapata F, Lindh R. J. Chem. Theory Comput. 2013;9:5404-5411 3. Barbatti M. WIREs Comput. Mol. Sci. 2011;1:620-633 P0022 Towards the Understanding of the Larger Phosphorescence Quantum Yield than Fluorescence in Dioxetanone Antonio Francés-Monerris(a,b) , Daniel Roca-Sanjuán(a) , Ignacio Fernández Galván(b) , Roland Lindh(b) (a) Instituto de Ciencia Molecular, Universitat de València, Valencia, Spain (b) Department of Chemistry-Angstrom, Theoretical Chemistry Programme, Uppsala University, Uppsala, Sweden Dioxetanone is a key chemical structure in many bioluminescent systems, such as the firefly luciferin, coelenterazine, etc.(1) The molecular structure of dioxetanone bears the chemiluminophore properties, providing a channel for a thermally activated chemical reaction that produces a product in an electronically excited state.(1) Even though the general aspects of the chemiluminescent mechanism have been determined,(2) the higher quantum yield of phosphorescence than fluorescence observed experimentally cannot be completely rationalized. Hence, according to previous works employing the complete-active-space second-order perturbation theory//complete-active-space self-consistent field (CASPT2//CASSCF),(2) no significant differences were found for the accessibility from the ground state (S0 ) to the two lowest-lying singlet (S1 ) and triplet (T1 ) excited states which are commonly associated to the emissive states. In other theoretical studies,(3) a concerted mechanism for the decomposition reaction of dioxetanone was proposed based on results obtained with the density functional theory (DFT) and time-dependent TD-DFT methods. The S1 state was found to have much higher energy than S0 and T1 , which was used to explain the preference for triplet emission rather than singlet emission. However, the theoretical approach employed was later proved to be not correct.(4,5) In the present contribution, we have revisited the previous CASPT2//CASSCF study by Liu et al.(2) focusing on the triplet-singlet ratio problem. Dynamic electron correlation has been taken into account for determining the geometries by using the CASPT2//CASPT2 approach.(6) Moreover, the relevance of the second triplet excited state (T2 ) in the chemi-excitation process has been studied. The new findings shed light on the higher probability for triplet than singlet emission in dioxetanone. References 1. Navizet I, Liu YJ, Ferré N, Roca-Sanjuán D, Lindh R. ChemPhysChem 2011;12:3064-3076. 2. Liu F, Liu YJ, De Vico L, Lindh R. J. Am. Chem. Soc. 2009;131:6181-6188. 3. Da Silva LP, Da Silva JCGE. J. Comput. Chem. 2012;33:2118-2123. 4. Roca-Sanjuán D, Lundberg M, Mazziotti DA, Lindh R. J. Comput. Chem. 2012;33:2124-2126. 5. Yue L, Roca-Sanjuán D, Lindh R, Ferré N, Liu YJ. J. Chem. Theor. Comput. 2012;8:4359-4363. 6. Roca-Sanjuán D, Aquilante F, Lindh R. WIREs Comput. Mol. Sci. 2012;2:585-603. P0023 Suitability of Macrolampis sp2 and Pyrearinus termitilluminans luciferases for light off bacterial biosensors of environmental toxicity Gabriele Verônica de Mello Gabriel(a) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar - Sorocaba), Sorocaba, São Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, São Paulo, Brazil Bioluminescence has been used for decades as a bioanalytical tool. Among its several applications its use in biosensors is increasing, including cellular toxicity biosensors. Water pollution is an environmental concern, therefore is of extreme interest the development of new biosensors. In this work we investigated the suitability of the Macrolampis sp2, a pH-sensitive luciferase, and Pyrearinus termitilluminans, a pH-insensitive luciferase, for bioluminescent bacterial light off biosensors of toxic agents such as metals and disinfectants. For this purpose, Escherichia coli were transformed with plasmids containing the cDNA of the above beetle luciferases, their bioluminescence analyzed by luminometry and CCD imaging in presence of toxic agents, and the sensitivity of this assay was compared with the naturally bioluminescent bacteria Vibrio fischeri from Biolux® kit. In living bacteria, bioluminescence of Macrolampis sp2 luciferase displays a flash like kinetics with a fast decay, being more suitable for fast assays with sensitive equipments, whereas P. termitilluminans displays a slower kinetics with a more intense and sustained luminescence being more suitable for less sensitive detection methods that require light integration. The luminometric assay is sensitivity enough to detect a minimum of 10 bacterial CFU/mL after 10s of signal integration, while in CCD assay a minimum of 100 bacterial CFU/mL could be detected by CCD imaging. The bioluminescent of E. coli showed sensitivity to different toxic agents such as metals, sanitizers and antiseptics with comparable sensitivity to the naturally bioluminescent bacteria V. fischeri. In general, these light off biosensors do not show specificity for any class of toxic agents, being potentially useful only as general toxicity biosensors. The exceptions are mercury and silver, for which these biosensors show special sensitivity, showing potential application in the detection of the presence of these metals in contaminated waters. Finally, in order to have a more stable assay for field measurements, we have also immobilized the bioluminescent bacteria in agarose in an Elisa plate. In this format, immobilized bacteria kept strong bioluminescence activity for 3 weeks when stored at 4 °C. (Financial support; FAPESP and CNPq). P0024 Conversation with a bioluminescent planktonic worm Anaïd Gouveneaux, Jérôme Mallefet Catholic University of Louvain, Louvain-la-Neuve, Belgium Tomopteris helgolandica is a transparent planktonic annelid (Polychaeta; Tomopteridae) able to produce yellow flashes (λmax  = 572 ± 2 nm) [1]. This cryptic species lives in the mesopelagic zone where the majority of organisms are visually adapted to the blue color dominating this environment [2]. Thus, it has been suggested that such an original bioluminescent signal could be used for intraspecific private communication [3]. But, is T. helgolandica able to perceive its own light? We know that 3-days larvae develop a pair of pigmented ocelli consisting of seven large rhabdomeric sensory cells topped by a lens [4]. The early stages are positively phototactic [5] but behavioral observations of adult specimens of the yellow-emitter T. septentrionalis have revealed photophobic responses to blue light flashes simulating dinoflagellate bioluminescence [6]. Through a similar approach, we have tested the behavioral effect of simulated bioluminescent signals on our model species. Isolated specimen were placed under infra-red lighting in a round aquarium and filmed by a coupled CCD video recording system. One camera was sensitive to the IR so we can track the animal moving. Simultaneously, an intensified camera only recorded the bioluminescent events. Manually controlled light signals were applied through a fake worm - provided with optic fibers reproducing the distribution pattern of T. helgolandica's photogenic organs - immersed in the seawater. We tested 0,2 s(-1) flashes and continuous signals as well as five different light colors (blue, green, yellow, orange and red). The 50h of video collected were analyzed using the video tracking software Ethovision XT (Noldus Information Technology). Spontaneous light emissions have been observed during physical contacts with the fake worm and during stressfull situation like emersion but they did not seem to appear in response to the simulated light signals. They did not demonstrate specific interest in the yellow light emission but seemed attracted by the continuous blue light signals. These results lead us to reassess the virtually admitted hypothesis of intraspecific communication. We are no w currently testing the hypothesis that T. helgolandica might use light signals against its predators as suggested by its responses to mechanical stimuli. Finally, independently to its own bioluminescence capabilities, it might take advantage of the bioluminescence emitted by its preys. References 1. Gouveneaux A, Mallefet J.. Physiological control of bioluminescence in a deep-sea planktonic worm, Tomopteris helgolandica. Journal of Experimental Biology 2013;216:4285-4289. 2. Warrant EJ, Locket NA. Vision in the deep sea. Biological Reviews 2004;79(3);671-712. 3. Dales RP. Bioluminescence in pelagic polychaetes. Journal of the Fisheries Board of Canada 1971;28(10);1487-1489. 4. Åkesson B. On the eyes of Tomopteris helgolandica (Tomopteridae, Polychaeta). Acta Zoologica 1964;45(3);179-189. 5. Åkesson B. The embryology of Tomopteris helgolandica (Polychaeta). Acta Zoologica 1962;43(2-3);135-199. 6. Buskey EJ, Swift E. Behavioral responses of oceanic zooplankton to simulated bioluminescence. The Biological Bulletin 1985;168(2);263. P0025 Improved method for chemiluminescent determination of activity of peroxidase-mimicking DNAzyme Anastasia Gribas(a) , Shulin Zhao(b) , Ivan Sakharov(a) (a) Lomonosov Moscow State University, Moscow, Russia (b) Guangxi Normal University, Guilin, China The optimization of experimental conditions for the chemiluminescent determination of peroxidase-mimicking DNAzyme formed at interaction of hemin and its aptamer EAD2 was performed. The effect of concentrations of hydrogen peroxide and luminol, acidity of the substrate solution and chemical nature and concentration of used buffer was estimated. Under optimized conditions a value of detection limit for the DNAzyme was 80 nM. Comparison of the conditions determined in this work with those reported previously showed that the optimization of the composition of substrate solution improved the sensitivity of the chemiluminescent determination of the DNAzyme. The obtained results open up promising perspectives for using the proposed method to improve the sensitivity of DNAzyme-based assays. Acknowledgement The authors thank the Russian Foundation for Basic Research (NK-13-04-91164/13 and 13-04-91164_GFEN_a) and the National Natural Science Foundations of China (Grant No. 21311120056) for financial support. P0026 Synthesis, Characterization of Lanthanides (III) Nitrate Complexes derived from Isonicotinic Acid Hydrazide and Studies on their fluorescence properties and Antimicrobial Activities Ahmed Hijazi, Ziyad Taha, Abdulaziz Ajlouni Jordan University of Science and Technology, Irbid, Jordan The work will describe the synthesis of Lanthanide (III) nitrate coordination complexes of hydrazones of N-(2-hydroxynaphthalen-1-yl) methylene) Isonicotinohydrazide with general formula [LnL 2 (NO3 )2. xH2 O]NO3 where [Ln = La, Pr, Sm, Nd, Er, Dy, Eu, and Gd L = N-(2-hydroxynaphthalen-1-yl) methylene) Isonicotinohydrazide. The spectroscopic methods such as IR, TGA-MS, (1) HNMR, (13) CNMR, Molar Conductivity Measurement, Elemental analysis were used to analyze and characterize ligand and complexes. The spectroscopic data show the 2;1 electrolytic nature of the complexes with a general chemical formula of [LnL 2 (NO3 )2. xH2 O]NO3 . Antioxidant and antimicrobial activities of both ligand and complexes were tested. The fluorescence properties of complexes were studied in detail. P0027 Theoretical study of pH dependence on photoluminescence of firefly luciferin Miyabi Hiyama(a) , Kenta Yamada(b) , Toshimitsu Mochizuki(a) , Hidefumi Akiyama(a) , Nobuaki Koga(c) (a) The University of Tokyo, Kashiwa, Chiba, Japan (b) Kyoto University, Kyoto, Japan (c) Nagoya University, Nagoya, Aichi, Japan Firefly luciferin (hereafter luciferin) is a substrate of a bioluminescence reaction and converted into oxyluciferin which creates bioluminescence. The key chemical reaction for firefly luminescence is known to be the oxidation of luciferin in luciferase protein. The spectroscopic information of luciferin is important for understanding such reaction. One cause for emission color change is the pH values of the solvent. The solvent pH value can change molecular structures and the chemical reaction pathway. The aim of our study is to elucidate the pathway of the photoluminescence process of luciferin in aqueous solution at different pH values based on theoretically estimated absorption spectra and free energy in the first excited states, S1 . [Figure: see text] The structures of luciferin and its conjugate acids and bases were optimized, and vibrational analyses at the B3LYP/aug-cc-pVTZ level were performed. The excitation energies and oscillator strengths were calculated by the time dependent density functional theory (TDDFT) method. The solvation effect in aqueous solution was taken into account by the polarizable continuum model. The concentrations of the chemical species in solutions with different pH values were estimated from the theoretical free energies. Figure 69 shows the theoretical absorption spectra at pH 1-10. The main peak at pH 10 corresponds to dianion, that at pH 8 corresponds to anion, and that at pH 2 corresponds to luciferin. Fig. 70 shows the relative free energies for dianion, phenol-anion, and luciferin in S1 state based on phenolate-anion. One important result is that the pathway for green emission at pH 8 is the emission from the equilibrium structure of S1 (S1 (eq) ) of dianion through the absorption of phenol-anion. However, the pathway for green emission at pH 2 is the emission from S1 (eq) of phenolate-anion through the absorption of luciferin. [Figure: see text] Not only the emission energy but also the free energy of the S1 states and the absorption energy are useful in understanding the photoluminescence pathway. Chemical species in acidic solutions may produce a different color emitter, which would be verified through future research. Reference 1. Hiyama M et al., Photochem. Photobiol. 2014 in press. 2. Hiyama M et al. Photochem. Photobiol. 2014;90:35-44. P0028 Atrazine and cyanuric acid detection with bioluminescent bacterial reporters. Anna HUA, Hervé GUEUNE, Mickaël CREGUT, Gérald THOUAND, Marie-José DURAND University of Nantes, Nantes, France Atrazine has been used worldwide as an herbicide for almost 30 years. Due to its low degradation in environments and its ecotoxicological properties, atrazine is forbidden in most European countries but still in use in the United States. Atrazine and other S-triazines compounds could be used as nitrogen souces by some microoganisms such as Pseudomonas sp ADP. In Pseudomonas sp ADP, atrazine is converted into cyanuric acid which is catalysed by three enzymatic steps encoded by the gene products of atzA, atzB, atzC. Thereafter, cyanuric acid degradation into NH3 and CO2 , are encoded by atzDEF operon which is under the control of the AtzR regulator. Monitoring in the environment atrazine as well as cyanuric acid could be done by severals methods including chemical analysis (chromatography) or immunoassays. Even if these methods are sensitive, there are expensive compared to bacterial bioassays. In this study we propose to evaluate the availability to dispose of specific bioluminescent bacteria to detect atrazine and cyanuric acid as biosensor. Methods; two vectors have been designed in Escherichia coli leading to two specific bacteria. 1/HG003 In order to detect cyanuric acid, the pBBluxatzD vector resulted from a transcriptional fusion of the atzR regulator and the atzD promoter with the luxCDABE genes of pBBluxMCS. A constitutive expression of AtzR was obtained by cloning atzR under plac promoter of pUC19. These vectors were transformed in E.coli TOP 10 leading to the bacteria E.coli pUCatzR pBBluxpatzD (HG003). 2/ HG004 The pUCatzABCR (this plasmid expressed the atzABC genes involved in the degradation of atrazine to cyanuric acid and the atzR gene under the control of plac) vector was transformed in E.coli pBBluxpatzD in order to obtain E.coli pUCatzABCR pBBluxpatzD (HG004). Results; bacteria were grown in Terrific Broth medium and freezed dried. After rehydration bacteria were exposed to the ch emicals. HG003 was exposed to cyanuric acid, after1.5 hours incubation, the bioluminescence was increased significantly. The detection limit was around 6 mg/L (in water), while no induction of bioluminescence was observed in the presence of atrazine. Bioluminescence of HG004 was observed after atrazine exposition (20 mg/L) and cyanuric acid but no induction was observed with other triazine compounds (irgarol, cymazine …). Perspectives; our results showed that using a part of atrazine degradation pathway and regulatory systems of Pseudomanas sp ADP, new bioelements have been developed. Exposition of bacteria with environmental samples is under consideration. P0029 Dynamic flash of light pattern on a luminous colony induced by oxygenation Hajime Karatani(a) , Hajimu Kawakami(b) , Yukihiro Nishikawa(c) (a) Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto, Japan (b) Department of Electronics and Informatics, Ryukoku University, Otsu, Japan (c) Department of Macromolecular Science and Engineering, Kyoto Institute of Technology, Kyoto, Japan Bacterial bioluminescence (BL) is arising from the luciferase reaction with reduced flavin mononucleotide (FMNH2 ), long-chain aliphatic aldehyde and molecular oxygen. The bacterial luciferase reaction is possibly associated with the respiratory activity.([1]) Once luminous bacteria have made a colony, they would exhibit BL as a cell population. In this study, the dynamic change in the colony BL, induced under oxygen rich conditions, has been characterized from a viewpoint of a collective action. For this purpose, strongly bioluminescent Photobacterium phosphoreum bmFP was used for the characterization. For comparison, a self-luminescent Escherichia coli, hovering the pETBlue-2 plasmid with a lux operon, was prepared and subjected to this characterization. The lux operon was isolated from P. phosphoreum bmFP and inserted into pETBlue-2 by Clontech In-fusion cloning reaction. An aliquot of the diluted luminous culture of P. phosphoreum bmFP was spread onto NaCl complete agar (1.0, 1.25 and 1.5w/v%) plate and a single colony was grown at 20 °C. For a self-luminescent E. coli, its colony was grown on 2YT agar (1.5w/v%) plate, containing 50 g/mL carbenicillin and 800 M isopropyl -D-1-thiogalactopyranoside, at 25 °C. The BL image of a colony was acquired at regular time intervals by a digital camera under oxygen- and argon- rich conditions. In the case of rather small colony (< 1 mm in diameter), a Nikon fluorescence microscope Eclipse E600W or Ti with no excitation light was used in combination with a Keyence color CCD VB-6000 or a Nikon color CCD DS-Ri1. The pixel values of time-series BL images were converted to the brightness images and then the brightness data at each pixel position were transformed into the frequency domain by the discrete Fourier transformation (DFT). The forefront of the colony usually showed the bright BL, independently of the colony size. By oxygenation, circular flash of light occurred in the area in the vicinity of the colony forefront and subsequently it moved toward the colony center while narrowing down the diameter. Such a rapid appearance circular BL seemed to be phenomenologically analogous to the oxygen triggered flash of light in the luminous liquid-medium under the oxygen-limited conditions. ([2], [3], [4]) The time required for the appearance of the circular BL followed by propagation toward to the colony center was roughly several seconds. By switching the ambient gas from oxygen to argon, the bright BL area reversely became darker from the vicinity of the center of the colony toward its forefront. The DFT analysis showed that the dynamic BL pattern induced by the oxygenation might be synchronized on the concentric circle area with one another. The colony of the lux gene overexpressing E. coli also showed a dynamic BL pattern in response to the oxygenation, although it was not so clear as compared with the colony of P. phosphoreum bmFP. The dynamic flash of light pattern mentioned above may be as a result of a collective action of the cells in a colony under the oxygen-rich conditions. Bacterial bioluminescence (BL) is arising from the luciferase reaction with reduced flavin mononucleotide (FMNH2 ), long-chain aliphatic aldehyde and molecular oxygen. The bacterial luciferase reaction is possibly associated with the respiratory activity.([1]) Once luminous bacteria have made a colony, they would exhibit BL as a cell population. In this study, the dynamic change in the colony BL, induced under oxygen rich conditions, has been characterized from a viewpoint of a collective action. For this purpose, strongly bioluminescent Photobacterium phosphoreum bmFP was used for the characterization. For comparison, a self-luminescent Escherichia coli, hovering the pETBlue-2 plasmid with a lux operon, was prepared and subjected to this characterization. The lux operon was isolated from P. phosphoreum bmFP and inserted into pETBlue-2 by Clontech In-fusion cloning reaction. An aliquot of the diluted luminous culture of P. phosphoreum bmFP was spread onto NaCl complete agar (1.0, 1.25 and 1.5w/v%) plate and a single colony was grown at 20 °C. For a self-luminescent E. coli, its colony was grown on 2YT agar (1.5w/v%) plate, containing 50 g/mL carbenicillin and 800 M isopropyl -D-1-thiogalactopyranoside, at 25 °C. The BL image of a colony was acquired at regular time intervals by a digital camera under oxygen- and argon- rich conditions. In the case of rather small colony (< 1 mm in diameter), a Nikon fluorescence microscope Eclipse E600W or Ti with no excitation light was used in combination with a Keyence color CCD VB-6000 or a Nikon color CCD DS-Ri1. The pixel values of time-series BL images were converted to the brightness images and then the brightness data at each pixel position were transformed into the frequency domain by the discrete Fourier transformation (DFT). The forefront of the colony usually showed the bright BL, independently of the colony size. By oxygenation, circular flash of light occurred in the area in the vicinity of the colony forefront and subsequently it moved toward the colony center while narrowing down the diameter. Such a rapid appearance circular BL seemed to be phenomenologically analogous to the oxygen triggered flash of light in the luminous liquid-medium under the oxygen-limited conditions. ([2], [3], [4]) The time required for the appearance of the circular BL followed by propagation toward to the colony center was roughly several seconds. By switching the ambient gas from oxygen to argon, the bright BL area reversely became darker from the vicinity of the center of the colony toward its forefront. The DFT analysis showed that the dynamic BL pattern induced by the oxygenation might be synchronized on the concentric circle area with one another. The colony of the lux gene overexpressing E. coli also showed a dynamic BL pattern in response to the oxygenation, although it was not so clear as compared with the colony of P. phosphoreum bmFP. The dynamic flash of light pattern mentioned above may be as a result of a collective action of the cells in a colony under the oxygen-rich conditions. References 1. Harvey EN. Bioluminescence. New York; Academic Press 1952;1-96. 2. Hastings JW. J. Cell. Comp. Physiol. 1952;39:1-30. 3. Lloyd D, James CJ, Hastings JW. J. Gen. Microbiol. 1985;131:2137- 40. 4. Karatani H, Yoshizawa S, Hirayama S. Photochem. Photobiol. 2004;79:120-5. P0030 Silver nanoparticle enhanced flow-injection chemiluminescence method for determination of hesperidin in pharmaceuticals Sayed Yahya Kazemi, Seyed Mohammad Abedirad Department of Basic Sciences,Sari Agricultural Sciences and Natural Resources University, Sari,Mazandaran, Iran Hesperidin (hesperetin 7-rhamnoglucoside) (Fig. 71), belongs to the flavanone type flavonoids, predominantly occurring in Citrus species. Hesperidin is thought to reduce capillary permeability and to have anti-inflammatory action, hence it is used to shrink hemorrhoids, reduce varicose veins and battle viral infections [1]. Hence, introduce the efficient method for determination of hesperidin is currently of interest. Chemiluminescence(CL) has been widely used as a rapid analytical technique in many basic research and practical applications, such as biology, pharmacology, environmental chemistry, clinical diagnosis, and food analysis, due to its low background nature, high sensitivity, simplicity, and low cost of instrumentation. Amongst the CL based technique, flow injection chemiluminescence (FI-CL) has caused considerable attention because of its convenient operation, rapid determination and accuracy [2-4]. In this,work we developed a simple, rapid and sensitive method for determination of hesperidin in pharmaceutical formulations. Ag nanoparticles was applied as mimic peroxidase for reaction of luminol and hydrogen peroxide.It was found that hesperidin could dramatically decreased CL signal. Under optimum condition and at flow rate of 2.7 mL min(-1) , a linear working range for hesperidin concentrations ranging from 3 × 10 (-8) to 5 × 10 (-6) mol L(-1) (r > 0.9888, n = 8), was obtained, with a detection limit of 1.8 × 10 (-9) mol L(-1) . The relative standard deviations were less than 3.1% (n =8) and recovery was 96%. The method was proved to be sensitive and selective and was applied to determine of hesperidin in pharmaceuticals with satisfactory results. [Figure: see text] References 1. Garg A, Garg S, Zaneveld LJD, Singla AK. Chemistry and Pharmacology of The Citrus Bioflavonoid Hesperidin. Phytother Res 2001;15:655-69. 2. Kazemi SY, Abedirad SM. Effect of glutathione on peroxyoxalate chemiluminescence of hypericin as the fluorophore. Spec Chim Acta A 2014;118:782-6 3. Zhang ZF, Cui H, Lai CZ, Liu LJ. Gold nanoparticle-catalyzed luminol chemiluminescence and its analytical applications. Anal Chem 2005;77:3324-9. 4. Iranifam M. Revisiting flow-chemiluminescence techniques; pharmaceutical analysis. Luminescence2013;28:798-820. P0031 Biological module of bioluminescent biosensor for environmental toxicity assay Victoria Lonshakova-Mukina(a) , Elena Esimbekova(a,b) , Valentina Kratasyuk(a,b) (a) Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Enzymes of luminous bacteria are actively used as to estimate metabolites and enzyme activity in biological samples, as to analyze integral toxicity in ecological monitoring [1-3]. We recently reported an immobilized multicomponent reagent based on coupled enzyme system NADH;FMN-oxidoreductase-luciferase from luminous bacteria [4]. However, the problem of keeping the reagent active during storage has not been solved definitely. Thus, the reagent can lose up to 80 % of its activity after storage at 4°C for 1 year. The goal of this work was to analyze the characteristics of the coupled enzyme system NADH;FMN-oxidoreductase-luciferase co-immobilized in starch gel with their substrates depending on the type and amount of stabilizer in order to develop a biological module of bioluminescent sensor combining high activity, stability during storage and use, and sensitivity to toxic substances within maximum permissible concentration (MPC). The obtained biomodule was a dried disc with a diameter of 6-7 mm; its dry weight was 1.5 ± 0.2 mg. We varied the concentration of the following enzyme stabilizers included in the biomodule; dithiothreitol (DTT), bovine serum albumin (BSA) and mercaptoethanol. The effect of stabilizers on biomodule characteristics was determined by the changes in the maximum light emission intensity. The largest increase in the biomodule activity caused by adding DTT or mercaptoethanol was observed when their concentration in the reagent was 0,1 mM. Changes in the biomodule activity during storage for 6 months at a temperature of 4(0) С were examined. It was shown that the activity of the control sample that didn't include any stabilizer decreased by 5 times in 6 months. Adding stabilizers to the biomodule makes it possible to reduce the decrease of activity during storage. The best result was achieved for the biomodule containing DTT at a concentration of 0,1 mM. Firstly, in this case the recession of the maximum luminous intensity during storage was less intensive as compared to the control sample, and after 6 months the residual luminous intensity was 80 % of the initial luminous intensity (Fig. 72). [Figure: see text] Secondly, among the obtained biomodules with different stabilizers, the biomodule containing 0,1 mM of DTT was the most sensitive to toxic substances (heavy metal salts, phenols, quinones). The biomodule has been shown to be more sensitive to quinones and the detection limit for them was less than their MPC. Finally, it was stable to various physical and chemical environmental factors, thus it can be used both in laboratory and field conditions. The developed biomodule can be integrated as a biological indicator into the bioluminescent biosensor for toxicity assay. The work was financially supported by the Russian Academy of Sciences (Program "Molecular and Cell Biology", grant No 6.8) and by the state contract between Ministry of Education and Science and Siberian Federal University, № 1762. Reference 1. Girotti S, Ferri EN, Fumo MG, Maiolini E. Anal. Chim. Acta. 2008;608:2-29. 2. Roda M, Guardigli E, Michelini E, Mirasoli M. Trends in Anal. Chem. 2009;28:307-322. 3. Kratasyuk VA, Esimbekova EN, Gladyshev MI, Khromichek EB, Kuznetsov AM, Ivanova EA. Chemosphere. 2001;42:909-915. 4. Esimbekova E, Kondik A, Kratasyuk V. Environmental Monitoring and Assessment. 2013;185:5909-5916. P0032 Structural properties of tryptophan microenvironment in bacterial luciferase Anna A. Deeva(a) , Elena V. Nemtseva(a,b) , Valentina A. Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Bacterial luciferase is widely used in analytical research studies wherein experimental conditions could vary causing structural transitions of the enzyme. To analyze protein structure and conformation the method based on protein intrinsic luminescence of three aromatic fluorophores - tryptophan (Trp), tyrosine (Tyr) and phenilalanine (Phe) - can be used [1]. In comparison with Tyr and Phe, Trp is very sensitive to polarity of the environment due to the large redistribution of electron density in the indole moiety after the absorption of photons [2]. The empirical analysis of a great number Trp fluorescence spectra in different proteins showed a correlation between spectral and structural properties allowing to divide Trp residues according to their micro-environment features into five spectral classes [3]; A, S, I, II, III. However a theoretical study based on the analysis of a crystal structure is more convenient for multi-tryptophan proteins like bacterial luciferase which spectrum is a sum of several components [4]. A bulk experimental data on Photobacterium leiognathi (Ph. l.) bacterial luciferase activity in different conditions is obtained at the moment. The aim of the study is to examine the main characteristics of each tryptophan environment in bacterial luciferase from Ph. l. to simplify the fluorescence analysis of this protein. As the crystal structure of the Ph. l. luciferase has not been solved yet, the structure of Vibrio harveyi (V. h.) bacterial luciferase (PDB entry; 3FGC) was used. The alignment of their sequences with BLAST showed ~61% homology of α-subunits and ~51% of β-subunits for these two luciferases. Firstly, differing amino acids located at the distance of 7.0 A from indole ring atoms of Trp residues in V. h. luciferase were mutated according to the alignment. Then an optimization of changed residues was made using molecular mechanics GROMOS force field implementation of Swiss-PdbViewer while remaining residues were kept fixed. The structural characteristics of new environment for seven Trp residues (Fig. 73) were analyzed; atoms O, N and S at the distance of 7.0 A from the center of indole ring and indole ring atoms, the number of atoms located in a sphere of radius 7.0 A, packing density, accessibility to water mole cules and relative polarity. [Figure: see text] Relying on the estimated parameters noticed above we defined the spectral class [3] for each Trp residue in the structure of the mutated luciferase. The results are represented in the Table 1. [Table: see text] Fluorophores of class S (αTrp40) are deeply buried inside a protein but the presence of potential hydrogen-bonding partners makes possible exiplexes formation and weak dipole-dipole interaction. The analysis of αTrp40 potential hydrogen bond donors showed that it might be partially quenched by SH group of αCys171. Trp residues belonging to the class I (330 - 333 nm) prevail over other classes, that corresponds to experimental fluorescence spectra for Ph.l. luciferase peaked at λmax = 330 - 334 nm with spectral width at half maximum Δλ = 55-60 nm. The environment of Trp residues belonged to class I has lower packing density than class S that means flexible environment facilitating the hydrogen-bonded exciplex formation and dipole relaxation during the lifetime of fluorophore excited state. αTrp194 belongs to class II implying the presence of the structured water near to indole ring. One of the Trp residues was found to be exposed to the solvent - αTrp277 (class III). Fluorophores of this class have spectral properties similar to the free Trp in solution. Thus, our research showed that structural changes of bacterial luciferase inner regions should be analyzed in the range <310 nm of fluorescence spectra to avoid the external tryptophan contribution. The changes of the αTrp277 microenvironment caused by substrate or quencher binding should be studied under the wavelength >390 nm. Acknowledgments The research was partially supported by the Project 1762 from The Ministry of Education and Science of the Russian Federation. References; 1. Kuznetsova IM, Yakusheva TA, Turoverov KK. Contribution of separate tryptophan residues to intrinsic fluorescence of actin. Analysis of 3D structure. FEBS Letters. 1996;452:205-210. 2. Burstein EA. The intrinsic luminescence of proteins is a method for studies of the fast structural dynamics. Molecular Biology. 1983;17:455-67. 3. Lakowicz JR. Principles of fluorescence spectroscopy. 3rd ed. Springer, 2006;530-37. 4. Reshetnyak YK, Koshevnik Y, Burstein EA. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. Biophys. J. 2001;81:1735-58. P0033 Gelatin and Starch Media Stabilize Bacterial Luciferase and Oxidoreductase Anna Bezrukikh(a) , Elena Esimbekova(a,b) , Valentina Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia The coupled enzyme system of luminous bacteria NADH;FMN-oxidoreductase-luciferase is used to produce the multi-purpose starch and gelatin immobilized reagents for bioluminescent analysis which meet the requirement of high stability during storage and use [1,2]. The objective of this work was to examine the effect of viscous gelatin and starch microenvironments on the coupled enzyme system when exposed to various physical and chemical environmental factors. We have studied the activity of the enzymes at different temperatures and their thermal inactivation with the additions of 1% and 5% gelatin, 2% starch and in buffer solution without additives within a temperature range from 10 to 43С. To study the effect of gelatin and starch on the stability of the coupled enzyme system in various ionic environments, we have used solutions with pH levels ranging from 5.9 to 8.0 and solutions with different ionic strengths from 0.01 to 0.4 moles, pH 6.8. The temperature optimum in the starch environment does not change in comparison with that of the control and constitutes 25С. In a gelatin environment the temperature optimum shrinks and shifts to lower temperatures due to the gelatin jellification observed under these conditions. The process of thermal inactivation includes two stages, which probably correspond to enzyme dissociation into subunits and their subsequent denaturation (Fig. 74). Comparison of the effective rate constants of thermal inactivation obtained in the starch environment, gelatin environment and in a buffer solution without additives demonstrates that the viscous environments of gelatin and starch does not affect the thermal inactivation of the enzymes. The exception is the case when gelatin is in gel form (i.e. when the temperature is lower than that of gelatin jellification). Under such conditions the coupled enzyme system is activated, while in buffer solution without gelatin it loses its activity. We also observed that the pH optimum of the coupled enzyme system does not change in the presence of gelatin, and when starch is added, the pH optimum shrinks and shifts to the acidic zone. The gelatin and starch environments have a stabilizing effect on the enzymes in the alkaline zone. Moreover, in gelatin and starch the optimal ionic strength of the solution shifts to the lower values. [Figure: see text] Thus the viscous microenvironments of both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under certain conditions. The results obtained in this work can help to improve the preparation procedure of gelatin and starch immobilized reagents for bioluminescent analysis and as a result to increase their stability and activity. The work was supported by the Russian Academy of Sciences (Program "Molecular and Cell Biology", grant No 6.8) and the Project 1762 from the Ministry of Education and Science of Russian Federation. 1. Esimbekova EN, Torgashina IG, Kratasyuk VA. Comparative study of immobilized and soluble NADH;FMN-oxidoreductase-luciferase coupled enzyme system. Biochemistry (Moscow) 2009;74(6);695-700. 2. Esimbekova E, Kondik A, Kratasyuk V Bioluminescent enzymatic rapid assay of water integral toxicity. Environ Monit Assess 2013;185(7);5909-5916. P0034 Bioluminescent Enzymatic Methods for Toxicological Safety Testing of Food Additives Anastasiia Asanova(a) , Elena Esimbekova(a,b) , Valentina Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Express evaluation of food additives safety is an actual task that can be solved using a bioluminescent assay. Currently only methods for food testing based on firefly bioluminescence to assess the quality of bacterial contamination are widely used [1]. The bioluminescent assays for determination of L-and D-lactate in beer and wheat grain infected with Fusarium also have been designed [2-3]. Thus, a great potential of bioluminescent bioassays for food safety testing is still not implemented. The purpose of this study was to develop a variety of bioluminescent approach for testing the toxicological safety of food additives. The new method was based on bacterial coupled enzyme system FMN;NADH-oxidoreductase-luciferase (R + L). The loss of light emission intensity of couple enzyme system in the presence of food additives has been estimated. Also the toxic effects of additives on the bioluminescence of the three triple enzyme systems R + L + trypsin, R + L + ADH (alcohol dehydrogenase) and R + L + LDH (lactate dehydrogenase) have been analyzed [4]. The following additives were tested; sodium benzoate (Е 211), potassium sorbate (Е 202) and sorbic acid (Е 200). The effects of nanomaterials such as Ag, Cu, Сu2О which have the prospect of introduction to the food technology also were identified. Results obtained by enzymatic tests were compared to the well-known tests based on survival and chemotaxis ciliates Paramecium caudatum, germinating of shoots and roots of cress " Cudriavyy" , survival of Daphnia magna, changes in the level of chlorophyll fluorescence of algae Scenedesmus spp. and foaming by the yeast Saccharomyces cerevisiae. The effects of the food additives on tests organisms were evaluated using parameters EC50 or LD50. The coupled enzyme system R + L and triple enzyme system with LDH showed a great sensitiv ity to the analyzed food preservatives. Values of EC50 were equal 0.03, 0.14, 0,008 and 0.66, 0.13, 0.07 mM for sodium benzoate, potassium sorbate and sorbic acid, respectively. The values of EC50 estimated by enzymatic tests were over two times less than that for the biological tests mentioned above. The maximum decrease in the relative activity of trypsin and ADH did not exceed 45 % in the studied concentration range of preservatives. It was shown that copper nanoparticles had a strong inhibitory effect on R + L. Moreover, the inhibitory effect increased in the presence of copper oxide (I). Values of EC50 were equal 4 μM and 1.5 μM for nanoparticles of copper and Cu2O respectively. Value of EC50 in the presence of nanoparticles of silver was 0.18 mM. Thus, bioluminescent enzymatic tests based on NADH;FMN-oxidoreductase-luciferase indicates toxicity of substances in the much lower concentrations than its actual level of the maximum availability in food products. These results have had the evidence that suggested bioluminescent enzymatic methods are high sensitive to various classes of food additives and it can be used in the safety evaluation of food additives assay. The work was financially supported by the Russian Academy of Sciences (Program "Molecular and Cell Biology", grant No 6.8) and by Project 1762 from The Ministry of Education and Science of the Russian Federation. 1. Stannard C, Gibbs P. Luminescence. 1986;1:3-10. 2. Girotti S, Muratori M, Fini F Et al. Eu. Food Res. and Tech. 2000;210:216-219. 3. Kratasyuk VA, Egorova OI, Esimbekova E.N., Et al. Applied Biochemistry and Microbiology. 1998;34(6);622-624. 4. Vetrova E, Esimbekova E, Remmel N. Et al. Luminescence. 2007;22(3);206-214. P0035 Optical correction of the results of bioassays based on bacterial bioluminescence in vitro applied to nanoparticles Albert A. Lisitsa(a) , Elena V. Nemtseva(a,b) , Valentina A. Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Recently wide spectrum of nanoparticles varying in composition, shape and size is extensively used in industries, as well as in medicine and pharmacology. That's why the determination of the nanoparticles hazard both for environment and for human health is becoming increasingly important. Some investigations point out that there is no evidence for 'nano-specific hazard' and conventional bioassays are useful and relevant to the evaluation of nanoparticle toxicity [1]. Luminous bacteria are often used as a sensor for determining the toxicity of various nanomaterials [2, 3]. Bioassays based on soluble and immobilized enzymes from luminous bacteria also were developed to evaluate the toxicity of different media [4, 5]. However, the use of bioluminescent signal is related to the problem of analysis of turbid or colored samples, whose optical properties can distort the results of biotesting (through reabsorption or scattering bioluminescence). The signal of bioluminescent bioassays can be corrected taking into account the spectral characteristics of the samples. The aim of this study was to evaluate the concentration range for silver, copper nanoparticles and fullerenes, at which the signal from bioassay based on bacterial luciferase must be corrected on the optical effects. Water solutions of the following particles were studied; silver nanoparticles (Ag NPs) (average particle size 10 nm) and copper nanoparticles (Cu(0) NPs) (average particle size of 43,8 ± 16,8 nm) synthesized in the Institute of Non-Ferrous Metals and Materials Science of SibFU (Krasnoyarsk, Russia), and the samples of hydrated fullerene (С60 FWS) (particle size 1,5-72 nm) provided by Institute of Physiological Active Compounds (Kharkiv, Ukraine). Absorption spectra were measured with the spectrophotometer Cary 5000i (Agilent Technologies), bioluminescence spectrum was measured with fluorescent spectrometer Aminco Bowman Series 2 (Thermo Spectronics). Correction coefficients k were calculated using the formula [2]; [Formula: see text] where g(λi ) -the portion of the emission intensity of bioassay at the wavelength λi in the total intensity at the range 420-630 nm, Di (λi ) - optical density of the sample at the wavelength λi and optical path length l; L - optical path length for quantum of bioluminescence through the sample. To find true value of bioluminescence intensity all experimental intensities should be multiplied to corresponding coefficients k. It was defined that the lowest concentration of the nanoparticles at which correction is necessary corresponds to a coefficient k = 1,1. The highest concentration, at which correction is still working, was taken these one corresponding k≈2,5. In the samples with k > 2,5 too high optical density prevents measuring adequate absorption spectra and the bioluminescence signal. The absorption spectra of nanoparticles solutions at different concentrations were measured in the wavelength range of bioluminescent emission (420-630 nm). The relative position of the absorption spectra of studied nanoparticles and emission spectra of bacterial bioluminescence in vitro is shown on the Fig. 75. [Figure: see text] Corresponding correction coefficients were calculated for all the measured spectra. From obtained k values the concentration range at which correction of bioassays signal is necessary and applicable were found for each nanoparticle (Table 1). Two types of bioassays (using soluble and immobilized enzymes) differ in the volume of analyzed sample and, hence, in optical path length L [4-5]. That's why different concentration ranges were obtained for them. [Table: see text] It was found that the minimal optical distortion in bioluminescent signal caused by C60 FWS; correction is necessary only at high concentrations (tens of millimoles/liter). The reason for this is small overlap of the absorption spectrum of C60 FWS with emission spectrum of bioluminescence (Fig. 75). For Ag NPs and Cu(0) NPs optical effects are significant; it's necessary to correct the bioassay results starting from concentration of tenth of millimoles/liter. These nanoparticles are known as broad-spectrum bactericidal agents and accurate assessment of their toxic effect is relevant. Acknowledgments; The research was partly supported by Project 1762 from The Ministry of Education and Science of the Russian Federation. References; 1. Donaldson K, Poland CA. Nanotoxicity; challenging the myth of nano-specific toxicity. Current opinion in biotechnology. 2013;Т. 24. - 4:724-734. 2. Bondarenko O et al. Sub-toxic effects of CuO nanoparticles on bacteria; kinetics, role of Cu ions and possible mechanisms of action. Environmental Pollution. 2012;169:81-89. 3. Aleshina ES, Bolodurina IP, Deryabin DG, Kucherenko MG. Сorrection of results of bioluminescenct analysis in view of optical properties of observed carbonic nanomaterials. Vestn.Orenburgsk. Gos. Univ. 2010;6:123-128. 4. Vetrova E, Esimbekova E, Remmel N, Kotova S, Beloskov N, Kratasyuk V, Gitelson IA. Bioluminescent signal system; detection of chemical toxicants in water. Luminescence 2007; 22(N3);206 - 214. 5. Esimbekova EN, Kondik AM, Kratasyuk VA. Bioluminescent enzymatic rapid assay of water integral toxicity. Environmental Monitoring and Assessment 2013;185(7);5909-5916. P0036 Bioluminescence in education; Contemporary bioluminescent practical training session for research activities in secondary schools Nadezhda Rimatskaia(a) , Oleg Sutormin(a) , Irina Sukovataya(a) , Valentina Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) RUSSIAN ACADEMY OF SCIENCES SIBERIAN BRANCH INSTITUTE OF BIOPHYSICS, Krasnoyarsk, Russia To expand the research component in school education the bioluminescent practical training session has been created. The training session is connected with the school curriculum and its aim is to develop pupils' research skills. There is lack of works in the school curriculum to ensure in full requirements of modern education, such as the development of research competence of pupils. This statement concerns the programs on the biology and ecology. We can partially solve the proposed problem using the phenomenon of bioluminescence - luminescence of living organisms. This natural phenomenon gives us a tool to visualize, i.e. to see many of the characteristics of the living organisms by relatively simple devices - the luminometers, which may be affordable to schools -. Very few living organisms have the ability to emit light, no more than several thousand species of the many millions that exist in the world. Most light-emitting organisms live in the sea. On land just some insects, worms and fungi glow. Among the achievements of modern biophysics, physicochemical biology and genetics is a discovery of the molecular mechanisms of bioluminescence and the ability to extract the enzymes that control the biochemical reactions emitting the light from luminescent organisms. The bioluminescence practical training session solves the problem of research competence of pupils on biology education in conditions of the practical absence of such practical activities in schools. The portable laboratory for modern school practical training session in biology and ecology was designed. The laboratory has a manual for the laboratory works, simplified and less expensive model of the device bioluminometer, the reagent kit for analysis (immobilized multicomponent enzymes with their substrates named " Enzimolyum" , FMN and others), dispensers and other accessories. The bioluminescent practical training session consists of a set of mini-research experiments. The laboratory works are designed to support the basic school biology course and can be used as demonstrations in biology and ecology classes. A special section of the training session includes lab works, which are offered as the basis for the school biological research in-depth studying of the subject, or within the modular learning on biology. The training session manual also provides a description of the luminous organisms and principles of bioluminescent analysis. The practical training session allows bringing closer school education to the modern biological technologies. The bioluminescent practical training session for pupils makes the basic course in biology interesting and enjoyable for pupils (through visualization of biological phenomena); promotes pupils' skills of self-searching, processing and analysis, helps to develop their creativity; introduces the modern methods of researching and testing to schoolchildren; allows to carry out the experiments with objects from the environment (snow, soft drinks, detergents, etc.) is easy to implement, and it does not contain harmful substances; may be considered as the foundation for environmental education and training of pupils. The practical training session is created for general and supplementary education of Krasnoyarsk Krai, Russia, CIS and foreign countries. The practical training session was successfully tested in Krasnoyarsk and Krasnoyarsk Kray; at the natural sciences schools in biophysics under the program " Gifted Children of Krasnoyarsk" , on " Science Festival" , " Festival of the natural sciences" , " The Natural Science Quest" , in regular classes with the pupils from several Krasnoyarsk schools. The analysis of the possibilities of implementing the modern school bioluminescent training session for the education system of the Krasnoyarsk Kray and Russia showed the necessity for its widespread implementation into schools as for scheduled classes on biology, ecology and chemistry, as well as for the development of the schoolchildren's research. We plan to bring the training session into the world market of educational materials for children. The research was supported by the Regional Autonomous public institution " Krasnoyarsk regional fund to support scientific and technical activities" P0037 Application of bioluminescent enzymatic method for assessment of the state of the soil. Nadezhda Rimatskaia(a) , Elizaveta Baigina(a) , Marina Kazanceva(a) , Devard Stom(c) , Valentina Kratasyuk(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Russian Academy of Sciences Siberian Branch Institute of Biophysics, Krasnoyarsk, Russia, (3) Irkutsk State University, Irkutsk, Russia Now monitoring of soil toxicity is a more difficult problem than monitoring of water and air environments because the soil is a multicomponent media. Its composition varies and contains a large number of polluting and interacting substances. Because of it the bioassay methods providing integral assessment of overall pollution loading have more advantages in comparison with the usual chemical analysis. We aimed to compare the bioluminescent enzyme-based method with standard methods of bioassay estimating the quality of soil. We applied a number of bioassay methods; bacterial coupled system NADH; FMN-oxidoreductase-luciferase (residual luminescence,%), Daphnia magna Straus (survival rate%), Cress Salad (germination,%), Paramecium caudatum (reaction chemo taxis%). We modeled the soil pollution by adding the model toxicants CdCl2 (MPC = 1 g/kg) and HgSO4 (MAC = 2 g/kg) into the reference soil sample. The model pollutants were added into the dry reference sample and then the standard water extract was prepared. The results are shown in the Table 1. The reference soil sample is non-toxic, that h is also confirmed by the chemical analysis. If model toxicants were added at the MPC the following methods are of the greatest sensitivity; the bioluminescent enzyme-based method, the analysis based on the survivability Daphnia magna Straus and analysis based on chemo taxis of Paramecium caudatum. Seven samples collected near Angarsk Petrochemical Company (APCC) and the Baikal. Lake from the upper horizon of soil were analyzed with Daphnia magna and conventional chemical methods. The bioluminescent enzyme method showed that the soil samples from Angarsk Petrochemical Company were toxic (residual luminescence was about 40%). The chemical analysis determined that toxicity of these samples was caused by Zn, Pb, Ni, Cu as their contents exceeding their MPC. Bioluminescent analysis is one of the most promising methods of fast environmental monitoring, as the bioluminescent system is very sensitive. When compared to conventional chemical analysis bioassay methods based on the luminescent bacteria give an integral assessment of toxicity. They are faster, easier, more accurate and sensitive than other bioassays based on Daphnia magna Straus, Paramecium caudatum, Cress Salad, etc. [Table: see text] P0038 Effect of radioisotope tritium on bioluminescence and mutations in luminous bacteria P. phosphoreum 1883 IBSO Oleg Guseynov(a) , Maria Selivanova(b) , Irina Litvinova(a) , Polina Karpenok(a) , Valeriya Guseynova(a) , Alena Petrova(a) , Nadezhda Kudryasheva(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Luminous bacteria and their enzymatic reactions were previously shown to be in proper use as bioassays to monitor toxicity in solutions of alpha- [1] and beta- [2-3] emitting radionuclides. Successive stages of activation (radiation hormesis) and inhibition (radiation toxicity) in bioluminescence (BL) kinetics were found in solutions of these radionuclides. The role of reactive oxygen species and intracellular components in the BL kinetic changes were discussed in [1, 4]. The current research is aimed at examining whether the changes in kinetics of light emission of P. phosphoreum exposed to beta-emitting radionuclide tritium are related to genetic mutations. The BL kinetics of 20-h bacterial culture grown under addition of tritiated water (HTO) or tritium-labeled amino acid valine was measured by TriStar Multimode Microplate Reader LB 941, Berthold Technologies. Due to the effective BL activation by tritium, the total BL quantum yield increased up to 140%. Genetic changes in amplicons of 16S ribosomal RNA gene were analysed using restriction fragment length polymorphism (RFLP) method with primers 500L and 1350R. This approach is fairly simple, can be performed rapidly and enables to discover differences in DNA sequences [5]. DNA extraction and PCR amplification we re performed by standard procedures [6]. PCR products were purified using the QIAGEN purification kit. Amplicons of about 900 base pairs were used for restriction analysis with endonucleases Hae III, Msp I, BspFN I, Taq I, BstHH I. Electrophoretograms were obtained on a 1.7% agarose gel at 85 V and analysed with the gel documentation system Gel Doc XR (Bio-Rad). The patterns of restriction fragments distribution in 16S rRNA PCR-RFLP analysis of P. phosphoreum with restriction endonuclease Taq I are shown in Fig. 76 where both variants (exposed to НТО and tritium-labeled valine) are compared with the control (P. phosphoreum without tritium treatment). The identical patterns for lanes 3, 4, 5 and 6 (fragments of about 460, 380 and 100 base pairs) indicate that no changes have occurred in endonuclease Taq I restriction sites. Performing 16S rRNA RFLP analysis with the other endonucleases did not show any differences between the control and exposed to tritium P. phosphoreum either. For comparison, different patterns of restriction fragments distribution for corresponding amplicons of other bacteria (Ralstonia eutropha B-5786 and Escherichia coli K-12) are displayed in Fig. 76. [Figure: see text] Thus, under the tested conditions of tritium treatment the total BL quantum yield increase in P. phosphoreum was not related to genetic mutations in the sites of endonucleases used detectable by 16S PCR-RFLP analysis. Acknowledgement Supported by the Russian Foundation for Basic Research, Grant No. 13-04-01305a. References 1. Alexandrova M, Rozhko T, Vydryakova G, Kudryasheva N. Journal of Environmental Radioactivity 2011;102:407-411. 2. Selivanova MA, Mogilnaya OA, Badun GA, Vydryakova GA, Kuznetsov AM, Kudryasheva NS. Journal of Environmental Radioactivity 2013;130:19-25. 3. Alexandrova MA, Badun GA, Kudryasheva NS. Luminescence 2012;27:95. 4. Kamnev AA, Tugarova AV, Selivanova MA, Tarantilis PA, Polissiou MG, Kudryasheva NS. Spectrochim Acta A; Mol. Biomol. Spectrosc. 2013;100:171-175. 5. Ibrahim A, Gerner-Smidt P, Sjo.Stedt A. Journal of Clinical Microbiology 1996; 34, 2894-2896. 6. Ciantar M, Newman HN, Wilson M, Spratt DA. Journal of Clinical Microbiology 2005;43:1894-1901. P0039 Bioluminescence Assay Based on Controlled Inhibitory Effect on Firefly Luciferase Ryoko Kurishiba(a) , Akihiko Ishida(b) , Hirofumi Tani(b) , Manabu Tokeshi(b) (a) Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Hokkaido, Japan (b) Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido, Japan Firefly luciferase (Luc) has been widely used as an analytical reagent in various bioluminescence (BL)-based assay technologies due to its fast response, high sensitivity, and specificity. Typically, the BL assay is based on the changes in Luc or ATP concentration in the presence of analytes. Recently, new type of BL assay based on controlled BL activity has been reported. For example, biotinylated Luc, BL activity of which depends on the binding of avidin, was used for competitive assay of biotin [1]. The assay using aminoluciferin derivatives is also considered to exploit the change of BL activity as a substrate in the presence or absence of target molecules [2]. This type of BL assay could be easily applied to homogeneous binding assay. In this work, we have designed a novel BL assay based on controlled inhibitor activity for Luc. A ligand-modified inhibitor is used in this assay. In the absence of target molecule, it inhibits BL reaction of Luc in competitive manner, while the presence of target cancels the inhibitory effect of it resulting in BL emission. To confirm availability of this method, we used biontinylated lauric acid (b-LA) as a ligand-modified inhibitor of Luc and streptavidin (SAv) as a target molecule. Lauric acid is a well-known competitive inhibitor of Luc, which competes against luciferin [3]. b-LA was synthesized from biotin N-hydroxysuccinimide ester and 12-aminolauric acid. We used two types of Luc, free Luc and Luc immobilized on sepharose beads. As we had expected, b-LA showed inhibitory effect on BL reaction with both of Luc because carboxylate group, essential for inhibitor, still remains in b-LA. Next, in the presence of SAv, different results were obtained between two Lucs. In the case of free Luc, BL remained inhibited in spite of the presence of SAv. This indicates that SAv-b-LA complex still has inhibitory effect on BL reaction. On the other hand, BL emission from immobilized Luc was recovered by the addition of SAv with depending on its concentration (Fig. 77). The difference in BL recovery between two Lucs can be simply explained by steric hindrance with the complex. From these results, our method is shown to be applicable to BL assay for target molecules and also competitive BL assay for ligand in the presence of target. This BL assay has an advantage over other BL assay based on controlled activity; there is no direct modification to Luc and substrate, thus the assay can be easily constructed. To carry out the proposed assay with free Luc, spacer between ligand and inhibitor, size of target molecule, and so on should be considered. We will test some antigens and antibodies in this assay with free Luc, and construct a competitive homogeneous immunoassay system using this method. [Figure: see text] Reference 1. Wu C, Kawasaki K, Ogawa Y, Yoshida Y, Ohgiya S, Ohmiya Y. Preparation of biotinylated Cypridina Luciferase and Its Use in Bioluminescent Enzyme Immunoassay. Anal. Chem. 2007;79(4);1634-1638. 2. Moravec RA, O'Brien MA, Daily WJ, Scurria MA, Bernad L, Riss TL. Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format. Anal. Biochem. 2009;387(2);294-302. 3. Ueda I, Suzuki A. Is There a Specific Receptor for Anesthetics? Contrary Effects of Alcohols and Fatty Acids on Phase Transition and Bioluminescence of Firefly Luciferase. Biophysical Journal 1998;75(2);1052-1057. P0040 Identification of quinone modified proteins in biological fluids using redox cycling based chemiluminescence assay Naotaka Kuroda(a) , Mohamed Elgawish(a) , Naoya Kishikawa(a) , Kaname Ohyama(a) , Kenichiro Nakashima(b) (a) Nagasaki University, Nagasaki, Japan (b) Nagasaki International University, Nagasaki, Japan Background Quinones encompass a broad discipline of biologically active compounds ranging from the endogenous mitochondarial electron transporter and vital medication for cancer to a variety of exogenous and carcinogenic xenobiotic encountered in the environment. Covalent modification of proteins is associated with the toxicity of many quinones, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Detection of adducted proteins accomplished by the use of radioactively labeled quinones or by employing adduct-specific antibodies for immunoblotting is labor-intensive, time consuming, and limited by the availability, quality, and specificity. We recently described a redox-cycling based chemiluminescence (CL) assay for simultaneous determination of menadione (MQ) and its conjugate with low molecular weight bio-thiols in rat plasma [1]. Herein we adapted this CL approach to selectively identify quinone-protein adducts either in vivo or in vitro. The suggested method exploits the unique redox cycling ability of quinone-protein adducts in presence of reductant, dithiotheritol (DTT), to liberate ROS which can be measured by luminol CL assay. Material and method The standard adduct was prepared by incubation of different concentration of MQ at molar ratio ranged from 1;1 to 1;50 with human serum albumin (HSA) at 37 °C for 24 hr. The excess MQ was removed by dialysis for 24 hr at 4°C. The adduct formation was confirmed by MALDI-TOF and spectrophotometric analysis. The CL assay was performed by mixing 50 µl of different concentration (50-200 µg/ml) of protein adduct with 50 µl of 100 μM luminol and 100 µl of 100 μM DTT. The CL response was measured for 5 min using Berthold luminometer. The selective identification of adducted protein in human serum and rat plasma was performed by HPLC-CL technique. Separation was performed on Shodex Asa hipak GS-520 7E (250 x 7.6 mm, Shodex, Japan) gel filtration column with 10 mM Tris-HCl buffer (pH 8) as mobile phase. The column eluent was mixed with 1.5 mM DTT and 1 mM luminol before CL detection. Results Under our investigation using MALDI-TOF analysis, we found the number of MQ complexed with HSA increased with increased molar ratio of HSA; MQ. For a 1;1 molar ratio of HSA; MQ, the number of MQ adducted was 1 occurred especially at the single free thiol; for 1;10 ratio, the number was 3; for 1;20, it was 4 and that for 1;50 it was 5 mainly occurred at ɛ-amino group of lysine residue which confirmed by the quenching of fluorescamine fluorescence. The CL assay is highly selective to identify not only MQ-HSA adduct but also other quinones-proteins adducts (Bovine serum albumin and β-lactoglobuline). The gel filtration chromatography (GFC)-CL assay successfully identified the adducted proteins, albumin, in complex mixtures of equimolar concentration of four proteins (HSA, cytochrome C, lyzozyme, and alcoholdehydrogenase) and human serum in vitro as well as in rat plasma in vivo after MQ administration. We also examined the fate of quinone adduct in rat plasma and found a time-dependent loss in protein adduct. This phenomenon could be attributed to the reversibility or the cellular degradability of adducted proteins. Overall, the redox-cycle based CL approach presented here could provide a reliable tool to identify the biological targets of quinones that encourage further studies of biological, pharmacological, and toxicological impact of quinones motif and how this might affect human health. Reference 1. Elgawish MS, Shimomai C, Kishikawa N, Ohyama K, Wada M, Kuroda N. Chem. Res. Toxicol. 2013;26:1409-1417. P0041 Development of bioluminescence endotoxin assay Akio Kuroda, Kenichi Noda Hiroshima University, Higashi-Hiroshima, Japan Endotoxin is lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, and causes fever or shock when it enters the human blood stream. The Limulus Amebocyte Lysate (LAL), the defense mechanism of horseshoe crab, has been used for endotoxin detection. LAL contains several enzymes that are activated in a series of reactions in the presence of endotoxin. The last enzyme activated in the cascade splits coagulogen, reading to gel formation or turbidity. There are three basic LAL test methodologies; gel-clot, turbidimetric, and chromogenic. In the chromogenic assay, the last enzyme splits a synthesized chromogenic substrate in response to endotoxin and releases chromophore, for instance para-nitro aniline, from the chromogenic substrate, producing a yellow color. However, these conventional methods have problems with regard to their measurement time or sensitivity. Here we synthesized modified firefly luciferin that could be splitted by the last enzyme and developed a new endotoxin detection method that combines LAL and bioluminescence. Then we applied a mutant firefly luciferase (Bioenex Inc. Japan), which has a luminescence intensity over 12-fold higher than that of the wild type, to the new endotoxin detection method. This new method increased the sensitivity of endotoxin detection by approximately 1,000-fold compared to the chromogenic method and enabled us to detect 0.0005 EU/ml of endotoxin within 15 min. P0042 The production of highly active recombinant 16.5 kDa-isoform of Metridia longa luciferase, the smallest copepod luciferase Marina Larionova(a,b) , Svetlana Markova(a) , Ludmila Burakova(a) , Eugene Vysotski(a) (a) Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia (b) Laboratory of Bioluminescence Biotechnology, Institute of Fundamental Biology and Biotecnology, Siberian Federal University, Krasnoyarsk, Russia Several isoforms of secreted coelenterazine-dependent luciferase which are responsible for a blue bioluminescence (λmax  = 480-485 nm) of marine copepod Metridia longa have been cloned. Two of them, MLuc164 [1] (or MLuc) and MLuc39 [2] have been successfully applied as sensitive and highly active bioluminescent reporters in vivo and in vitro assays. The main advantage of secreted luciferase as a reporter is its no-lysis protocol that allows the conduction of live cell assays, multiple assays on the same cells and suits well for development of high-throughput screening technologies. Here we report expression, purification and characterization of another secreted isoform of Metridia luciferase, MLuc7 that has a greater bioluminescent activity as compared to MLuc164 and MLuc39 isoforms, and perhaps is more perspective as a bioluminescent reporter. The mature MLuc7 isoform without N-terminal signal peptide for secretion is a 16.5-kDa protein and, to date, the smallest of the known natural luciferases. The MLuc7 sequence is actually a highly conservative region of the copepod luciferase sequences and almost corresponds in size to the truncated MLuc164 luciferase with a high bioluminescent activity [3]. Our attempts to produce a significant amount of the functionally active recombinant MLuc7 luciferase using Escherichia coli host system and various expression vectors were unsuccessful. The expression of MLuc7 luciferase in E. coli cells at high levels resulted in the formation of inclusion bodies and yielded large amounts of the protein requiring renaturation and refolding to be functionally active. These recombinant MLuc7 samples obtained from the inclusion bodies were characterized by heterogeneity and propensity to aggregate. The refolded MLuc7 from E. coli cells differed from the correctly folded MLuc7 secreted by CHO mammalian cells in properties. With the use of the Bac-to-Bac baculovirus expression system only, we were able to produce substantial amounts of pure correctly folded MLuc7 luciferase with a high bioluminescent activity. The MLuc7 luciferase was expressed in Sf9 insect cells with its own natural signal peptide for secretion and purified from the culture media with a yield of high purity protein of ~3 mg/L. This MLuc7 luciferase expressed in the insect cell system is a monomeric protein showing ~4-fold greater bioluminescence activity than the luciferase expressed and purified from E. coli. The close correspondence of the experimental mass of Mluc7 luciferase purified from insect cells with that calculated from amino acid sequence indicates that luciferase does not undergo post-translational modifications, and the cleavage site of the signal peptide for secretion is at VQA-NP, as predicted from sequence analysis. This work was supported by RFBR grant 12-04-00131 and the "Molecular and Cellular Biology" Program of the Russian Academy of Sciences. References; 1. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES. Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem. 2004;279:3212-7. 2. Borisova VV, Frank LA, Markova SV, Burakova LP, Vysotski ES Recombinant Metridia luciferase isoforms; expression, refolding and applicability for in vitro assay. Photochem Photobiol Sci. 2008;7:1025-31. 3. Markova SV, Burakova LP, Vysotski ES. High-active truncated luciferase of copepod Metridia longa. BBRC 2012;417:98-103. P0043 Rapid bioluminescent detection of Salmonella cells using firefly luciferase-antibody conjugates and polysterene microparticles Galina Lomalina(a) , Alena Istrate(a) , Natalia Rudenko(b) , Natalia Ugarova(a) (a) Lomonosov Moscow State University, Moscow, Russia (b) Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia The ability to produce stable and active conjugates of the firefly luciferase with antibodies is very important for the bioluminescence-based enzyme immunoassay. Conjugation of firefly luciferase with antibody through NH2 -groups of the enzyme resulted in a great loss of the luciferase activity [1]. Thermostable mutant of Luciola mingrelica firefly luciferase with His6 -tag at the C-terminus with high enzyme activity was used in this work [2]. It retains 70 % of the activity after 2 days incubation at 3 7ºС that is sufficient for most common applications of the firefly luciferase. This mutant contains two surface SH-groups (Cys62, and Cys164) and these residues are the most likely targets of conjugation, because they are located 20 Å away from the luciferase active site, and their substitutions are shown not to lower the luciferase activity and stability [3]. The free SH-groups of the luciferase were used for conjugation with monoclonal anti-Salmonella antibodies (Sal). Antibodies were modified with a bifunctional coupling agent, SPDP, whereas the luciferase was used in unmodified form. Pyridyl disulfide groups were first introduced to the Sal according to the method of Carlson et al [4] slightly modified by us. Then, the reaction of the activated Sal with luciferase was performed in 0.02 mol/L Na-phosphate, pH 7.4, containing 0.15 mol/L NaCl at 4°C during 15 h. The optimal Luc/Sal molar ratio was equal to 1;2. The conjugates were purified by metal-chelate chromatography. We obtained Luc-Sal conjugates which showed 71 ± 3 % of the initial luciferase activity. The linear dependence between the bioluminescence intensity and luciferase concentration was observed between 10(-14) and 10(-7) mol/L luciferase The molecular weight of the Luc-Sal conjugate was ~230 kDa that corresponded to Luc-Sal conjugate with 1;1 stoichiometry. The Luc-Sal conjugates were very stable; after two months of storage at 0ºC its retained more than 90% of the luciferase activity and thermal stability at 37ºС was similar to that of the non-modified enzyme. The Luc-Sal conjugates were used for bioluminescent detection of Salmonella cells. The detection limit of the bioluminescent "sandwich"-type enzyme immunoassay of Salmonella is known to be high as 5∙10(5) CFU/mL without a preliminary cultivation of the cells. To improve analytical characteristics of the bioluminescent detection of Salmonella cells, we used monodisperse polystyrene microparticles (PS) (d = 240 nm) whose working surface area was increased approximately by a factor of 100 compared to the surface of a polystyrene microplate well. We coated the surface of polystyrene microparticles with Pluronic F108-PDS [5], a triple block-copolymer composed of two terminal relatively hydrophilic polyoxyethylene blocks and hydrophobic polyoxypropylene central block. During the adsorption, the hydrophobic block of Pluronic covers the hydrophobic surface of microparticle and a brush-like structure on the surface is formed. It prevents a nonspecific adsorption of proteins as well as their denaturation and inactivation. The Pluronic F108 molecules have long side chains containing in total 265 polyoxyethylene units that prevents a direct interaction of the luciferase and antibody molecules with polystyrene matrix. The side polyoxyethylene chains of Pluronic F108-PDS contained pyridyl disulfide groups (PDS) which were used to bind covalently Sal to microparticles. The NH2 -groups of the Sal were activated with SPDP, while the pyridyl disulfide bonds of PS-F108-PDS were reduced to SH-groups. After this the interaction between PS-F108-PDS and activated Sal resulted in formation of microparticles linked with Sal antibodies (PS-F108-Sal). The diameter of the PS-F108-Sal microparticles increased up to 294 ± 7 nm in accordance with the size of the conjugated molecules and did not change within one month of storage at 4°C indicating that desorption of Pluronic from the microparticle surface did not occur. We used the PS-F108-Sal microparticles as a capturing agent and the Luc-Sal conjugates as a detecting agent in bioluminescent enzyme immunoassay of Salmonella cells. The dependence of bioluminescence intensity on the Salmonella cell concentration is shown in Fig. 78 (curve 1). The detection limit was equal to 2.7 x 10(3) CFU/mL, that is ~100 times lower than the detection limit of Salmonella immunoassay based on the use of polystyrene microplates. This essential improvement of the detection limit is explained not only by an increase in the binding surface area. Furthermore, the Sal antibodies are connected with the polystyrene surface through a long spacer that provides the mobility of antibodies and makes them more accessible to the bacterial cells. This can explain the fact that the fast and efficient binding of Salmonella cells to the PS-F108-Sal microparticles was observed after 1 h incubation at room temperature, whereas in the case of microplates the reaction required incubation at 37ºC. The flexible polyoxyethylene chains of Pluronic shielded the hydrophobic surface of microparticles and prevented an interaction of the proteins with polystyrene matrix. It explains a very low nonspecific adsorption of detecting Luc-Sal conjugate on PS-F108-Sal microparticles (less than 0.5 %). It is important to underline that the bioluminescence intensity depends linearly on the cell concentration in the large range from 10(3) to 10(6) CFU/mL. To assess the specificity of the method, we have used E. coli cells (strain K12). The Salmonella and E. coli cells have a similar structure of the cell wall. When E. coli cells were assayed according to the protocol developed, the bioluminescent signal was close to the background level and did not depend on the cell concentration (Fig. 77, curve 2). It confirms the specificity of the method based on the high specificity of Sal antibody and sensitivity of bioluminescent detection. Acknowledgements This work was financially supported by the Russian Foundation for Basic Research (No. 08-04-00624 and 11-04-00698). References 1. Murphy MJ, Squirrell DJ. Covalent coupling of firefly luciferase to antibodies. In; Campbell AK, Kricka LJ, Stanley PE, editors. Bioluminescence and Chemiluminescence; Fundamental and applied aspects. Chichester; John Wiley and Sons, 1994;301-4. 2. Koksharov MI, Ugarova NN. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng Des Sel 2011;24:835-44. 3. Modestova YA, Lomakina GY, Ugarova NN. Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase. Biochemistry (Moscow) 2011;76:1147-54. 4. Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. N-succinimyl 3-(2-peridyldithio) propionate, a new heterobifunctional reagent. Biochem J 1978;173:723-7. 5. Fromell K, Hulting G, Ilichev A, Larsson A, Caldwell K. Particulate platform for bioluminescent immunosensing. Anal Chem 2007;79; 8601-7. [Figure: see text] P0044 Bioluminescence helps Malaria Research; Exploiting new Luciferases to improve analytical performance of Antimalarial screening assays Luca Cevenini(a) , Elisa Michelini(a,c) , Maria Maddalena Calabretta(c) , Grazia Camarda(b) , Giulia Siciliano(b) , Bruce Branchini(d) , Aldo Roda(a) , Pietro Alano(b) (1) Dept of Chemistry, University of Bologna, Bologna, Italy (2) Istituto Superiore di Sanità, Roma, Italy (3) INBB, Roma, Italy (4) Connecticut College, NEw LONDON (CT), USA In the renewed efforts to combat malaria, substantial improvement of bioanalytical tools are needed to tackle the key step of parasite transmission at different stages. Success of the most effective antimalarial drug artemisinin relies on the unique ability to kill early asexual parasites, but reports of P. falciparum reduced susceptibility to this drug urgently calls for drug screening assays to identify novel effective antimalarials. P. falciparum gametocytes represent the sexual forms of the parasites that develop within the host erythrocytes. The lack of reliable assays to screen compound libraries for gametocytocidal activity prompted us to develop novel reporter cell-based assays exploiting the multiplexing and imaging potential of luciferases [1,2]. First, novel red and green-emitting luciferases expressed for the first time in malaria parasite have been cloned under the control of gametocyte specific promoters and successfully integrated in the parasite genome. Four luciferases were selected thanks to their high signal to background ratios (two orders of magnitude compared to conventional wild-type luciferase) and glow type emission kinetics. We report for the first tim e a single cell bioluminescence imaging assay for transgenic gametocytes performed with an optical microscope (40X magnification) equipped with an ultrasensitive EM-CCD camera. Then we developed a dual reporter assay (DR-GAM) designed to identify compounds blocking gametocyte development at an early or a mid-late stage of maturation, measuring activities of two luciferases emitting at different wavelengths driven by gametocyte-specific promoters. Transgenic parasites were used in validation assays using artemisinin derivatives; preliminary data suggest that the selected luciferases are likely to represent promising novel tools in malaria drug screening and discovery. This assay will be used to screen the " malaria box" of 400 newly identified antimalarial compounds from Medicines for Malaria Venture. 1. Ekström L, Cevenini L, Michelini E, Schulze J, Thörngren JO, Belanger A, Guillemette C, Garle M, Roda A, Rane A. Testosterone challenge and androgen receptor activity in relation to UGT2B17 genotypes. Eur J Clin Invest. 2013 Mar;43(3);248-55. 2. Cevenini L, Michelini E, D'Elia M, Guardigli M, Roda A. Dual-color bioluminescent bioreporter for forensic analysis; evidence of androgenic and anti-androgenic activity of illicit drugs. Anal Bioanal Chem. 2013 Jan;405(2-3);1035-45. P0045 Spin-orbit coupling effects in chemiluminescence Boris Minaev(a,b) , Hans Agren(a) , Valentina Minaeva(b) (a) Royal Institute of Technology, Stockholm, Sweden (b) Bohdan Khmelnitsky National University, Cherkasy, Ukraine Chemiluminescence's reaction paths can be considered as the reversed photochemistry. Many photochemical reactions start from the triplet excited state and are terminated at the stable singlet ground state of the diamagnetic products. A great number of well-known reactions of such type proceed through the spin-flip intersystem crossing (ISC) process. This process is governed mainly by spin-orbit coupling (SOC) matrix elements between triplet (T) and singlet (S) excited states. In a similar way one can anticipate that ISC and SOC are of crucial importance in chemiluminescence because of its reversed character. Bioluminescence as a special form of chemiluminescence (where light emission is due to enzymes catalyzed reactions) depends on SOC effect in a great extent, since the spin-flip ISC processes are very typical for many enzymes [1]. They can differ in chemical structure of enzymes (luciferases), substrates (luciferins) or in quantum yields (from 0.1 for marine bacteria to 1 for fireflies) [2]. The bioluminescent emitters of all luminous creatures represent the S and T states of nπ* type in heterocyclic compounds [3], where SOC effects are well known as being induced by one-center SOC integrals [4]. In the present talk we start with consideration of the well known chemiluminescence's reaction of thermal decomposition of tetramethyl-1,2-dioxetane [2-4]. The singlet ground state (S0 ) reaction of dioxetane starts with the thermal cleavage of the O-O bond producing a biradical •OCR2 CR2 O• in the (1) Dσσ state (R = CH3 ). This is a biradical with two bitopic centers; thus, a number of quasi-degenerate S and T states are there at this stage. The triplet state of the (3) Dπσ symmetry is the most important among them [4]. The biradical in this state dissociates into two carbonyl molecules, one of which being in the triplet nπ* state. The starting (1) Dσσ state crosses the (3) Dπσ state many times at the biradical stage which provides a good chance for the ISC process. The main message of our talk is that the SOC matrix element between these two states is relatively high since it includes a large one-center SOC integral at the oxygen atom (about 46 cm(-1) ). A number of similar examples of ISC processes are considered including photochemical analogous like the Paterno-Buhi reactions. The Ca(2+) -regulated photoproteins are responsible for bioluminescence of marine coelenterates [5]. Addition of the calcium ions to the Ca(2+) -regulated photoproteins initiates light emission. (By these reasons the photoproteins are used as calcium ion-indicators in the field of biological imaging). The photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain, and the oxygen "pre-activated" substrate, 2-hydroperoxycoelenterazine, which is non-covalently (but tightly) bound inside the protein within a hydrophobic cavity [5]. The bioluminescence originates from an oxidative decarboxylation of the protein bound substrate being triggered by the Ca(2+) ions. The reaction generates a protein bound product (coelenteramide) in the (3) nπ* excited state. The excited coelenteramide relaxes to its ground singlet state emitting the blue light (465-495 nm). Position of the phosphorescence band depends on the substituent in the flavin cycle [5]. In all cases studied the chemi-excitation occurs because the ISC process is induced by the 2p atomic orbital rotation at one oxygen center which creates a torque. Thus the strength of the SOC effect is a driving force which determines the route of chemi-excitation at the stage of the intermediate biradical dissociation. 1. Minaev BF. Environment Friendly Spin-Catalysis for Dioxygen Activqtion. Chem. Chemical Tech. 2010;4:1. 2. McCapra F. Methods Enzymol. 2000;305:3. 3. Wilson T, Hastings J. Annu. Rev. Cell Dev. Biol. 1998;14:197. 4. Minaev B, Agren H. EPA Newsletter 1998;65:8. 5. Belogurova NV, Alieva RR, Kudryashova NS. J. Mol. Struct. 2009;924-926:148. P0046 Cloning and characterization of luciferase from a Melanesian luminous click beetle Yasuo Mitani, Kazuki Niwa, Yoshihiro Ohmiya National Institute of Advanced Science and Technology, Tsukuba, Ibaraki, Japan Luminous click beetle is distributed almost exclusively in Central and South America. Only one genus has been known in Melanesia, but the description of Melanesian species has been fragmentary. Melanesian click beetle's luciferase and phylogenetic relation to the other click beetles still remain unknown. We collected a living luminous click beetle, Photophorus jansonii in Fiji (Fig. 79). It has two spots on the pronotum which emits green yellow light, and has no ventral luminous organ. In this study, we cloned a luciferase gene from the Fijian click beetle by using RT-PCR. The deduced amino acid sequence showed high identity of ~85% to the luciferases derived from other click beetle species. We expressed the Fijian click beetle's luciferase by an E. coli expression system and characterized its biochemical properties. The Km for D-luciferin and ATP were 173 M and 270 M, respectively. The luciferase was pH-insensitive and the spectrum measured at pH 8.0 showed a peak at 559 nm, which was in the range of green yellow light as seen in the luminous spot of the living Fijian click beetle. The Fijian click beetle's luciferase was assigned to the Elateridae clade by a phylogenetic analysis, and its position was between Central and South American click beetles (Fig. 80). [Figure: see text] [Figure: see text] P0047 Development of artificial chromosome vector-based luciferase assay system Yoshihiro Nakajima(a) , Kazutoshi Murotomi(a) , Mayu Yasunaga(a) , Tetsuya Ohbayashi(b) , Mitsuo Oshimura(c,d) (a) Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa, Japan (b) Research Center for Bioscience and Technology, Tottori University, Yonago, Tottori, Japan (c) Graduate School of Medical Sciences, Tottori University, Yonago, Tottori, Japan (d) Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan Bioluminescent reporters, which emit light by oxidizing its substrate luciferin, have become an essential tool for studying various aspects of biological functions, including gene expression, posttranscriptional modification and protein-protein interactions, because the sensitivity and range of the linear response are superior to those of other reporters, including β-galactosidase, chloramphenicol acetyltransferase, and fluorescent proteins. In particular, luciferases are used as sensitive probes to monitor gene expression, quantitatively, and longitudinally in living cells, explant tissues, and in vivo. Fluorescent proteins have contributed immensely to the advancement of cell biology and are used as powerful probes to monitor an extensive array of entities, ranging from single molecules to whole organisms. However, fluorescent reporters require exogenous illumination to emit light, making them unsuitable for the long-term quantitative monitoring of gene expression because the reporter is bleached and the subject can suffer phototoxic damage caused by repetitive exogenous illumination. Recent advances in luciferase technology allow us to monitor the expression of multiple genes simultaneously when luciferases are used that induce differently colored emission spectra, namely, green-emitting and red-emitting beetle luciferases that act on a single bioluminescent substrate (multi-color luciferase assay system) [1]. In general, however, generation of stable cell lines or transgenic mice carrying multiple promoter - luciferase gene cassettes need long time and complicated procedures. To overcome the technical limitation, we utilize an artificial chromosome vector in which multiple transgenes can be introduced into the vector by site-specific recombination [2, 3]. To verify capability of the vector, we generated fibroblast stable cell line expressing green- and red-emitting luciferases under the control of mPer2 and mBmal1 promoters, canonical clock genes, respectively. We successfully monitor longitudinal antiphasic bioluminescence oscillations, consisting with intrinsic gene expression profile. The result indicates that the artificial chromosome vector serve as an effective tool for generating cell line and for monitoring multiple gene expressions. 1. Nakajima Y, Ohmiya Y. Expert. Opin. Drug Discov. 2010;5:835-49. 2. Yamaguchi S et al. PLoS ONE. 2011;6:e17267. 3. Takiguchi M et al. ACS Synth. Biol. 2012 P0048 Bioluminescence immunoassay using luciferase-encapsulated liposome as a label Yusuke Nakatani(a) , Chiaki Shido(a) , Akihiko Ishida(b) , Hirofumi Tani(b) , Manabu Tokeshi(b) (a) Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Hokkaido, Japan (b) Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido, Japan Enzyme immunoassay is widely used for analytical practice, such as clinical diagnostics. In the past few decades, various luciferase-based immunoassay platforms have been reported. [1, 2] However, increasing demand for detection of ultratrace analytes, highly sensitive immunoassay based on bioluminescence is required. Enzyme-encapsulated liposome is one of the most promising labels responding to the request. Encapsulating in liposomes and conjugating them to antibodies or antigens give us several advantages; many enzymes can be labeled at once, and no need for direct modification of enzyme can prevent denaturation of enzyme. Additionally, enzymes in liposome have been reported to show higher activity and/or stability than in bulk aqueous media. [3] In spite of these facts, there is few reports on bio/chemiluminescence enzyme-encapsulated liposomes for immunoassay. [4] In this work, we have prepared firefly luciferase(Luc)-encapsulated liposomes and then applied them to immunoassay. Thermostable Luc [5] was purified from Escherichia coli as a fusion protein with glutathione S-transferase. Biotinylated Luc-encapsulated liposomes were prepared by two methods, simple hydration and freeze-thaw methods. The liposomes obtained were extruded via a 100-nm polycarbonate filter to make size-controlled unilamellar liposomes, and then Luc-encapsulated liposomes and free Luc were separated by gel-filtration chromatography. The composition of the liposome was dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); cholesterol; N-biotinyl-cap-phosphatidylethanolamine = 30; 29.9; 40; 0.1 (mol). Immunoassay of C-reactive protein (CRP) was carried out as follows (Fig. P0048;1). Anti-CRP antibody (10 µg/mL), a mixture of 5% skim milk and 0.1% Tween 20, CRP (0 ~ 1 µg/mL), anti-CRP antibody (10 µg/mL), streptavidin (6 µg/mL), and appropriately diluted biotinylated Luc-encapsulated liposomes were added to the wells of the microtiter plates in order. Washing and incubation was done at each step. BL measurement was carried out by adding substrate solution containing lysis buffer to the well. For comparison, biotinylated Luc was made by using commercially available biotinylation kit, and used as a label for ELISA. We firstly studied Luc encapsulation into the liposomes. Comparing the two method for liposome preparation, by using the freeze-thaw method, Luc activity encapsulated in liposomes was found to be 5 times higher than that by the simple hydration one. In addition, no activity loss was observed until three cycles of freeze-thaw, thus we chose this method for preparation of Luc-encapsulated liposomes. Moreover, it was found that Luc was more stable in liposomes than in aqueous media. These results indicate Luc-encapsulated liposomes could be useful as a label for immunoassay. As applying Luc-encapsulated liposomes to immuno-sorbent assay, we found very high blank BL signal due to nonspecific adsorption of the liposomes. Thus, optimization was made in terms of liposomal size, phase transition temperature (Tm ) of lipids, and blocking reagent. After then, we decided to use the lipids with high Tm , DMPC (Tm  = 25°C) and DPPC (Tm  = 41°C), with small liposomal size (100 nm). We also found skim milk was the best blocking reagent compared to bovine serum albumin, casein, and synthetic polymers tested. Finally, the limit of detection (LOD) defined as the concentration giving BL of blank + 3σ was 9 pg/mL of CRP (Fig. P0048;2) that is much superior to that obtained by using biotinylated luc (50 ng/mL of CRP). In conclusion, we found Luc-encapsulated liposomes can be a promising label for highly sensitive immunoassay. [Figure: see text] [Figure: see text] References 1. Minekawa T, Ohkuma H, Abe K, Maekawa H, Arakawa H. Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin-luciferase bioluminescence. Luminescence 2011;26:167-171. 2. Wu C, Irie S, Yamamoto S, Ohmiya Y. A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase. Luminescence 2009;24; 131-133. 3. Kuboi R, Yoshimoto M, Walde P, Luisi PL. Refolding of Carbonic Anhydrase Assisted by 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Liposomes. Biotechnol. Prog. 1997;13:828-836. 4. Suita T, Tani H, Kamidate T. Application of Horseradish Peroxidase-Encapsulated Liposomes as Labels for Immunodotblotting. Anal. Sci. 2000;16:527-529. 5. Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, and Southworth TL. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal. Biochem. 2007;361:253-262. P0049 The C-terminal tyrosine deletion in mitrocomin increases its bioluminescent activity Liudmila Burakova(a,b) , Pavel Natashin(a,b) , Svetlana Markova(a,b) , Elena Eremeeva(a,b) , Eugene Vysotsky(a,b) (a) Institute of Biophysic, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia (b) Siberian Federal University, Krasnoyarsk, Russia Hydromedusan Ca(2+) -regulated photoproteins from various organisms, being highly identical (76-64%) in amino acid sequences [1] and spatial structures [2], are frequently found in several isoforms [3, 4]. All of these photoproteins, except mitrocomin, contain Pro residue at C-terminus, which is important for bioluminescence because its deletion or replacement, e.g. in aequorin, destroys the luminescence capacity [5]. In case of mitrocomin, this C-terminal residue is tyrosine [6]. The cause of that has not been established so far. In the present study, we quantitatively characterize an effect of deletion of the C-terminal tyrosine on bioluminescence properties of mitrocomin. The studies were performed for one of the mitrocomin isoforms (KF882009) which we recently cloned with a functional screening [4] from the cDNA expression library prepared from the jellyfish Mitrocoma cellularia . The mutant with deletion of C-terminal tyrosine was constructed by site-directed mutagenesis. The C-terminal Tyr, in contrast to Pro in other hydromedusan photoproteins, was shown not to be crucially important for mitrocomin bioluminescence function since its deletion even increases the specific bioluminescence activity 1.6-fold. In addition, the mutation does not affect coelenterazine binding as the apparent dissociation constant of mutant apo-mitrocomin-coelenterazine complex is similar to wild-type mitrocomin, the rate of an active photoprotein formation from apoprotein, oxygen, and coelenterazine, as well as the yield of an active photoprotein (~10-20%). However, the deletion of C-terminal Tyr results in a shift of the light emission spectrum to a shorter wavelength (λmax  = 458 nm) as compared to wild-type mitrocomin (λmax  = 473 nm), the twofold decrease in bioluminescence reaction decay kinetics, and a 5-fold increase of Ca(2+) -independent luminescence, thus indicating that mutant mitrocomin appears to be less stable than wild-type photoprotein. It is noteworthy that fluorescence spectrum of mutant mitrocomin exactly coincides with that of the wild-type photoprotein (λmax  = 472 nm). Basing on nucleotide sequence of mitrocomin gene, we hypothesize that TAT codon for Tyr could arise from stop codons TAA or TAG through a single-nucleotide random mutation either A→T or G→T. As C-terminal Tyr does not drastically influence bioluminescent function, most likely, such mutation would survive during evolution. This work was supported by RFBR grant 12-04-00131 and the 'Molecular and Cellular Biology' Program of the Russian Academy of Sciences. References 1. Vysotski ES, Markova SV, Frank LA. Calcium-regulated photoproteins of marine coelenterates. Mol Biol 2006;40:355-367. 2. Natashin PV, Markova SV, Lee J, Vysotski ES, Liu JZ. Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins. FEBS J 2014;[doi; 10.1111/febs.12715]. 3. Prasher DC, McCann RO, Longiaru M, Cormier MJ. Sequence comparisons of complementary DNAs encoding aequorin isotypes. Biochemistry 1987;26:1326-1332. 4. Markova SV, Burakova LP, Frank LA, Golz S, Korostileva KA, Vysotski ES. Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria; cDNA cloning, expression, and characterization of novel recombinant protein. Photochem Photobiol Sci 2010;9:757-765. 5. Nomura M, Inouye S, Ohmiya Y, Tsuji FI. A C-terminal proline is required for bioluminescence of the Ca(2+) -binding photoprotein, aequorin. FEBS Lett 1991;295:63-66. 6. Fagan TF, Ohmiya Y, Blinks JR, Inouye S, Tsuji FI. Cloning, expression and sequence analysis of cDNA for the Ca(2+) -binding photoprotein, mitrocomin. FEBS Lett 1993;333:301-305. P0050 Chemiluminescence property of schiff base ligand bissalicylideneiminato and their Ni and Cu complexes Omeleila Nazari, Mahboube Jozaei, Mohammad Javad Chaichi, Mahjoube Ehsani University of Mazandaran, Babolsar, Mazandaran, Iran Introduction Luminol is a well known chemiluminescent reagent because of its essential applications on analysis of metal ions, hydrogen peroxide and some biological compounds such as hemin [1]. Addition of schiff base compound as catalyst can enhance the CL reactions and increase the intensity of the emission light [2]. The schiff base ligand complexes like bissalicylideneiminato(salen), Fig. 81, are particularly interesting for luminescence studies, because of their good chemical stability, intense absorption coefficients and high photoluminescence quantum yields [3, 4]. Sample r (1/s) f(1/s) M(µv) J(µv) It (µv) Ligand(10(-4) ) 1.1E3 6.4E4 1.9E7 1.8E7 2.0E7 Ligand(10(-5) ) 1.2E4 9.6E3 1.2E6 5.2E4 1.1E6 [Figure: see text] Expremental The CL reaction was monitored by means of a Sirius single Tube Luminometer (Berthold, Germany).The desired solution was made in a glass cell, using 0.3 ml luminol(10(-2) M in 0.15 M NaOH), 20μl of samples (include salen ligand and their Ni and Cu complexes) in 10(-4) , 10 (-5) and 10 (-6) M concentrations in methanol and 50 µl of hydrogen peroxide(10(-2) M) as a final reagent and the CL spectrums (intensity versus time) were recorded immediately after mixing. Subsequently the data were used in non-linear least squares curve fitting program KINFIT [5]. Result and discussion The CL spectrums showed that addition of all ligand and complexes have enhancing effect on luminol CL. The effect of the ligand as an enhancer catalyst was strong and more than the complexes. The experiments showed that the metal ion itself could not have very significant effect. For kinetic parameters evaluations of CL spectrum data, we use a simplified model based on a consecutive irreversible first-order reactions [6]. The integrated rate equation for the CL intensity versus time is; [Formula: see text] It is a light intensity at time t, M is a theoretical maximum level of intensity if all the reactants completely converted to chemiluminescence-generating material, r and f are respectively, the first order rate constants for the rise and fall of the burst of CL. The model permits an estimate of intensity at the maximum (J) from the reaction after the collection of only a part of the emitted light. The parameters was given by the following formula; [Formula: see text] The kinetic parameters thus obtained for all experiments are summarized in Table 1 based on non-linear least-squares fitting program KINFIT. [Table: see text] Refrences 1. Zhang XR, Baeyens WRG, Campaña AM. Recent developments of luminol based chemiluminescence analysis. Biomedical Chromatography 1999;13(2);169-170. 2. Smanmoo C, Yamasuji M, Sagawa T, Shibata T, Kabashima T, Yuan DQ, Kai M. Di imine ligand as a novel chemiluminescence enhancer of luminol containing compounds. Talanta 2009;77(5);1761-1766. 3. Cozzi PG, Dolci LS, Garelli A., Montalti M, Prodi L, Zaccheroni N. Photophysical poperties of Schiff-base metal complexes. New Journal of Chemistry 2003;27(4);692-697. 4. Taha ZA, Ajlouni AM, Al Momani W. Structural, luminescence and biological studies of trivalent lanthanide complexes with N, N'-bis (2-hydroxynaphthylmethylidene)-1, 3-propanediamine Schiff base ligand. Journal of Luminescence 2012;132(11);2832-2841. 5. Dye JL, Nicely VA. A general purpose curve fitting program for class and research use. Journal of Chemical Education 1971;48(7);443. 6. Hadd AG, Seeber A, Birks W. Kinetics of two path way in proxyoxalate chemiluminescence. Journal of Organic Chemistry 2000;5(9);2675-83. P0051 Quantum yield and spectrum of D-aminoluciferin bioluminescence reaction Kazuki Niwa(a) , Dai-ichiro Kato(b) , Mika Maenaka(b) , Yoshihiro Ohmiya(a) (a) National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan (b) University of Hyogo, Himeji, Japan Qquantum yield (QY) value and luminescence spectrum of bioluminescence reaction using D-aminoluciferin, that is an analogue of native D-luciferin, were examined in the presence of luciferases from Luciola cruciata and its mutants. For the purpose of the quantitative analyses, an integrating sphere spectroradiometer system was employed and calibrated using a spectral irradiance standard lamp that is traceable to the national standards of radiometry. Although the luminescence spectra from native D-luciferin with the mutant luciferases varied widely, the spectra from D-aminoluciferin were almost identical (Fig. 82), that the luminescence peak wavelength were from 583 to 593 nm. QY values of D-aminoluciferin were also determined and they varied from 20 to 36%. We previously reported that QY values of native D-luciferin varied from 15 to 60% depending on the enzyme luciferases and that the QY values correlated to the luminescence peak wavelength [Niwa et al., Photochem. Photobio., 2010, pp1047]. Correlations between QY values and luminescence peak wavelengths of D-aminoluciferin were agreed with those of native D-luciferin (Fig. 83). Hydroxyl group of D-luciferin is considered to play an important role for the bioluminescence color determination, as the effect of hydrogen bonding network with environmental amino acid residues. Our result supports that the hydroxyl group contributes to the color determination, because D-aminoluciferin does not have hydroxyl group to make appropriate hydrogen bonding network. [Figure: see text] [Figure: see text] P0052 First-Principles Investigation on Optical Properties of Firefly Luciferin Anion Yoshifumi Noguchi(a) , Miyabi Hiyama(a) , Hidefumi Akiyama(a) , Nobuaki Koga(b) (1) Institute for Solid State Physics, the University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, Japan (2) Graduate School of Information Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan A mechanism of firefly bioluminescence, involving the multistep processes that occur in the firefly body, is an attractive topic in biophotochemistry or related research fields. Actually, numerous theoretical and experimental investigations have been conducted over the last few decades. These studies have produced a common understanding that firefly bioluminescence, especially regarding the color and the strength of the light is sensitively affected by environmental conditions though each one plays no key role because any influence is negligible on the total effect. Therefore, any comprehensive treatment to provide a systematic explanation would be required. In theoretical viewpoint, considering experimental environmental condition is challenging issue. Instead, a possible approach concerning firefly bioluminescence starts from maximally simplified model, isolated firefly luciferin in vacuum in this case, and accumulates the reliable knowledge in step-by-step. Along this line, we will investigate optical properties of firefly luciferin anion in vacuum by using all-electron first-principle GW + Bethe-Salpeter method based on many-body perturbation theory beyond the framework of density functional theory (DFT) [1]. A three-step calculation is employed in this study. First is a ground state calculation by using a conventional DFT calculation. The LDA wave functions and Kohn - Sham orbital energies obtained here are used for constructing a GW self-energy operator in the second step of the calculation. Promising single excited spectra related to (inverse) photoemission spectra are obtained in this stage. In order to take the excitonic effect into account in present calculation, we solve Bethe-Salpeter equation within GW approximation (GWA) in the last stage. This procedure is called GW + Bethe-Salpeter method. The present calculation is carried out with all-electron mixed basis approach in which the wave functions are expanded as a linear combination of atomic orbitals (AOs) and plane waves (PWs) (see Fig. 84). Owing both use of AOs and PWs, the whole electronic states from core electron states localizing around nucleus to free electron states above the vacuum level are accurately described with small basis sets. Figure 85 shows GW quasiparticle energies and LDA Kohn-Sham orbital energies calculated for the isolated firefly luciferin anion in vacuum. The whole valence levels and 50 empty levels are plotted here. As consistent tendency in general, the HOMO-LUMO gap, where HOMO is the highest occupied molecular orbital and LUMO is the lowest unoccupied molecular orbital, improves from too small (or underestimated) LDA value (=1.32 eV) to GW one (=4.15 eV). Since the whole empty levels, including LUMO level, are located on the Rydberg states, the Rydberg excitation appears at low photon energy range in the photoabsorption process. The present method reproduces the lineshape of the experimental spectra well, in particular corresponding to the Rydberg excitation, however, overestimates the overall peak positions by about 0.4-0.5 eV. In this presentation, we will compare BSE-calculated photoabsorption spectra with those by time-dependent density functional theory (TDDFT) calculation with localized AOs as well as by experiments [2], and discuss theoretical treatment of Rydberg excitation in detail. [Figure: see text] [Figure: see text] 1. Noguchi Y, Hiyama M, Akiyama H, Koga N, in preparation. 2. Støchkel K, Milne BF, Nielsen SB. J. Phys. Chem. A 2011;115:2155. P0053 Bioluminescence resonance energy transfer (BRET) image analysis of Ras-Raf interaction in live cells using Nanoluc luciferase and Venus yellow fluorescent protein and bioluminescence microscopy Katsunori Ogoh(a) , Takeo Takahashi(a) , Atsushi Miyawaki(b) , Hirobumi Suzuki(a) (a) OLYMPUS Corporation, Hachioji, Tokyo, Japan (b) RIKEN Brain Science Institute, Wako, Saitama, Japan NanoLuc (Promega) is a new luciferase originated from deep sea shrimp and emits blue bioluminescence utilizing imidazopyrazinone substrate. Since NanoLuc is approximately 150 fold brighter than firefly luciferase, a bioluminescent image of cells expressing NanoLuc can be captured within a sub-second exposure time by a bioluminescence microscope, LV200 (Olympus) equipped with an electron multiplying (EM)-CCD camera (ImagEM, Hamamatsu Photonics). In this study, we used NanoLuc and a yellow-emitting mutant fo Aequorea GFP (Venus) as the donor and acceptor of bioluminescence resonance energy transfer (BRET), respectively, to image activation of Ras, a small G protein that transduces signaling from receptor tyrosine kinase to Raf (MAP kinase kinase kinase) at the inner part of cell membrane. H-Ras and Ras-binding domain (RBD) of Raf-1 were fused to Venus and NanoLuc, respectively. HeLa cells were co-transfected with the Venus-Ras and RBD-NanoLuc plasmid DNAs. After substrate administration, bioluminescent images through the donor (435-495 nm) and BRET (530 nm long pass) channels were captured alternately using LV200 equipped with ImagEM. This experimental system allowed us to image Ras-Raf interaction with a temporal resolution of 10 seconds. Immediately after stimulation with EGF (epidermal growth factor), an increase in BRET signal occurred on the plasma membrane (PM). While the PM-associated BRET increase due to Ras-Raf association faded out gradually, we clearly identified another increase in BRET signal appearing inside the cytoplasm at approximately 10 min after EGF stimulation. The latter activation of Ras involved endomembranous structues, such as Golgi apparatus and recycling endosome. To map the Ras activation locations more precisely, BRET-imaged samples are being subjected to immunocytochemical experiments that use organelle-specific antibodies. In this way, we demonstrate that BRET imaging using NanoLuc with the help of LV200, and ImagEM is a powerful means for studying spatiotemporal pattern of cellular events in live cells. P0054 Isolation of an enzyme involved in fungal bioluminescence Tatiana Pereira(1) , Cassius Stevani(a) , Hans Waldenmaier(a) , Anderson Oliveira(b) (a) IQ USP, São Paulo, SP, Brazil (b) ICT UNIFESP, São José dos Campos, São Paulo, Brazil Bioluminescence (BL) is known since Antiquity (1, 2), but only on the 19(th) century the light emitted from decaying trees was attributed to saprophytic fungi (1). Although BL has been studied for more than a century the detailed reaction pathway involved in fungal bioluminescence remains unknown. The involvement of enzymes in fungal BL postulated in the 1960's (3) was only confirmed in 2009 by an independent group (4), who also verified that the mechanism of all four lineages of luminescent fungi share the same type of luciferin and luciferase (5). In this work it is described the isolation of one enzyme involved in the bioluminescence pathway must probably reductase. Extracts prepared with cultivated mycelium of Neonothopanus gardneri were homogenized and fractionated by centrifugation, precipitation with ammonium sulfate, followed by two different columns chromatography steps. The light emission from each fraction was monitored by using a luminometer and the classical cold/hot extract procedure (4) and to measure the amount of protein, it was used a spectrophotometer and the Bradford method (6). The reductase specific activity was calculated from the observed formation rate constant divided by the amount of protein (detail in Fig. 86). As expected, the specific activity increased along the puri fication process (Fig. 86). The fraction obtained by the affinity chromatography was submitted to a native PAGE, which confirmed the presence of the reductase in one of the bands. [Figure: see text] References 1. Desjardin DE, Oliveira AG, Stevani CV. Fungi bioluminescence revisited. Photochem. Photobiol. Sci. 2008;7:170-182 2. Waldenmaier HE, Oliveira AG, Stevani CV. Thoughts on the diversity of convergent evolution of bioluminescence on earth. Int. J. Astrobiol. 2012;1:1-9 3. Airth RL, Foerster GE. The isolation of catalytic components required for cell-free fungal bioluminescence. Arch. Biochem. Biophys. 1962;97(3);567-573 4. Oliveira AG, Stevani CV. The enzymatic nature of fungal bioluminescence. Photochem. Photobiol. Sci. 2009;8:1416-1421 5. Oliveira AG, Stevani CV. Evidence that a single bioluminescent system is shared by all known bioluminescent fungal lineages. Photochem. Photobiol. Sci. 2012;11:848-852 6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72(1-2);248-254 P0055 Spectral characteristics of the fluorescence in scale worms (Polychatea, Polynoidae) Maria Plyushcheva(a) , Katiane Pereira da Silva(b,c) , Daniel Martin(d) , Paulo de Tarso Cavalcante Freire(c) , Fyodor Kondrashov(a,e) , Alejandro Goñi(b,e) (1) CRG, Barcelona, Spain (2) ICMAB-CSIC, Belaterra, Spain (3) Universidade Federal do Ceará, Fortaleza, Brazil (4) CEAB CSIC, Blanes, Spain, (5) ICREA, Barcelona, Spain Among the segmented marine worms, the species of the sub-family Polynoidae are commonly known as scale-worms because their dorsum is covered by ornamented scales or elytra. In some cases, an area of the epithelium of the lower face of the elytra was reported to emit light flashes upon stimulation with ultraviolet (UV) light [1]. The origin of the bioluminescence in polynoids is a photo-protein that reacts specifically to the presence of superoxide anions called polynoidin [2]. Several sources of fluorescence were observed in all studied "bioluminescent" species. First source is so called "photogenic area", corresponding to cells of the ventral epithelium, around the place where elytra are attached to elytrophore [2]. From the dorsal side all elytra of scale worms are covered by tubercles. Under UV excitation, tubercles show bright bluish fluorescence in both "bioluminescent" and "non-bioluminescent" species (see Fig. 87). The third source of fluorescence in "bioluminescent" species is located at the dorsal side of elytra in dorsal epithelial cells. Non-bioluminescent species have the same fluorescent system but the ability to emit light is a question of morphological organization. The presence of polynoidin in "non-bioluminescent" species suggests that the ability to produce bioluminescence has been lost as a consequence of morphological adaptation [3]. Here we have measured and analyzed the spectral characteristics of the light emission in three species, bioluminescent Harmothoe areolata (HA), Harmothoe imbricata (HI) and non-bioluminescent Lepidonotus squamatus (LS); the former one from the Mediterranean Sea and the remaining ones from the White Sea. The focus of this work concerns the fluorescence or photoluminescence (PL), i.e., light emission after excitation by light absorption at shorter wavelengths, in-vivo as well as in-vitro experiments. Spectra of the tubercles on freshly off-lopped scales were measured at room temperature using eight different laser lines for excitation from the violet (405 nm) to the red (632 nm). An example of the spectral changes in the PL emission observed by varying the excitation wavelength is shown in the left panel of Fig. 88 for Harmothoe areolata. Spectra were collected with a high resolution LabRam spectrometer using a confocal optical microscope and corrected for the spectral response of the grating and detector. The main task consisted in disentangling the contributions from different chromophores of the protein by a careful lineshape analysis using purposely defined functions composed of Gaussian peaks. We were able to spectrally separate, for the first time, the contributions of the different chromophores to the PL emission, by paying special attention to the spectral changes upon variation of the excitation wavelength. [Figure: see text] [Figure: see text] The right panel of Fig. 88 shows a representative spectrum of Harmothoe areolata (black solid curve) obtained with 405-nm excitation. The red solid curve represents the result of a lineshape fit using the four chromophore lineshape functions (color lines) depicted in the figure. The colors roughly correspond to the wavelength of the maximum emission for each chromophore, indicated by their values in eV in Fig. 88 (right panel). These four chromophores appear to be common to all three species, whereas HI and LS posses each an additional characteristic chromophore strongly emitting in the far red and orange spectral range, respectively, which determines the particular color of the fluorescence of that worms. The light emission associated with each chromophore comprises a set of equidistant Gaussian peaks of the same width and characteristic intensity ratios, as expected for electronic - optical transitions between different vibrational levels in chromophores containing aromatic rings and/or conjugated carbon-carbon bonds [4]. These results constitute the basis to understand the spectral changes of the fluorescence of the polynoidin protein upon variation of temperature and/or pressure, with the ultimate goal of unraveling the interplay between light emission and morphology. References; 1. Nicol JAC, Mar J. Biol. Ass. UK 1958;37:33-41. 2. Bassot JM, Nicolas M. T.. Histochem. Cell Biol. 1995;104:199-210. 3. Plyuscheva MV, Martin D.. Proc. of the 9th Int. Polychaete Conf. Zoosymposia 2009;2:379-389. 4. Atkins PW. Physical chemistry (Oxford University Press; Oxford, 1998). P0056 Thioesterification activity and the biological function of the enigmatic luciferase-like enzyme of Zophobas morio (Coleoptera; Tenebrionidae) Malpighian Tubules Rogilene Prado(a) , Dai-Ichiro Kato(c) , Mario Murakami(b) , Vadim Viviani(a) (a) University of São Carlos, Sorocba, São Paulo, Brazil (b) Brazilian Biosciences National Laboratory, Campinas, São Paulo, Brazil (c) University of Hyogo, Himeji, Hyogo, Japan It has been suggested that beetle luciferase evolved from fatty acyl-CoA synthetase, a member of adenylate-forming enzyme superfamily that catalyze the activation of fatty acids through adenylation at the expense of ATP and the subsequent transfer and thioesterification of the carboxylic group to CoA for different metabolic purposes. We recently cloned and characterized a luciferase-like enzyme from the Malpighian tubules of the non-bioluminescent Zophobas morio mealworms (Coleopetra; Tenebrionidae). This enzyme is serving as protein model to study the origin and evolution of beetle luciferases since this enzyme is a CoA-ligase able to produce weak luminescence in the presence of the firefly D-luciferin substrate. However, the natural biological function and substrate of this enzyme in the Malpighian tubules remains unknown. Through a spectrophotometric assay, the thioesterification activity of this luciferase-like enzyme was measured using different carboxylic substrates. The results show that this luciferase-like enzyme is able to catalyze the thioesterification of different groups of carboxylic substrates including long and short-chain fatty acids, aromatic acids, amino acids and L-luciferin, indicating that this luciferase-like enzyme has a broader repertory of substrates than fatty acyl-CoA synthetases. This observation suggests that beetle protoluciferases may have arisen from CoA ligases with broader substrate repertories, and not necessarily from the more specific fatty acyl-CoA synthetases. Furthermore, whereas this enzyme can thioesterify CoA with L-luciferin, it is unable to catalyze the efficient thioesterification of D-luciferin isomer. Thus, the lack of efficient thioesterification reaction with D-luciferin may have sterically favored an otherwise lower oxidative reaction, resulting in the emergence of the oxygenase activity of protoluciferases, leading to the evolution of luciferases. The presence of this enzyme in Malpighian tubules, its association with endoplasmic reticulum, the broader repertory of carboxylic substrates, and the oxygenase activity with fortuitous metabolites or xenobiotics such as D-luciferin suggest that this enzyme could be a new type of xenobiotic-ligase (Financial support; Fapesp 2012/02161-9, 2012/04857-1; CNPq). P0057 Bioluminescence in firefly oocytes; is there a metabolic function? Rogilene Prado(a) , Pablo Nunes(b) , Fabio Abdalla(a) , Vadim Viviani(a) (a) University of São Carlos, Sorocaba, Brazil (b) São Paulo State University, Rio Claro, Brazil Bioluminescence, which is found in many organisms, is biologically important and used for different communicative functions such as defense, offense and sexual attraction. The major function of light emission in fireflies is for communication during courtship, but firefly eggs, larvae and pupae are also able to emit light. Furthermore, we previously reported that the fat body of larval fireflies is also weakly bioluminescent, providing evidences that the photogenic tissue of lanterns evolved from the fat body, specifically the trophocytes. It is believed that larvae and pupae use the bioluminescence to keep predators away. Generally, bioluminescence is not considered metabolically essential at all, therefore, the function of the light emission from eggs is a mistery. Through the use of immunofluorescence with rabbit anti-firefly luciferase-polyclonal antibodies and CCD imaging we investigated the bioluminescence in ovarioles and eggs removed from virgin females of Aspisoma sp (Lampyridae). Fat body and lanterns were used as control. Fat body, lantern, eggs and ovarioles were incubated with rabbit anti-firefly luciferase followed by incubation with goat anti-rabbit secondary antibody conjugated to Cy5 (molecular probes®). Immunofluorescence using confocal microscopy revealed the occurrence of luciferase in cells of the germarium and vitellarium regions of whole ovariole. These cells coat the early and mature oocytes. As expected, the lanterns were strongly marked and the fat body was weakly marked indicating presence of luciferase. The CCD imaging confirmed the presence of luciferase and luciferin once the bioluminescence is naturally originated from ovariole. The classical communication functions of bioluminescence are clearly not applicable to non-fertilized eggs or oocytes. These results indicate that primary germ cells are able to emit light and that bioluminescence may have a metabolic function in this case (Financial support; Fapesp 2012/06121-9). P0058 Screening for Histone Deacetylase (HDAC) Active Compounds Tobias Pusterla(1) , Franka Maurer(a) , EJ Dell(a) , Sheraz Gul(b) , Gesa Witt(b) (a) BMG LABTECH GmbH, Ortenberg, Germany (b) European Screening Port GmbH, Hamburg, Germany Histones are small basic proteins around which DNA in a stacked configuration is bound to build chromatin prior the replication procedure. After translation, histone side chains are often modified by acetylation, deacetylation, methylation, ADP-ribosylation, and phosphorylation. Histone deacetylation is carried out by enzymes that build a complex with histones. These complexes target specific promoters to repress transcription through deacetylation of histones in specific nucleosomes. HDAC inhibitors (HDACi) have been used to treat neurological symptoms, as well as cancers, parasitic and inflammatory disease. In order to find compounds that are potential inhibitors of HDAC activity (Class I and II), a chemiluminescent Proof-of-Concept Screen was performed on the PHERAstar FS microplate reader from BMG LABTECH. In order to measure the activity of HDAC class I and II, the HDAC-Glo™ I/II assay from Promega was used. In this assay, an acetylated peptide is offered as an HDAC substrate. After deacetylation, a protease cleaves the deacetylated substrate and releases aminoluciferin that is immediately consumed by a luciferase. The resulting luminescent signal is proportional to the HDAC activity. To test the assay for inhibitors, a subset of the LOPAC library was screened for active compounds against HDAC. Usually, promising hits are defined to show 90 % or higher inhibition. Indeed, a few hits were found during the compound screen. Some of them showed also up in the Counter Screen, indicating false negatives that are either active against the protease in the developer solution or against the luciferase. This shows that Counter Screens are very useful especially if the assay includes a coupled enzyme system to detect target activity. This shows that the PHERAstar FS can be used for screening assays as well as for assay development. P0059 Smartphone embedded enzymatic chemical Luminescence-Based Biosensor for Point-Of-Need Application Aldo Roda(a,c) , Luca Cevenini(a) , Donato Calabria(b) , Maria Maddalena Calabretta(c) , Elisa Michelini(a,b) (a) Dept. of Chemistry, University of Bologna, Bologna, Italy (b) CIRI Health Sciences & Technologies, Bologna, Italy (c) INBB, Roma, Italy We present for the first time the use of a smartphone to image and quantify biospecific enzymatic reactions coupled with bio-chemiluminescence [1] to detect analytes in saliva. To this end we implemented a portable disposable minicartridge into a smartphone. The production of photons by a chemical reaction facilitates the device configuration which simply consists of a nitrocellulose disk in which the analyte biospecific enzyme is co-immobilized with the light producing enzymes; e.g., bacterial luciferase/diaphorase to monitor all the NAD(H) dehydrogenases and peroxidase/H2 O2 coupled with oxidase enzymes. The minicardridge is placed in contact with the smartphone objective via a plano-convex lens (6mm diameter) that allows to focus the spot image at a distance of 12mm from the phone objective. A disposable PDMS minicartridge has been designed and fabricated using low-cost 3D printing technology. A microfluidics channel system allows to introduce the sample, substrates and buffer required for the enzymatic-coupled reaction which occurs in a custom home-made small dark box to shed from ambient light. Two o more parallel microfluidic channels can be designed to allow a multiplex format or to use one spot as a reference for calibration purposes. Suitable phone applications are used to control exposition time (20-30s) in order to achieve the needed detectability and for data handling. As a proof of concept the serum and saliva lactate is measured using lactate dehydrogenase coupled with bacterial luciferase system allowing to measure as low as 0.1 mmol/L of lactate. This approach can be exploited in endurance sports for lactate monitoring. Therefore the use of smartphone-based biosensors with bio-chemiluminescence detection could find broad applications for the detection of several analytes of clinical interest (e.g., glucose, ethanol, cholesterol) in samples such as saliva and blood. 1. Michelini E, Cevenini L, Mezzanotte L, Roda A. Luminescent probes and visualization of bioluminescence. Methods Mol Biol. 2009;574:1-13. P0060 Evaluation of the antiradical capacity of Baccharis oxyodonta extracts using luminol chemiluminescence Gustavo Yuri M. Viegas(a) , Cinthia Indy Tamayose(b) , Marcelo J. P. Ferreira(a) , Oriana A. Fávero(a) , Wilhelm J. Baader(b) , Paulete Romoff(a) (a) Escola de Engenharia e Centro de Ciências e Biológicas e da Saúde, Universidade Presbiteriana Mackenzie, São Paulo - SP, Brazil (b) Instituto de Química, Universidade de São Paulo, São Paulo - SP, Brazil Antioxidants are defined as substance that can prevent, delay or repair oxidative damages in biological systems as they interfere in the production and destruction of reactive oxygen species, due to their capacity to reduce reactive oxygen species. Several methods for determining the total antioxidant capacity of pure substances and mixtures have been developed which utilize a specific radical reaction which is inhibited by the addition of a potentia l antioxidant compound.([1]) The chemiluminescent oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) by hydrogen peroxide in the presence of a transition metal catalyst in aqueous alkaline medium occurs with the involvement of free radicals, including radical species formed by the oxidation of the luminol monoanion by the transition metal catalyst, and several reactive oxygen species (ROS) like superoxide anion and hydroxyl radical.([2],[3]) The addition of compounds able to scavenge free radical species will lead to the reduction of the radicals and thereby to the inhibition of light emission. Thus, the suppression area of the emission intensity curve is a measure of the antiradical capacity of a sample as it is proportional to the number of radical quenched by the additive.([4]) The antiradical capacity of complex mixtures can be expressed conveniently in relation to the standard antiradical Trolox® as Trolox® percentage (%Trolox®). In this sense we proposed a screening test for antiradical activity of extracts, separation phases and chromatographic fractions, using the antiradical capacity as a preliminary parameter for potential biological activity.([4]) In the present work we report the antiradical activity of hydroalcoholic extracts obtained from the aerial parts of male and female individuals of Baccharis oxyodontha collected in Campos do Jordão, São Paulo. Baccharis genus is the largest genus in the family Asteraceae, with over 500 species distributed throughout the North and South American continents, of which 120 occur in Brazil. Many of them are used in folk medicine to treat a wide variety of diseases, such as liver and kidney disorders, diabetes and inflammatory processes.([5]) The plants of this genus are usually dioecious herbs and shrubs, which means that they produce a male and a female plant. The antiradical capacity of extracts was determined using the luminol chemiluminescence antiradical assay, as well as conventional antiradical assays. The hydroalcoholic extracts of Baccharis oxyodonta aerial parts were submitted to HPLC-MS/MS analysis which leads to the identification of eighteen chlorogenic acid derivatives, being 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid the major compounds, along with four flavonoids. Chlorogenic acids (CGA) are a family of esters formed between certain trans cinnamic acids and (-)-quinic acid [1L-1(OH)-3,4/5-tetrahydroxycyclohexanecarboxylic acid], which possess a broad spectrum of pharmacological properties, including antioxidant, hepatoprotectant, antibacterial, antihistiminic, and other biological effects. The hydroalcoholic extracts obtained from male and female species show similar capacities in the luminol assay, and the %Trolox® values were determined as 37.4 ± 0.1 and 39,13 ± 0,03 %, respectively. In fact, the chemical compositions of the extracts are very similar. Additionally, the chemiluminescent luminol assay proved to be considerably more sensitive as compared to the DPPH and ABTS assays. Financial support; Fundo Mackenzie de Pesquisa, FAPESP, Capes, CNPq. 1. Fedorova GF, Trofimov AV, Vasil'ev RF, Veprintsev T. L.. ARKIVOC 2007;163:2007. 2. Augusto FA, de Souza GA, de Souza Jr SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. 3. Baader WJ, Stevani CV, Bastos EL. In The Chemistry of Peroxides, Rappoport Z., ed. Chichester; Wiley & Sons, 2006, vol. 2, chapter. 16, 1211. 4. Bastos EL, Romoff P, Eckert CR, Baader WJ. J. Agric. Food Chem. 2003;51:7481. 5. Abad MJ, Bermejo PJ. Arkivoc 2007;7:79. P0061 DNA barcoding of Japan's bioluminescent organisms Darrin Tyler Schultz, Yuichi Oba Nagoya University, Nagoya City, Aichi Prefecture, Japan Japan has been called a " treasure box" of bioluminescent organisms(1) , with 58 described terrestrial bioluminescent species and several dozen more species of bioluminescent snails, fish, squid, and other taxa in the surrounding waters (2,3) . While some species' luciferase genes have provided important research tools in the past, there are many species whose biochemistry eludes characterization. A DNA barcode library for Japan's bioluminescent organisms will provide a starting point for biochemical studies of uncharacterized species, as well as help clarify the taxonomy of several contested species. This ongoing study will be represented by more than 50 specimens of molluscs, 40 specimens of fishes, 50 specimens of fungi, 200 specimens of arthropods, and 200 specimens of annelids. Each specimen represents a different locale. In addition, sympatric nonluminous species, and nonluminous members of several cryptic species complexes were included in this analysis. The ITS barcode sequence for fungi, and the COI barcode sequence for other taxa will be uploaded to the Barcode of Life Datasystems (BOLD) database under the project name Japanese Bioluminescent Organisms Barcoding Project (JBOBP)(4) in conjunction with the Japanese Barcode of Life Initiative (JBOLI). Our group is actively seeking out further samples from all taxa, including specimens of bioluminescent organisms from other countries for comparative studies. 1. Haneda, Y. On the luminous organisms (Hakkou Seibutsu no Hanashi). Tokyo; Hokuryukan (In Japanese), 1972 2. Oba Y et al. The Terrestrial Bioluminescent Animals of Japan. Zoo. Sci 2011;28(11);771-789 3. Haddock SHD, et al. Bioluminescence in the Sea. Annu Rev Mar Sci 2010;2; 443-493. 4. JBOBP. http;// P0062 Role of 270 and 271 residues on function of Lampyris turkestanicus luciferase Raheleh Shakeri(a) , Saman Hosseinkhani(b) , Sussan Kabudanian Ardestani(a) (a) Institute of Biochemistry and Biophysics, University of Tehran,, Tehran, Iran (b) Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran The mutation of Leu300 to Arg in Lampyris turkestanicus leads to inactivation of the enzyme. Modeling studies had shown that Arg300 side chain have ion interactions with side chains of glutamate 270 and 271 that lead to very low activity of the enzyme. It seems that low activity is a result of high rigidity of the enzyme structure. According to these reasons, the roles of these residues are investigated on function of luciferase. Mutations were created using Quick Change Site-Directed Mutagenesis on pET28a plasmid containing cloned luciferase. Mutations were confirmed by standard sequencing. Mutant plasmids were transformed to competent E.coli BL21 cells for protein expression and purification using affinity chromatography. Luciferase activity was measured by adding enzyme solution to a complex containing 50 mM Tris, 5.0 mM luciferin, 40 mM ATP and 50 mM MgSO4 . The mutant forms of luciferase, surprisingly, were almost fully inactivated before purification. Low activity was observed after purification of mutants. SDS-PAGE analysis of purified mutant proteins showed decreased expression of mutant luciferases relative to native luciferase. The Km values of mutants for luciferin was almost the same as that of wild-type luciferase. It may be suggested part of low activity is due to decreased expression. It seems, these mutations have decreased mRNA stability or translation of the luciferase. Further experiments are underway to identify the effects of these mutations on level of expression, structure and function of the enzyme. Reference 1. Mortazavi M, Hosseinkhani S. Design of thermostable luciferases through arginine saturation in solvent-exposed loops. Protein Eng Des Sel. 2011 Dec;24(12);893-903. 2. Amini-Bayat Z, Hosseinkhani S, Jafari R, Khajeh K. Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase. Biochim Biophys Acta. 2012 Feb;1824(2);350-8. P0063 Determination of iron in G3 corrosion-resistant alloy tubing and coupling by flow injection analysis with chemiluminescence detection Xiaodong Shao(a,b) (a) CNPC Tubular Goods Research Institute, No. 89 Jinyeer Road, Xi'an 710077, China (b) CNPC Key Laboratory for Petroleum Tubular Goods Engineering, No. 89 Jinyeer Road, Xi'an 710077, China With the increase desire for energy, the worldwide search f or new sources of hydrocarbons is turning to the gas and oil wells containing high hydrogen sulfide. The maximum temperature in these wells can be up to 320 °C, and the maximum pressure is up to 100 MPa. Meanwhile, the partial pressure of hydrogen sulfide and carbon dioxide is very high. During exploitation, the elemental sulfur may be separated out. In order to meet the exploitation requirements of these wells with high temperature, high pressure and high partial pressure of hydrogen sulfide and carbon dioxide, the oil country tubular goods must adopt alloy materials with excellent resistance to high temperature and corrosion, good strength, plasticity, toughness, metallurgical stability, workability and solderability. The application scope in industry of nickel corrosion-resistant alloy is increasing, for example, in aerospace, nuclear power and ship products. In addition, many nickel corrosion-resistant alloys have excellent heat resistance, and become the ideal selection for resistance to corrosion and high temperature [1]. Therefore, the nickel corrosion-resistant alloy oil country tubular goods have excellent resistance to corrosion. This is because that the formed passive film on surface isolate the further contact between matrix and aggressive medium, protecting the matrix. It is not only has lower metal loss, but also can be subject to local corrosion, especially for resistance to hole corrosion or crevice corrosion, intergranular corrosion and stress corrosion. Thus, nickel corrosion-resistant alloy oil country tubular goods will be widely used in the exploitation of acid gas and oil wells. Continuous improvement of metallurgy and manufacturing technology promotes the development of nickel corrosion-resistant alloy. The content of chemical elements in nickel corrosion-resistant alloy directly influences the properties of alloy materials. In order to control the quality of nickel corrosion-resistant alloy products, the contents of trace impurities and alloy elements in nickel corrosion-resistant alloy should be determined. In alloy-making processes, the content of alloyed elements such as nickel, iron, manganese, chromium, molybdenum, vanadium, and so on, is required to be strictly controlled because it fundamentally determines the performance of nickel corrosion-resistant alloy. It is well known that iron is an important element in nickel corrosion-resistant alloy. Therefore, a rapid and precise analytical method for the determination of iron is essentially required for the production control of nickel corrosion-resistant alloy. In recent years, various chemical analysis techniques have distinct advantages in sensitivity, reproducibility, simplicity, cost effectiveness, flexibility and rapidity. A range of analytical techniques have been used for analysis of iron in various samples including UV-vis spectrophotometry, ion chromatography, atomic absorption spectrometry, inductively coupled plasma atomic emission spectrophotometry, and voltammetry [2-4]. Spectrophotometric methods occupy special position due to their simplicity, less expensive instrumentation and high sensitivity. A number of chromogenic reagents, such as o-phenylenediamine with hydrogen peroxide, p-anisidine with N, N-dimethylaniline, and ammonium thiocyanate have been reported for the determination of iron. Chemiluminescence refers to the emission of light from a chemical reaction, which can occur in solid, liquid or gas systems. The fundamentals of chemiluminescence have been comprehensively reviewed in a number of textbooks and articles in recent years. Two main categories of chemiluminescence reaction have been described in the literature, direct and indirect [5]. Direct chemiluminescence can be represented by; [Formula: see text] where A and B are reactants and [I](*) is an excited state intermediate. The luminol reaction is an example of this form of chemiluminescence. In certain cases where the excited state is an inefficient emitter, its energy may be passed on to another species (a sensitizer, F) for light emission to be observed. This is called "indirect chemiluminescence" and is exemplified by the peroxyoxalate (light stick) reaction; [Formula: see text] Progress in flow injection chemiluminescence analysis has received much attention in chemical analysis due to the high sensitivity, rapidity and simplicity of this method [6]. The chemiluminescence method with different chemiluminescence systems has been exploited for the determination of iron. The emission of light observed when a solution containing luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione or, more simply, 3-aminophthalhydrazide) and hydrogen peroxide. Luminol-hydrogen peroxide reaction as a classical chemiluminescence system is reported by different research groups, which is based on the oxidation of luminol by hydrogen peroxide. In this work, it is found that iron (III) remarkably catalyzed the chemiluminescence reaction between luminol and hydrogen peroxide, and leading to fast chemiluminescence. On the basis of this a simple, sensitive and rapid procedure is developed for the determination of iron (III). The increment of chemiluminescence intensity produced is directly proportional to the iron (III) concentration, and the linearity is obtained in the range of 6.0 ng mL(-1) -700.0 ng mL(-1) (R(2)  = 0.9993), with the limit of detection (LOD) is 2.0 ng mL(-1) (3×σnoise ). The relative standard deviations (RSD) were less than 3.0% (n = 5). At the flow rate of 2.0 mL min(-1) , a complete determination of iron (III), including sampling and washing, could be completed in 0.5 min, offering the sampling efficiency of 120 h(-1) accordingly. The proposed procedure was satisfactory for the application to determine iron in G3 corrosion-resistant alloy tubing and coupling samples. Acknowledgments This work was financially supported by CNPC Key Laboratory for Petroleum Tubular Goods Engineering. The author gratefully acknowledges the CNPC Tubular Goods Research Institute, and China National Quality Supervision, Testing and Inspection Center of Oil Tubular Goods. References 1. Guo X, Li J, Zhou B, Influence of shear temperature on chip formation of turning GH4169. J. Shanghai Jiaotong University 2009;43:79-83. 2. Senee K, Saisunee L, Napaporn Y. A simple and green analytical method for determination of iron based on micro flow analysis. Talanta 2007;73:46-53. 3. Divjak B, Franko M, Novic M. Determination of iron in complex matrices by ion chromatography with UV-Vis, thermal lens and amperometric detection using post-column. J. Chromatogr. A 1998;829:167-174. 4. Ugo P, Moretto L, Rudello D, Birrel E, Chevalet J. Trace iron determination by cyclic and multiple square-wave voltammetry at nafion coated electrodes. applicationto pore-water analysis. Electroanalysis 2001;13:661-668. 5. Barni F, Lewis S W, Berti A, Miskelly G M, Lagoa G. Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta 2007;72:896-913. 6. Powe AM, Fletcher KA. Molecular fluorescence, phosphorescence, and chemiluminescence spectrometry. Anal. Chem. 2004;76:4614-4634. P0064 A practical method for the determination of niobium in API X100 SAW pipe based on the reaction between potassium ferricyanide and luminol Xiaodong Shao(a,b) (a) CNPC Tubular Goods Research Institute, No. 89 Jinyeer Road, Xi'an 710077, China (b) CNPC Key Laboratory for Petroleum Tubular Goods Engineering, No. 89 Jinyeer Road, Xi'an 710077, China With the rapid development of economy, the demands of oil and gas are further increased. Pipeline system is the main form of transportation for natural gas. To improve transportation efficiency, reduce project investment, long distance transportation of oil and natural gas pipeline has the trend to high strength steels. The use of high strength line pipe steels is beneficial for the reduction the cost of gas transmission pipelines by enabling high pressure transmission of large volumes of gas. The high strength line pipe steels can also improve the security of long-distance transmission effectively. Thus, the high strength line pipe steels will become the preferred ma terials for modern natural gas transmission pipeline [1]. In particular, high strength line pipe materials with the yield strength of X80 or higher have been developed over the past few decades around the world. With the Second West-East Natural Gas Transmission Pipeline Project accomplished in china, an X100 operation trail will be constructed recently. With a much wider anticipation of X100 linepipe being recognized increasingly, a lot of researches that conducted to speed up the process of industry production and pipeline construction, focus on chemical composition design, steel rolling technology, strength and toughness of material, the effect of strain ageing, field weld-ability, strain-based design or strain capacity of linepipe and so on [2]. And X100 line pipe has been developed in Japan Nippon Steel, Europipe and other companies. China West-East Natural Gas Transmission Pipeline Project has been completed for the X70 grade submerged-arc welding helical (SAWH) and submerged-arc welding longitudinal (SAWL) steel pipe, the Second West-East Natural Gas Transmission Pipeline Project mainly steel pipe is the X80 SAWH and SAWL steel pipe. The alloying of steel with several metallic elements plays an important role for improving the mechanical and chemical properties such as strength, toughness, heat resisting, and corrosion resistance. In steel-making processes, the content of alloyed elements such as niobium, chromium, nickel, molybdenum, vanadium, and titanium and so on, is required to be strictly controlled because it fundamentally determines the performance of steel materials. Therefore, a rapid and precise analytical method is essentially required for the production control of manufactured steels. It was well known that niobium was an important element in the high strength line pipe steels. Niobium is the valuable alloying agents and coexists in a number of industrially important alloys and steels. From the literature, numerous analytical methods were employed for the determination of niobium in different types of matrices including high-performance liquid chromatography [3], atomic emission spectrophotometry [4], electroanalytical method and spectrophotometry [5]. Spectrophotometric methods occupy special position due to their simplicity, less expensive instrumentation and high sensitivity. Similarly, spectrophotometric methods also play an important role in niobium determination. Progress in flow-injection chemiluminescence analysis has received much attention in chemical analysis due to the high sensitivity, rapidity, and simplicity of this method [6]. It is well known that the reaction between luminol and potassium ferricyanide could emit chemiluminescence, this reaction has been applied in different research fields. In this paper, it was observed that niobium (V) could remarkably inhibit the chemiluminescence reaction between luminol and potassium ferricyanide, based on which a simple, sensitive as well as rapid procedure was designed to determine niobium (V). The decrement of chemiluminescence signal was linear with the niobium (V) concentration over the range from 10.0 ng mL(-1) to 800.0 ng mL(-1) (R(2)  = 0.9991) with relative standard deviations of less than 3% (n = 5) and the detection limit was 3.0 ng mL(-1) (3×σnoise ). At a flow rate of 2.0 mL min(-1) , a complete determination of niobium (V), including sampling and washing, could be completed in 0.5 min, offering the sampling efficiency of 120 h(-1) accordingly. The proposed method has been applied satisfactorily to the determination of niobium (V) in API X100 SAW steel pipe samples with the improved accuracy and precision. The satisfactory performance in the determination of niobium in API X100 SAW steel pipe demonstrated that the method was practical and suitable not only for quality control analysis but also for product analysis, confirming the promise for X100 SAW steel pipe research. Acknowledgments This work was financially supported by CNPC Key Laboratory for Petroleum Tubular Goods Engineering. The author gratefully acknowledges the CNPC Tubular Goods Research Institute, and China National Quality Supervision, Testing and Inspection Center of Oil Tubular Goods. References 1. Li H. Hot topics in study and application of steel line pipe for natural gas transportation. China Mechanical Engineering 2001;12:349-352. 2. Nafisi S, Arafin MA, Collins L, Szpunar J. Texture and mechanical properties of API X100 steel manufactured under various thermomechanical cycles. Materials Science and Engineering; A 2012;531:2-11. 3. Ryozo N, Shozo S, Shozo S. Determination of niobium, titanium and zirconium by high-frequency plasma torch emission spectrometry and its application to steel. Anal. Chim. Acta 1974;70:265-274. 4. Li D, Hu X, Wang H. Separation and simultaneous determination of niobium and tantalum in steel by reversed-phase high-performance liquid chromatography using 2-(2-pyridylazo)-5-diethylamino phenol as a pre-column derivatizing reagent. Talanta 2004;63:233-237. 5. Burns DT, Chimpalee D. Spectrophotometric determination of niobium after extraction with thiocyanate and 1-naphthylmethyltriphenylphosphonium chloride into microcrystalline 1,4-dichlorobenzene. Anal. Chim. Acta 1992;256:307-310. 6. Powe AM, Fletcher KA. Molecular fluorescence, phosphorescence, and chemiluminescence spectrometry. Anal. Chem. 2004;76:4614-4634. P0065 Determination of Perphenazine in pharmaceuticals using chemiluminescence reaction of luminol catalyzed by myoglobin Bahram Shohreh(a) , Seyed Mohammad Abedirad(b) , Sayed Yahya Kazemi(b) (a) Department of Animal Science,Sari Agricultural Sciences and Natural Resources University, Sari,Mazandaran, Iran (b) Department of Basic Sciences,Sari Agricultural Sciences and Natural Resources University, Sari,Mazandaran, Iran, (3) Department of Basic Sciences,Sari Agricultural Sciences and Natural Resources University, Sari,Mazandaran, Iran Perphenazine is chemical known as 4-[3-(2-chlorophenothiazin-10-yl)propyl]-1-piperazineethanol which belongs to the phenothiazine family of drugs. It is used as neuroleptic, antiemetic and antidepressive. Moreover, it is found to be effectual in the treatment of Parkinson's disease [1] In these regards, introduce simple, reliable and highly sensitive method which can be established for the determination of perphenazine has great attention in medical and life sciences. The attractiveness of chemiluminescence (CL) as an analytical tool lies primarily in the simplicity of detection since most samples have no unwanted background luminescence and no optical filters are required to separate the excitation wavelengths and scatter. Hence, it is widely used for determination of various analytes with low detection limit and wide linear range [2]. Myoglobin (Mb) is a small (MW ~17.5 kDa) and stable heme protein designed to reversibly bind dioxygen. It is primarily found in mammalian skeletal and cardiac muscle tissue and is the protein responsible for oxygen storage and transport within these tissues. The molecule contains a single iron protoporphyrin or heme moiety and is thus classified as a metalloprotein It has been proven that MB could catalyzed CL reaction as catalyst [2]. Herein, we developed a flow-based chemiluminescence method for determination of promazine in pharmaceuticals. It was found that promazine could inhibit dramatically decreaced CL signal from reaction of luminol and potassium ferricyanide in the presence of myglobin as the catalyst. Under optimum condition, a linear working range for promazine concentrations ranging from 1 × 10 (-7) to 5 × 10 (-5) mol L(-1) (r > 0.9912, n = 8), was obtained, with a detection limit of 8 × 10 (-9) mol L(-1) . The relative standard deviations were less than 2.8% (n =8) and recovery was 97%. The method was proved to be sensitive and selective and was applied to determine of promazine in pharmaceuticals with satisfactory results. [Figure: see text] 1. Pacini L, Limatola C, Palma E, Spinedi A. Effects of pherphenazine on the metabolism of inositol phospholipids in SK-N-BE(2) human neuroblastoma cells. Biochem Pharmacol 1994;48:1655-7 2. G ao X, Liu Y, Song Z. Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature .Luminescence 2007;22:88-91 P0066 A transcriptional profile of Arachnocampa Flava (Diptera; Mycetophylidae) laterns est Library Jaqueline Rodrigues Silva(a,b) , Danilo Trabuco Amaral(a,b) , Therese Wilson(c) , John Woodland Hastings(c) , Vadim Viviani(a,b) (a) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil (b) Graduate School of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar-Sorocaba), Sorocaba, São Paulo, Brazil (c) Department Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA The Oceanic fungus-gnats from Arachcnocampa spp (Diptera; Mycetophylidae) produces its own bluish bioluminescence (484 nm) by lanterns derived from Malpighian tubules. In these species, the bioluminescence is used to attract flying preys to threads of mucus and silk. Although some studies showed that its bioluminescence system involves a luciferin, a luciferase and ATP, the biochemistry and molecular biology of its bioluminescence remains largely unknown. Previously we constructed a cDNA library from the Australian A. flava lanterns. In order to better understand the molecular physiology and the origin of its bioluminescence, we analyzed the EST library. About 600 EST were analyzed. Among the gene products, we found proteins involved with nutrient reservoir (7%), oxidoreductases (6%), binding proteins (18%) such as ATP binding, RNA binding and ions binding, hydrolases (7%), transferases (6%) and transporters (5%). AMP-ligases were also abundant, around 1% of transcripts. The transcriptional profile shows some similarity with those of the Malpighian tubules of the dipteran Drosophila and with the coleopteran Zophobas morio (Tenebrionidae) mealworm, especially the presence of xenobiotic detoxification and excretion enzymes, like glutathione S-transferases, alcohol dehydrogenase and aldehyde dehydrogenase. The abundance of AMP-ligases in both the lanterns of Arachnocampa flava, the presence of luciferase-like enzymes in the Malpighian tubules of Zophobas mealworm, and the involvment of ATP in Arachnocampa bioluminescence, suggest a possible link between these enzymes, detoxification and the origin of bioluminescence in Arachnocampa spp. (Financial support; FAPESP and CNPq) P0067 Transcriptional survey of the Malpighian tubules of Zophobas morio (Coleoptera; Tenebrionidae) Jaqueline Rodrigues Silva(a,b) , Danilo Trabuco Amaral(a,b) , Rogilene Aparecida Prado(a,b) , Vadim Viviani(a,b) (a) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil (b) Graduate School of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar-Sorocaba), Sorocaba, São Paulo, Brazil The Malpighian tubules play a key role in osmorregulation in insects. Although a transcriptional analysis was done for the Malpighian tubules in Drosophila melanogaster (Diptera) far more extensive roles than ion and water transport were found. The Malpighian tubules also are involved in excretion of a broad range of organic solutes and xenobiotics. However, functional genomic analysis has not been carried yet for any Coleoptera. Recently, our group cloned a luciferase-like enzyme from the Malpighian tubules of Zophobas morio larvae, a close relative of Tribolium castaneum (Coleoptera; Teneobrinidae). This enzyme is an AMP / CoA-ligase which displays weak luminescence activity with firefly D-luciferin, a xenobiotic for this non-bioluminescent insect. Using the cDNA library previously constructed from Malpighian tubules of larval Zophobas morio, we have randomly isolated, partially sequenced and analyzed ca 540 clones, obtaining a first transcriptional profile of the most representative expressed genes, associating them to their possible biological functions. A high percentage of mitochondrial genes were found which is consistent with the high metabolic activity required by this organ during the formation of primary urine. Common transcripts included enzymes involved in osmoregulation such as solute transporters and ATPases, and in detoxification and excretion such as cytochrome P450, glutathione S-transferase, and short chain dehydrogenase (SDR). In comparison, the transcriptional analysis of Drosophila melanogaster the Z. morio Malpighian tubules also showed high expression of organic and inorganic solutes transporters, gene products related to xenobiotcs metabolism, several transcription factors, gene products involved in signaling pathways and many novel genes which could be related to other Malpighian tubules functions. The interesting presence of AMP-CoA ligases with luciferase/oxygenase activity toward the xenobiotic firefly D-luciferin, suggest their potential involvement in new important excretive/detoxificative functions in Malpighian tubules physiology via adenylation/thioesterification and detoxification via oxidation. (Financial support; FAPESP and CNPq). P0068 BRET-based homogeneous competitive immunoassay of progesterone Daria Smirnova, Jeanne Samsonova, Natalia Ugarova Lomonosov Moscow State University, Moscow, Russia Bioluminescence resonance energy transfer (BRET) is widely used in different approaches, most often for the investigation of protein-protein interactions. Homogeneous competitive immunoassay is one of perspective but insufficiently explored option of BRET application in analytical biochemistry and biotechnology [1]. The purpose of the work was to develop a BRET-based homogeneous immunoassay for a low molecular antigen. Here we propose a rapid and sensitive immunoassay of low molecular weight antigen, progesterone (P4), based on the BRET phenomenon occurred between a bioluminescent enzyme, luciferase (donor), and a fluorescent dye (acceptor) included into the structure of the specifically interacted pair "antigen-antibody". The thermostable mutant of Luciola mingrelica luciferase (Luc) with maximum of bioluminescence at 550 nm was used as a bioluminescence donor. The P4 derivative with free carboxyl group preliminary activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide was covalently coupled to the enzyme via Lys amino groups. Molar excess of P4 derivative to the enzyme ranged from 5 to 60. After 2 h incubation unbound P4 derivative was removed by gel filtration on G-25 micro spin-column. The retained bioluminescent activity of the enzyme in Luc-P4 conjugates depended on the ratio Luc;P4 in the reaction mixture. While P4 excess increased from 1;5 to 1;60 bioluminescent activity of Luc decreased from 60 to 10% of the original enzyme. To prevent Luc inactivation during synthesis the enzyme was first mixed with ATP and Mg(2+) in saturating concentrations and then covalent coupling reaction was performed between the protected Luc and the activated P4 derivative [2]. This approach lead to increase of retained Luc activity to 55, 70 ± 5, 86 ± 2, 85 ± 5, and 85 ± 5% for Luc;P4 ratio 1;60, 1;40, 1;20, 1;10 and 1;5, correspondingly. The formation of specific complexes between all Luc-P4 conjugates and anti-progesterone antibodies was confirmed in heterogeneous ELISA. Luc-P4 conjugates immobilized on the surface of microtiter plate were titrated against anti-progesterone antibodies. It was shown that Luc-P4 conjugates with Luc;P4 ratio 1;60 and 1;40 had a similar ability to form specific complex with antibodies. This ability slightly decreased for Luc-P4 conjugate with 1;20 ratio and for Luc-P4 conjugates with 1;10 and 1;5 ratio changes were profound. The fact indicated that maximum antigen incorporation of P4 to Luc is achieved at 20-40 molar excess of coupling antigen in the reaction mixture. In terms of bioluminescence activity and antibody-binding ability the Luc-P4 conjugate produced with the use of 20 molar excess of P4 was used as an optimal BRET-donor for further experiments. As a BRET-acceptor Alexa fluor 610-x dye (emission max at 630 nm) conjugated with anti-progesterone antibodies (Fl-Ab) was used. The dye has an optimal spectral overlap between the Luc bioluminescence spectra and the dye excitation spectra and higher quantum yield compared to conventional dyes as Cy3 or Cy3.5 [3]. Coupling reaction between antibodies and the dye was performed via free amino groups of antibodies. Excess of unbound dye was removed by gel filtration on G-25 micro spin-column. Dye incorporation ratio was evaluated spectrophotometrically at 609 nm. Fl-Ab conjugates with the dye incorporation ratio from 3 and higher were obtained and tested for the BRET measurement. Specific interaction between Luc-P4 and Fl-Ab lead to the BRET phenomenon, i.e. to the reduction of light emission at 550 nm and the increasing of light emission at 630 nm. The BRET-index was calculated as the ratio of the light emission at 630 nm to the light emission at 550 nm. Linear relationship was observed between BRET-index and the amount of dye bound to antibody. However dye incorporation ratio higher than 10 influenced the ability of fluoro labelled antibodies to bind P4 (Luc-P4). For further experiments the Fl-Ab conjugate with maximum incorporation ratio of dye (10) which retained antigen binding ability was used. If the BRET-acceptor (Fl-Ab) concentration was increased the BRET-index value increased too up to a saturation level. On the contrary, while the BRET-donor (Luc-P4) concentration was increased the BRET-index value decreased (Fig. 90). Addition of free P4 inhibited the specific binding between Luc-P4 and Fl-Ab which resulted in BRET-index drop (Fig. 91). For the P4 analysis an incubation of P4-Luc and Fl-Ab in the presence of different concentrations of free P4 was followed by addition of substrate solution, measurement of luminescence and the calculation of the BRET-index. To improve assay sensitivity a few parameters of the BRET-based immunoassay of P4 were optimized. We considered concentration and volume of P4-Luc, Fl-Ab and P4, temperature and time of incubation, and order of reagents addition. The best sensitivity of analysis was achieved at the lowest concentration of Luc-P4 which could be authentically detected by a luminometer (2 nM). The decrease of Fl-Ab concentration improved the detection limit of P4 assay but constricted the dynamic range of BRET-index. As an optimal the concentration of Fl-Ab equal to 50 nM was chosen to provide a dynamic range of BRET-index up to 80 % of the maximum binding at given concentration of Luc-P4 conjugate (2 nM). It was also found that the decreased temperature caused the BRET-index drop; while dynamic range of the BRET-index at 37°C and room temperature was the same it was restricted at 4 °C. The calibration curves of P4 assay obtained for incubation at 37°C and room temperature were identical, however, elevated background level was observed at 37°C. Incubation of reagents mixture for 15 min was sufficient to achieve equilibrium in binding and therefore to provide maximum BRET signal. An order of reagents addition did not have much influence on the assay sensitivity. The increased volume of P4 standard solution was used to improve the assay sensitivity. Different Luc-P4 conjugates were tested in BRET-based analysis of P4 under finally optimised conditions (Fig. 91). Reduction of Luc;P4 ratio from 1;20 to 1;5 lead to profound decrease of dynamic range of BRET-index and therefore to reduced accuracy of P4 detection. Among all tested conjugates the Luc-P4 conjugate with ratio 1;20 showed the best sensitivity along with the highest dynamic range of the BRET-index (Fig. 91, curve 1). The developed BRET-based homogeneous competitive immunoassay allows P4 determination within 0.3-100 ng/ml in short time (≤ 20 min) at room temperature. BRET-based immunoassay of a low molecular weight antigen could find a wide range of applications by substitution of the current pair of specific reagents by another one. References 1. Bacart J, Corbel C, Jockers R, Bach S, Couturier C, et al. The BRET technology and its application to screening assays. Biotechnol. J. 2008;3:311-4. 2. Squrrell DJ, Murthy J.. Luciferase labelling method. US Patent 5837465, 1998. 3. Yamakawa Y, Veda H, Kitayama A, Nagamune T. Rapid Homogeneous Immunoassay of Peptides Based on Bioluminescence Resonance Energy Transfer from Firefly Luciferase. J.Biosci. Bioeng. 2002;93:537-42. [Figure: see text] Experiment conditions; Luc-P4 conjugate (conjugate with Luc;P4 ratio 1;20) concentrations - 2 (1), 7 (2), 20 (3), 70 (4), 200 (5) nM, incubation - 30 min at 37°C. BRET- index was calculated as I630 /I550 for Luc-P4- I630 /I550 for Luc. [Figure: see text] Luc;P4 molar ratio taken for a conjugate synthesis; 1;20 (1), 1;10 (2), 1;5 (3), Luc with no P4 added (4).Assay conditions; Luc-P4 - 2 nM, Fl-Ab - 50 nM; an order of reagents addition and volume ratio - Luc-P4; free P4;Fl-Ab -1;10;1, incubation - 15 min at room temperature. P0069 Effect of pH on thermal stability of bioluminescent coupled enzyme system NAD(P)H;FMN-oxidoreductase-luciferase Maria Sumarokova, Irina Sukovataya, Valentina Kratasyuk Siberian Federal University, Krasnoyarsk, Russia Bioluminescent enzyme systems based on bacterial luciferases offer a unique and general tool for analysis of the many analytes and enzymes in the environment, research and clinical laboratories and other fields [1-3]. Development of physico-chemical basis of bioluminescence assay, extension of the scopes of bioluminescence assay, increase of enzymes activity and stability are great importance now. The use of enzymes of bioluminescent coupled enzyme system NAD(P)H;FMN-oxidoreductase-luciferase is complicated by their instability under different treatments; high temperatures, extreme pH values​​, etc. Usually, the study of the influence of various physical and chemical factors on the enzymes is performed less frequently investigated the influence of several complex factors. In earlier reports [4-5], we described the effects of organic solvents on catalytic activity of bacterial luciferases isolated from bacteria and possibility of thermostabilization and increase of activity of the coupled enzyme system NADH;FMN-oxidoreductase-luciferase in viscous aqueous-organic mixtures. In the present study, the effect of pH on the thermal stability of bioluminescent coupled enzyme system NAD(P)H;FMN-oxidoreductase-luciferase have been investigated in pH range 5-9. The stabilities of the bioluminescent coupled enzyme system towards temperature and pH were evaluated with the activities (maximum reaction rate - I0 ) determined at different temperatures using a five minutes incubation time. The thermal stabilities of the bioluminescent coupled enzyme system were evaluated with the activities determined at different temperatures (15, 20, 25, 30, 37°C). For thermal stability study was carried out a series of experiments to determine the residual enzymes activity depending on the time and inactivation rate constant in the relevant buffer. It was showed that the effects of temperature on the activity of the coupled enzyme system NADH;FMN-oxidoreductase-luciferase were pH dependent. The increasing pH of the reaction medium leads to decrease of the residual enzymes activity depending on the time for each pH value for each pH value. It was found that in all pH range studied enzyme inactivation follows the first order kinetics (Fig. 92) because the dependence in semi-logarithmic coordinates was straight. We have the same results for another temperature, such as 15, 20, 25, 30 and 37 ºC. Thus, the coupled enzyme system is inactivated in accordance with the first order reaction kinetics for each pH value. It was determined that thermoinactivation of coupled enzyme system has linear nature and occurs with nondissociative mechanism. When the activities of enzyme system were determined at different pHs with temperatures, which is higher than temperature optimum, such as 30 ºC and 35 ºC, the residual activities of coupled enzyme system was the same at pH 7,48 and 8. [Figure: see text] Inactivation rate constants for each pH value were calculated. It was shown that with increasing pH value lead to an increasing of inactivation rate constants. Growth of inactivation rate constants shows that thermal stability of coupled enzyme system decreases. These results can considered as a good feature for usage in practice and for solution of problem of the protein-solvent interaction as a general problem concerning the understanding of enzyme catalysis mechanisms. The work was financially supported by the Russian Academy of Sciences (Program "Molecular and Cell Biology", grant No 6.8) and by the state contract between Ministry of Education and Science and Siberian Federal University, № 1762. References 1. Kratasyuk VA, Esimbekova EN, Gladyshev MI, Khromichek EB, Kuznetsov AM, Ivanova EA. The use of bioluminescent biotests for study of natural and laboratory aquatic ecosystems. Chemosphere 2001;42;909-915. 2. Deryabin DG, Aleshina ES. Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors. Applied Biochemistry and Microbiology (Moscow) 2008;44;292-296. 3. Mariscal A, Peinado MT., Carnero-Varo M, Fernandez-Crehuet J. Influence of organic solvents on the sensitivity of a bioluminescence toxicity test with Vibrio harveyi. Chemosphere 2003;50;349-354. 4. Sukovataya IE, Tyulkova NA. Kinetic analysis of bacterial bioluminescence in water-organic media. Luminescence 2001;16;271-273. 5. Sutormin OS, Sukovataya IE, Kratasyuk VA. Thermal stability of coupled enzyme system NADH;FMN-oxidoreductase-luciferase in solvents of different viscosity. Luminescence 2012;27:162. P0070 Effect of structured microenvironments on stability of coupled enzyme system ADH;FMN-oxidoreductase-luciferase Oleg Sutormin(1) , Irina Sukovataya(a) , Valentina Kratasyuk(a,b) (a) Institute of modern biology and biotechnology of SibFU, Krasnoyarsk, Russia (b) Institute of Biophysics Siberian Branch of RAS, Krasnoyarsk, Russia Most of the enzymes, within the cell, aren't operated in conditions that correspond to the classical Michaelis- Menten equation, because the cell's conditions are a heterogeneous medium (hyaloplasm), but not a dilute solution, therefore enzymes catalyze in the heterogeneous medium. Kinetics of biochemical reactions in hyaloplasm are caused by a complex combination of factors, among which are important structural characteristics of the microenvironment of enzymes and substrates. From this point of view, much more important to investigate the functioning of enzymes in heterogeneous or structured environments not in the water to understand of the cell working as a whole system. The heterogeneous or structured environments could be organic solvents as glycerol or sucrose. The aim of this work was fund effect heterogeneous environments on coupled enzyme system NADH;FMN-oxidoreductase-luciferase. The influence of the viscosity of the reaction medium on the kinetic parameters of the coupled enzyme system of bioluminescence bacteria NAD(P)H;FMN-oxidoreductase-luciferase was investigated. It was showed that in an experimental model of coupled bioluminescent enzyme system with sucrose the reaction rate inhibited to a lesser extent compared with glycerol. The value of the total yield of luminescence and a constant decay of coupled bioluminescent enzyme system do not depend on the viscosity of the reaction media. The influence of the viscosity of the reaction medium on the thermal stability and thermal inactivation kinetics of the coupled enzyme system of bioluminescence bacteria NAD(P)H;FMN-oxidoreductase-luciferase was investigated. It was showed that the increasing viscosity of the reaction medium leads to increase of thermal stability of the coupled enzyme system. Sucrose stabilizes long-lived intermediate of bioluminescent reaction. The fluorescence spectra of coupled bioluminescent enzyme system (NAD(P)H;FMN- oxidoreductase and luciferase) in the different viscosity and temperature above the optimum was investigated. It was shown that the increasing in viscosity leads to concentration quenching of the fluorescence intensities. The largest contribution to the fluorescence spectrum includes the " internal" tryptophan residues. The fluorescence spectra obtained for the luciferase and NAD(P)H;FMN-oxidoreductase was showed lack of conformational changes in the structure of proteins in the viscous medium simulated various concentrations of glycerol and sucrose at the extreme temperature The research was supported by Project 1762 from The Ministry of Education and Science of the Russian Federation P0071 E-Tools for Bioluminescence Lev Sukovatyy, Irina Sukovataya, Danila Khudonogov Siberian Federal University, Krasnoyarsk, Russia Development of educational programs based on bioluminescence phenomenon implemented within the project " Bioluminescent Biotechnologies" at the laboratory established in the Institute of Fundamental Biology and Biotechnologies (IFB&BT) of Siberian Federal University under the supervision of the leading scientist Prof. O. Shimomura [1]. Development of practical skills in up-to-date scientific facilities, awareness of lab equipment and advanced methods for learning and research automated testing takes place on the basis of hardware and software complexes [2]. Program-simulators such as Adobe Captivate [3] help teach the user to operate on complex experimental devices, check readiness for the user to operate the contemporary experimental equipment. Adobe Captivate is an electronic learning tool which can be used to author software demonstrations, software simulations, branched scenarios, and randomized quizzes in. swf format. It can also convert Adobe Captivate generated. swf to. avi which can be uploaded to video hosting websites. Adobe Captivate allows to generate robust software simulations with multiple learning modes in a single recording session, including a demonstration of the procedure, a simulation for practicing the steps, and an assessment. Adobe Captivate software was used to create a program-simulator for the different type of devices, which used in practical training courses [4] on bioluminescence in academic programs for Bachelor and Master degree levels. These e-tools employment is especially urgent, because give possibility easily create show-me demos, interactive let-me-try application simulations, and test-me assessments for both educational and research path in up-to-date e-space too. The latter is especially urgent as the graduate's competency in the ability to implement experimental research based on unique computer-aided scientific equipment. Development of practical skills, scientific search engines and bio-physical investigation research planning with graduate students is one of topical tasks currently. Therefore, another substantial area for ICT applications is e-tools oriented at training and re-training research staff of a new type, including graduate students for Life Science. [Figure: see text] Active employment of e-Science technologies allows a researcher to fundamentally improve existing scientific experimental methodology, being facility-assisted and integrated with information and computer-aided measuring techniques. The work was financially supported by the state contract between Ministry of Education and Science and Siberian Federal University, № 1762, 2011-2013. Reference 1. Web-site of the laboratory " Bioluminescent Biotechnologies" - http;// 2. Foster I. Service-Oriented Science. Science 2005;308:814-817. 3. Web-site of Adobe Captivate - http;// 4. Frank LA, Eremeeva EV, Petushkov VN et al. editors. In; Shimomura O. The Nobel Laureate, I. I. Gitelson. Special biophysical training courses; biology, physics and chemistry of bioluminescence; textbook. Academician of RAS. Krasnoyarsk; Siberian Federal Univ., 2012. - 218 p. ISBN 978-5-7638-2728-6. P0072 Development of firefly luciferase mutants with low reactivity to dATP in pyrosequencing system Shigeya Suzuki(a) , Eriko Nasu(a) , Keiko Gomi(b) (a) Kikkoman Biochemifa Company, Noda, Chiba, Japan (b) Kikkoman Corporation, Noda, Chiba, Japan [Introduction] In prosequencing system, pyrophosphate produced in an extension reaction is converted to ATP by ATP sulfrylase and pyruvate orthophosphate dikinas e (PPDK). However, luciferase cannot completely discriminate between ATP and dATP used as substrates for extension of DNA strands. Therefore, in the current pyrosequencing systems, deoxyadenosine α-thiotriphosphate (dATPαS) is used instead of dATP. dATPαS has low activity in DNA extention. To develop the pyrosequencing system with high efficiency, improvements in substrate selectivity of lucifarase are important. [Materials and Methods] LUC-H (Kikkoman) is Luciola lateralis luciferase to convert the 217th amino acid to leucine (L) and 490th to lysine (K). 344A is luciferase to convert the 344th amino acid to alanine (A) in LUC-H. ATP/dATP ratio measurement reagents were prepared containing 1 microg/mL each luciferase. In ATP measurement, 0.1 mL of 1 x 10(-7) M ATP was added to 0.1 mL of ATP/dATP ratio measurement reagent. In dATP measurement, 0.1 mL of 1 x 10(-5) M dATP was added. The ATP production from pyrophosphate is catalyzed with PPDK using AMP and phosphoenolpyruvate (PEP) as the substrates, which are inactive for luciferase. Therefore, AMP-PPDK-based pyrosequencing system is suitable for highly sensitive DNA sequencing.(1)) DNA extension measurement reagent (including PPDK (Kikkoman), apyrase, D-luciferin, PEP, AMP) were prepared containing LUC-H or 344A. 2 microL of 5 x 10(-6) M template DNA (caagcttggc actggccgtc gttttacaac) and 2 microL of 5 x 10(-6) M primer DNA (gttgtaaaac gacgg) were added to 0.1 mL of DNA extension measurement reagent followed by the addition of 2 microL of 5,000 U/mL Exo-Klenow Fragment (Cloned) and 94 microL ultrapure water. 0.03 mL of 1 x 10(-5) M dCTP or dATP were added and luminescence was measured with the Lumat LB9507 (Berthold). The single-base DNA was synthesized by dCTP and not synthesized by other nucleotides. [Results and discussions] In measurements of reactivity to ATP and dATP, the ATP/dATP ratio for LUC-H was 160. In contract, the ATP/dATP ratio for 344A was 7,200. In DNA extension with LUC-H, luminescence of dATP was higher than that of dCTP (Fig. 94a). However, any luminescence of dATP was hardly observed in 344A (Fig. 94b). These results indicated that the dATP reactivity of luciferase was successfully improved. 344A is possible to hold the background luminescence attributable to dATP to low levels and to perform highly accurate nucleic acid analyses in pyrosequencing. [Figure: see text] References 1. Zhou G, Kajiyama T, Gotou M, Kishimoto A, Suzuki S, Kambara H. Enzyme system for improving the limit in pyrosequencing. Anal Chem. 2006 Jul 1;78(13);4482-9. P0073 Mechanistic understanding of the bacterial bioluminescence Chaitanya Tabib(a) , Thomas Bergner(a) , Silvia Lang(b) , Karl Gruber(b) , Ellen Zechner(b) , Peter Macheroux(a) (a) Institute of Biochemistry, Technical University, Graz, Austria (b) Institute of Molecular Bioscience, Karl-Franzens University,, Graz, Austria Bioluminescence is the production of light by living organisms using enzyme-catalyzed reactions as a key factor to release the energy. The yellow-green light emitted by fireflies is a well-known example. The bioluminescent reaction is catalyzed by "luciferase", an enzyme employing FMN as a redox cofactor to drive the mono-oxygenation of the aldehyde substrate to the acid product. The free energy released during the oxidation of the aldehyde gives rise to an excited state FMN-4a-hydroxide, which in-turn serves as the "luciferin" in bacterial bioluminescence (1, 5), as shown in the following reaction; The light emission in bacteria is catalyzed by a heterodimer consisting of a 40 kDa α-subunit and a 37 kDa β-subunit. The genes for bacterial light production are present as an operon, luxCDABEG; luxA and luxB encode the α and β subunits of luciferase; luxC, luxD, and luxE specify the enzymatic components of a fatty acid reductase complex necessary for synthesis and recycling of the aldehyde; and luxG encodes a flavin reductase (2). Many strains of Photobacteria also carry an extra gene, termed LuxF, having a lux operon gene order of luxCDABFEG. Sequence similarity to luxB suggests that luxF has arisen by gene duplication, however, its role in bacterial bioluminescence is obscure especially because only free-living but not symbiontic photobacteria appear to exhibit the luxF insertion in their lux-operon (3, 6). Its main function supposedly is to bind the myristylated FMN (myrFMN, a possible side product of the luciferase reaction), which is thought to bind sufficiently tight in the active site of luciferase thus leading to inhibition of the bioluminescence reaction (4). The generation of 6-(3'-(R)-myristyl)-FMN (myrFMN) in the genus Photobacterium is a largely unexplored phenomenon in bacterial bioluminescence. In the present study, we have developed a method for the isolation of myrFMN from Photobacterium leiognathi S1. Isolated and purified myrFMN was then used to determine its binding affinity to recombinantly produced apo-LuxF and luciferase. We could show by isothermal microcalorimetry that binding to apo-LuxF (Kd  = 80 nM) was fifty times tighter than to luciferase (Kd  = 4.0 μM). In addition, we exploited this tight binding of myrFMN to recombinant apo-LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria including strain in the genera Photobacterium, Vibrio and Aliivibrio. This analysis revealed that myrFMN is present in all photobacterial strains tested suggesting that myrFMN production is independent of the occurrence of luxF. Similarly, finding of trace amounts of myrFMN in Vibrio and Aliivibrio indicates that myrFMN generation is not restricted only to the genus Photobacterium. In order to determine the source of myrFMN we are currently in the process to knock-out certain genes in P. leiognathi to analyze the role of luciferase and LuxF in the generation of myrFMN. References; 1. Dunlap PV. Bioluminescence, microbial. In Schaechter M (ed.), Encyclopedia of Microbiology, 3rd edition. Oxford; Elsevier, 2009;45-61. 2. Ast JC, et al.. Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan. Journal of Bacteriology 2007;189:6148-6158. 3. Raibekas AA. Green flavoprotein from P. leiognathi; purification, characterization and identification as the product of the lux G(N) gene. Journal of bioluminescence and chemiluminescence 1991;6:169-176. 4. Moore SA, et al. Structural refinement of the non-fluorescent flavoprotein from Photobacterium leiognathi at 1.60 A resolution. J. Mol. Biol. 1995;249:195-214. 5. Kurfurst M.S. et al. Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction. Proc Natl Acad Sci USA 1984;81:2990-2994. 6. Lee CY, et al. The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli. Eur J Biochem 1991;201:161-167. P0074 Photoluminescence of Lanthanide Metal Complexes with dmethylpyridine-2,6-dicarboxylate (dmpc) ligand Ziyad Taha, Abdulaziz Ajlouni, Ahmed Hijazi Jordan University of Science and Technology, Irbid, Jordan A series of lanthanide metal complexes with the general formula [Ln(dmpc)(NO3 )2 )(H2 O)2 ]NO3 (Ln = Nd, Dy, Sm, Pr, Gd, Tb, La, Eu and Er) were prepared by direct reaction between the hydrated nitrate metal and dimethylpyridine-2,6-dicarboxylate (dmpc) ligand in a 1;1 molar ratio in ethylacetate-chloroform mixture. The luminescence properties of the ligand and its complexes in solid state and in methanol, DMF and DMSO solutions were investigated. The Tb, Eu, Sm and Dy complexes exhibit characteristic luminescence of Tb(III), Eu(III), Sm(III) and Dy(III) indicating efficient energy transfer from dmpc ligand to the Ln(III) ion. Scavenging activities of dmpc ligand and its Ln(III) complex on DPPH• radical were investigated in DMSO at a different concentration range. P0075 Effect of reductants on oscillatory chemiluminescence of fluorescein catalyzed by horseradish peroxidase Hirofumi Tani(a) , Ai Masuyama(b) , Akihiko Ishida(a) , Manabu Tokeshi(a) (a) Faculty of Engineering, Hokkaido University, Sapporo, Hokkai do, Japan (b) Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Hokkaido, Japan Oscillatory chemiluminescence (OscCL) is one of the most interesting chemical reactions related to physiological behaviour of the life. However, OscCL is relatively few reported [1] as well as its application to analytical chemistry since the reaction systems and conditions are very limited. Previously, we have found OscCL of fluorescein (FL) - H2O2 reaction catalysed by horseradish peroxidase (HRP) under relatively high H2 O2 concentration range [2]. The oxidation reaction in this OscCL would be affected by the presence of reductants, and thus resulting change in the oscillation pattern could be exploited as new parameters for analysis of the reductants. In this work, we studied the effects of various reductants on the OscCL response curve in terms of intensity, period, number of peak, and decay, and explored the possibility of the application to analytical chemistry. The OscCL reaction was simply performed by injecting H2 O2 into the mixture of fluorescein and HRP buffered by MES at pH 6.0. The reductants tested were ascorbate (Asc), glutathione (GSH), and 2-mercaptoethanol (ME). They were premixed with fluorescein/HRP. In the CL response curve obtained, peak height, peak spacing, and peak number were measured as CL intensity, period, and frequency, respectively. Firstly, we have studied the condition of oscillation. In terms of H2 O2 , OscCL was obtained at the concentration range from 25 to 320 mM (Fig. 95b), and at higher and lower concentrations of the range, CL with single peak obtained (Fig. 95a). In OscCL, the first peak was observed immediately after injection of H2 O2 , and as repeating CL emission the peak height and spacing were gradually decreased. After optimization of HRP, FL, and MES concentrations and pH from point of view of oscillation persistence, OscCL response curve with 15 well-defined peaks for 10 min was obtained. In the presence of Asc, the oscillation pattern remained unchanged but the appearance of the first peak was delayed. The delay time was dependent on Asc concentration. The delay should be due to scavenging radical species in the reaction by Asc. The presence of GSH has changed OscCL response, in which intensity, period, and frequency were all increased (Fig. 95c). In case of ME, frequency was increased while intensity almost unchanged. The use of ethylene glycol without reductive SH group did not show the change in the OscCL pattern. Thus, the substances with reducibility were found to alter the OscCL pattern. Additionally, the manner of changes was depending on the species and the concentration. This indicates the combined use of the parameters, delay, intensity, period, and frequency in the OscCL, could be applicable to the analysis of reductants. [Figure: see text] References 1. Iranifam M, Segundo MA, Santos JLM, Lima JLFC, Sorouraddin, MH. Oscillating chemiluminescence systems; state of the art. Luminescence 2010;25(6);409-418. 2. Segawa T, Suehara S, Kamidate T, Watanabe H. Oscillatory chemiluminescence during peroxidation of umbelliferone catalyzed by horseradish peroxidase. Bull. Chem. Soc. Jpn 1994;67(5);1301-1305. P0076 Use of bioluminescent enzyme system to detect antioxidant activity of fullerenol С60 Оy (ОН)х Anna Tarasova(a,b) , Nadezhda Kudryasheva(b) , Ekaterina Kovel(a) , Grigoriy Churilov(c) , Natalia Vnukova(c) , Victoria Isakova(c) , Irina Osipova(c) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia (c) Institute of Physics SB RAS, Krasnoyarsk, Russia Fullerenols are water-soluble polyhydroxylated fullerene derivatives. Physiological activity is an important property of fullerenols; their antioxidant ability is of specific interest for researchers. The study is aimed at revealing the antioxidant properties of fullerenol С60 Оy (ОН)х in solutions of model inorganic and organic oxidizers - potassium ferricyanide (complex salt of iron) and 1,4-benzoquinone. Toxicity of oxidizers' solutions was assessed by the bioluminescent assay based on a coupled enzyme system involving NADH;FMN-oxidoreductase from Vibrio fischeri and bacterial luciferase from Photobacterium leiognathi. Possibility to measure both oxidative and general toxicities is an obvious advantage of this bioassay. The oxidative toxicity (OxT) is attributed to redox activity of toxic compounds, while the general toxicity (GT) considers, in a nonadditive way, all interactions of exogenous compounds with components of the bioluminescent enzymatic assay system - redox reactions, hydrophobic and polar interactions. The basic peculiarity of the enzymatic assay is its specificity to oxidizers that can compete with FMN in reduction by NADH and, hence, inhibit the luminescent reaction. In this case, the delay period (T0.5 ) appears in bioluminescent kinetics [1]. The oxidizers were shown not only to decrease the bioluminescent intensity (I(max) ) [2], but increase the bioluminescence delay period (T0.5 ) [1-3], too. Fullerenes (С60 ) were synthesized by carbon helium high-frequency arc plasma at atmospheric pressure [4]. Fullerenol С60 Оy (ОН)х (х = 22-24, у = 2-4) was produced by fullerene С60 hydroxylation in concentrated nitric acid with the following hydrolysis of the polynitrofullerenes [5]. The structure of polyhydroxylated fullerenes was characterized by means of infrared and X-ray photoelectron spectroscopies [6]. Concentrations of fullerenol (< 2.5 · 10(-3) g/L) inhibiting the bioluminescence intensity by less than 10% were applied in bioluminescence measurements. To characterize the changes in general toxicity of the oxidizer solutions under the exposure to fullerenol (F), the detoxification coefficients, DGT , were calculated as; DGT=IFrel/Irel, where IFrel and I(rel) are relative bioluminescent intensities in the oxidizer solutions in the presence and absence of fullerenol, respectively. To characterize the changes in oxidative toxicity of the oxidizer solutions under the action of fullerenol, the bioluminescence delay periods in the presence and absence of fullerenol ((T0.5 )F and T0.5 , respectively) were compared; detoxification coefficients, DOxT , were calculated as; DOxT  = T0.5 /(T0.5 )F . The values of DGT , DOxT  > 1 showed the decrease of general and oxidative toxicities of the oxidizer solutions under the exposure to the С60 Оy (ОН)х , while DGT , DOxT  ≈ 1 - the absence of the fullerenol effect. Characteristics of fullerenol detoxification efficiency were revealed. The fullerenol decreased general toxicity (DGT  > 1) of the oxidizer solutions at concentrations < 10(-3) g/L. The decrease of oxidative toxicity (DOxT  > 1) was observed at concentrations lower than 2∙10(-5) g/L. The oxidative toxicity did not change (DOxT  ≈ 1) at fullerenol concentrations ranging from 10(-3) to 4∙10(-5) g/L. Detoxification seems to be a result of the formation the oxidizer-fullerenol complexes in water solutions followed by the redox neutralization of oxidizers by С60 Оy (ОН)х . References; 1. Vetrova EV, Kudryasheva NS, Kratasyuk VA. Photochem Photobiol Sci 2007;6:35-40. 2. Tarasova AS, Fedorova ES, Kudryasheva NS. Luminescence 2012;27:163-164. 3. Tarasova AS, Stom DI, Kudryasheva NS. Environ Toxicol Chem 2011;30:1013-1017. 4. Churilov GN, Kratschmer W, Osipova IV, Glushenko GA, Vnukova NG, Kolonenko AL, Dudnik AI. Carbon 2013;62:389-392. 5. Isakova VG, Goncharova EA, Bayukov OA, Churilov GN. J Appl Chem 2011;64:1093-1097. 6. Li J, Zhang M, Sun B, Xing G, Song Y, Guo H, Chang Y, Ge Y, Zhao Y. Carbon 2012;50:460-469. P0077 The importance and use of the ab initio and semiempirical computational methods for understanding the paths of chemiexcitation. A dioxetane thermolysis revisited Rostislav Vasil'ev, Yuri Tsaplev, Aleksei Trofimov Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia Chemiluminescence is the result of a sequence of elementary processes. A reactant, Rg, is activated on collisions with fast species from the tail of the Maxwellian distrib ution. If the vibrational energy is greater than the activation energy Ea , Rg is converted into a transition state, TS, which is half-way between Rg and a product, Pr, and may be close to the excited Pr (Pr*, in T1 or S1 electronic state). Thus, the chemiexcitation constitutes a "horizontal" radiationless TS→Pr* transition. The structure of TS, reaction coordinate that transfers the system to Pr*, the energy barriers or gaps for this transition, etc. are of peculiar importance for the chemiexcitation mechanism. All these data can be obtained by the computational methods of quantum chemistry, which is an extension of quantum mechanics into the realm of chemistry. The basis of quantum mechanics is the wave or Schrödinger equation (SE) postulated by an analogy with the classical Hamilton equation. The SE is not a simple algebraic expression, but a linear partial differential equation. Its solving should yield the energy E, a system of energy levels, and a very important wave function Ψ, which may be used for calculating various parameters and properties. However, the Schrödinger equation may be precisely solved only for hydrogen atom. For molecules the number of variables involved is astronomical and only approximate computations are possible. There exist two main groups of approaches to solving computationally the Schrödinger equation. A. Ab initio methods invoke a minimal number of approximations, using high-level mathematics. Hartree-Fock (HF) theory seeks the solution assuming that an electron is moving in a mean field of the other electrons. Full configuration interaction (CI) introduces the electron correlation by mixing an optimum amount of different electronic configurations. This approach allows, in principle, to calculate the excitation energies (i.e. the energy differences between the ground and excited states) provided that both states are equally balanced. Density functional theory (DFT) (1,2) describes a system of electrons via its density and not via its many-body wave function. It provides strategies at much lower costs than other techniques and improves upon the HF solution by expanding the wave function in a Taylor series. The exchange-correlation functional can include terms accounting for both exchange energy and the electron correlation that is omitted from HF theory. It is used for ground rather than for excited states. B. Semiempirical methods (AM1, PM3, PM6 and PM7) (3) solve the SE via parameterization against experimental data. Instead of calculating some integrals, the known thermochemical data are implemented. The average error (AE) between calculated (PM6) and experimental heats of formation for 4492 species is 8.0 kcal/mol. For 1373 compounds involving only H, C, N, O, F, P, S, Cl, Br, the PM6 AE is 4.4 kcal/mol. The equivalent AE for other methods were; RM1; 5.0, B3LYP 6-31G*; 5.2, HF 6-31G*; 7.4 kcal/mol. However, the AE is very high for simple diatomic molecules, such as N2 and O2 . An example; thermolysis of dioxetanes. The above-mentioned semiempirical methods were applied to a "classical" case of thermolysis of 1,2-dioxetanes (DO) (4,5). In this context, the enigmatic feature is high (up to 0.6) yield of the spin-forbidden chemiexcitation of triplet carbonyl products. For six alkyl and phenyl substituted DO, Ea (calc) was found coinciding with Ea (exptl) at the accuracy of 1.5 - 2.0 kcal/mol. The semiempirically modeled structure of DO is a plane square. TS is intermediate between DO and two carbonyl products. The O-O bond in TS is very weak but, nevertheless, not zero, as is evident from the calculated bond-lengths and bond-orders data. The TS remains to be plane, its structure may be called "biradicaloid" rather than biradicalic and, consequently, a concerted mechanism is more probable than a widely discussed stepwise path through a biradical. The potential energy surfaces of the S0 and T1 states in TS are close and, in compliance with the Hund rule, T1 is lower than S0 , which increases the rate constant kisc of the S0  → T1 intersystem crossing. Besides, there is a shallow well (2-4 kcal/mol deep, the so called "Eyring Lake") on the top of TS. The well prolongs the lifetime, τ. Thus, the yield of the T1 product chemiexcitation, Φ* = kisc τ, may be high even when a spin-forbidden S0  → T1 transition is slow. In summary, quantum chemical computations are of peculiar importance for chemistry. However, their results should always be taken with care. The SE has not been deduced "from the first principles" - they simply do not exist in quantum physics. Funding by the Russian Academy of Sciences is gratefully appreciated. References 1. Geerlings P, De Proft F, Langenaeker W. Conceptual Density Functional Theory. Chem Rev 2003;103:1793 - 1873. 2. Yue L, Roca-Sanjuán D, Lindh R, Ferré N, Liu YJ. Can the closed-shell DFT methods describe the thermolysis of 1,2-dioxetanone? J Chem Theory Computation 2012;8:4359-63. 3. Stewart JJP. Optimization of parameters for semiempirical methods VI; more modifications to the NDDO approximations and re-optimization of parameters. J Mol Model 2013;19:1-32. 4. Adam W, Trofimov AV. Contemporary trends in dioxetane chemistry. In; The Chemistry of Peroxides, Chapter 15 (Ed. Rappoport Z). V.2, Part2, Patai Series; The Chemistry of Functional Groups. Chichester; Wiley, 2006;1171-1209. 5. Vasil'ev RF. Changes of structure and energy on the route from dioxetane to carbonyl products. A quantum chemical study. J Biolumin Chemilumin 1998;13:69-74. P0078 Macrolampis Firefly Luciferase; A New Dual Reporter Gene for Simultaneous Ratiometric Intracellular Ph Sensing and Gene Expression Gabriele V. Gabriel, Vadim R. Viviani Laboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCAR), Sorocaba-SP, Brazil Firefly luciferases are widely used as bioluminescent reporter genes, for bioimaging and biosensors. For simultaneous analysis of different gene expression and cellular events, luciferases and GFPs emitting distinct bioluminescence colors have been coupled with each promoter making dual and multicolor reporter systems. Firefly luciferases bioluminescence spectrum is pH-sensitive, undergoing a typical large red shift at acidic pHs. Noteworthy, no one has ever considered to take advantage of the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH changes, an important indicator of cell homeostasis and physiology. Here we report for the first time the use of Macrolampis and other firefly luciferases for ratiometric intracellular pH-sensing. A relationship between the ratio of intensities of bioluminescence spectra at 550 nm and 610 nm and pH was obtained, and successfully used to estimate intracellular pH in live E.coli. The results show the potential of Macrolampis firefly luciferase as a novel reporter gene for simultaneous analysis of intracellular pH and gene expression or ATP analysis, providing the first dual reporter system employing a single luciferase gene. (Financial support; FAPESP 2011/23961-0 and CNPq) P0079 Chemiluminescent Determination of Hydrogen Peroxide using Fe(III) -TAML activator, a Potent Mimicking Enzyme Marina M. Vdovenko, Alexandra S. Demiyanova, Kirill E. Kopylov, Ivan Yu. Sakharov Lomonosov Moscow State University, Moscow, Russia Efforts to replace native peroxidase with its low molecular weight alternatives have stimulated a search for peroxidase mimetics. Herein we describe the oxidation of luminol with hydrogen peroxide catalyzed by commercial available Fe(III) -TAML activator 1a, which was showed to be more active catalyst than hemin. At Fe(III) -TAML activator 1a use in chemiluminescent assay for H2 O2 determination the limit value (3σ) was 5 x 10(-8) M, whereas in the presence of hemin the detection limit was significantly higher and equal to 6 x 10(-7) M. The linear ranges (R(2)  = 0.98) of the assay were 6 x 10(-8) - 1 x 10(-6) M and 6 x 10(-7) - 1 x 10(-6) M H2 O2 for Fe(III) -TAML 1a and hemin, respectively. The CV values for Fe(III) -TAML 1a-based assay measured within the working range varied from 1.0 to 3.7 % (n = 4), whereas in the case of hemin - 5.0 to 9.7 % (n = 4). Moreover, the sensitivity of Fe(III) -TAML 1a-based method was 56 times higher than that of hemin-based method. The obtained results open good perspectives to apply Fe(III) -TAML activator 1a in CL analytical methods instead of hemin, traditionally used peroxidase mimetic. The authors thank the Russian Foundation for Basic Research (NK-13-04-91164/13) for financial support. P0080 Chemiluminescent determination of reactive oxygen species using Pholasin luminophore in birds and insect phagocytes Libor Vojtek(a) , Pavel Dobes(a) , Lucie Prokopova(a) , Jitka Vinklerova(b) , Michal Vinkler(b) , Pavel Hyrsl(a) (a) Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic (b) Department of Zoology, Charles University in Prague, Vinicna 7, 12844 Praha 2, Czech Republic Phagocytosis is one of the most important innate immunity mechanisms which prevents organism against pathogens overcoming the natural barriers. There are several methods widely used for evaluation of phagocytosis efficiency. One of them is to measure the level of reactive oxygen species (ROS) produced by phagocytes - respiratory burst of phagocytes. ROS production can be elicited by addition of activators such as lipopolysaccharide (LPS), zymosan, starch particles, phorbol-12-miristate-13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanin (fMLP). Produced ROS cause oxidation of luminophore which subsequently emits the energy in form of light measured by luminometers. Luminol is the most widely used luminophore. On the other hand evaluation of respiratory burst using this method on birds or insect samples is not possible due to a low production of ROS and low sensitivity of luminol. In birds is production limited by absence of myeloperoxidase, enzyme responsible for the main production of ROS and hypochlorous acid. Purpose of this work was to find more sensitive luminophore and optimize chemiluminescent (CL) measurement of oxidative burst for birds' and insects' samples. As more sensitive (with app. thirty times higher luminescent signal than luminol) was found the luminophore Pholasin - photoprotein extracted from bioluminescent mollusc Pholas dactylus. For CL measurement of oxidative burst in birds we combined this luminophore with Salmonella enterica and Escherichia coli LPS; an ideal activators that give fast and stable enhancement of ROS production (Fig. 96). Data acquired by this assay can be subsequently compared to results of other experiments by evaluation of peak of the reaction (maximum intensity of respiratory burst in counts per second, CPS) or integral of the reaction. So far there are four studies of heterophil respiratory burst determination in avian immunology with the use of Pholasin and the first study on great tits (Parus major). As Pholasin proved its applicability for measurement of ROS level in avian blood, we tried to optimize this assay also for insect haemolymph, where the level of ROS is very low and luminol as luminophore is not sensitive enough. We found out that haemocyte suspension must be diluted 100x or 1000x to reduce antioxidant capacity of Galleria mellonella haemolymph. Unlike avian blood we did not detected any influence of PMA on ROS production and more likely is the reaction limited by the amount of Pholasin and peroxidases present. With the increased amount of Pholasin and presence of horse radish peroxidase (HRP) we observed faster response with significantly higher peak confirming presence of ROS in G. mellonella (Fig. 97). As Pholasin reacts mainly with hydrogen peroxide we suggest implementing its addition for buffering antioxidant activity of haemolymph and the use of HRP for conversion of other ROS than hydrogen peroxide that are also produced by haemocytes. Taken together we proved that Pholasin can be used as luminophore for detection of ROS produced by insects and avian blood cells. High antioxidant activity of insect haemolymph affecting the system will be completely inhibited once we find the proper balance between haemolymph dilution (diluting the antioxidant effect) and concentrations of Pholasin, hydrogen peroxide and HRP. Our research was supported by research grant NAZV-KUS 2012 (QJ1210047) and European Social Fund and state budget of Czech Republic (CZ.1.07/2.3.00/30.009). [Figure: see text] [Figure: see text] P0081 Emission Properties of Firefly Bioluminescence Studied by the Methods with Usual ATP Injection and Photolysis of Caged-ATP Yuki Yanagisawa(b) , Takeshi Kageyama(b) , Naohisa Wada(a) , Masatoshi Tanaka(b) , Shin-ya Ohno(b) (a) Toyo University, Itakura-Machi/Gunma, Japan (b) Yokohama Nat'l University, Hodogaya-Ku/Yokohama, Japan The time-resolved spectrum and the rise time course of firefly bioluminescence initiated by ATP injection into the HEPES buffer containing luciferin, luciferase(Luc) and Mg2+ were first measured with time resolution of 10 ms. In order to compare those with the time-resolved bioluminescence spectrum and the time course of its emission intensity triggered by photolysis of caged-ATP, we developed the new method of measuring the primary reaction; the exact time of ATP injection is monitored on the time course of emission intensity as a pulse produced by a ns-laser pulse coincident with the ATP injection. The shape of the time course of emission intensity is significantly dependent on pH; both time constans of rise and decay emission curves increase with lowering pH from 7.8 to 6.8. The observed time course of emission intensity is composed of two components; the formation process of luciferyl-adenylate dominates its shape of emission after reactant homogenization. In the case of the usual ATP injection, increment of [H+] might reduce the affinity of ATP binding at the catalytic center of Luc. Contrarily, in the case of the bioluminescence triggered by photolysis of caged-ATP, the diffusion-limited reaction dominates its shape of emission independent on pH [1]. The time-resolved spectra can be decomposed into two Gaussians peaked at 2.0 eV and 2.2 eV. The intensity of 2.2 eV component decreases with lowering pH, being consistent with previous report [2]. Spectral line shapes do not change from the initiation of the enzymatic reaction up to 200s for both methods of ATP injection. After 200s, the 2.0 eV component decays slower than the 2.2 eV component in the case of the photolysis of caged-ATP, which may be caused by the product of photolysis still remaining in the catalytic center of Luc. While above results are not influenced regardless of the concentration ratio [Ln]/[Luc], we could not examine the conclusion by Ando et al. [2] that the intensity of the band at 2.2 eV decreases with lowering pH contrary to that of the band at 2.0 eV almost independent of pH by measuring the absolute quantum yield of bioluminescence. References 1. Kageyama T, et al. Photochem. Photobiol. 2011;87:653-658. 2. Y Ando, et al. Nature Photonics 2008;2:44-47. P0082 Study on the possible ecological functions of fungal bioluminescence Hans Waldenmaier(a) , Adão Domingos(c) , Ana Martins(c) , Cassius Stevani(b) (a) University of Sao Paulo - Biochemistry, Sao Paulo, SP, Brazil (b) University of Sao Paulo - Chemistry, Sao Paulo, SP, Brazil, (3) Reserva Betary, Iporanga, SP, Brazil The last significant investigation of the ecological relationship between bioluminescent mushrooms and animals was performed over 30 years ago by Sivinski, who used bioluminescent mushrooms in sealed glass vials that were covered with an adhesive glue to capture arthropods that were attracted to the fungal lights 1. Sivinski proposed several nonexclusive hypotheses explaining the occurrence of bioluminescence in fungi at the interspecies level. Since Sivinki's work the issue has not been reexamined, although there has been a call for more experiments 2. In this work, we also performed glue-trap experiments, but our updated method utilize artificial acrylic mushrooms illuminated with green LED lights and controls without illumination. Moreover, we also performed direct ob servational studies of bioluminescent mushrooms with infrared video. Experiments were performed in the Brazilian Atlantic rainforest in Sao Paulo state (Mata Atlântica biome), and the transitional palm forests of Piauí (Mata dos Cocais biome). Experiments began in 2012 and are on going. As reported previously, we found greater number of arthropods on the luminescent glue-traps than controls. More specifically the orders Archaeognatha, Coleoptera, Collembola, Diptera, Hemiptera, Neuroptera, and Orthoptera were captured on the illuminated glue-traps in statistically greater numbers than controls (Fig. 98, pnn). There was no evidence of any group of arthropods being repelled by the luminescence. We also noticed the glue traps were only capable of capturing smaller arthropods, larger and stronger arthropods and potentially other larger animals were able to release themselves from the glue-traps and escaping detection, this issue plagued our glue-trap study. In order to gain more accurate observations of the ecology of bioluminescent fungi we have been using infrared video technology. Arthropods are able to perceive UV-blue-green light, and have corresponding light receptors 3. The attraction of arthropods to the IR light should not be an issue with the IR study, the longest wavelength that could be detected by any insect is ca. 630 nm 3. The IR video has provided us a much better picture of what is occurring at the actual mushroom. We could not only identify the animals around the mushrooms, but also observe what they were doing. One general trend we have observed in both biomes is that cockroaches feed on the mushrooms, at the same time arachnids are attracted to the light and feed upon the cockroaches. This is reminiscent of the "burglar alarm" effect observed in the ecology of dinoflagellate bioluminescence 4. 1. Sivinski JM. Psyche. 1981;88:383-390. 2. Bermudes D, Petersen RH, Nealson KH. Mycologia 1992;84(5);799-802. 3. Briscoe AD, Chittka L. Annu. Rev. Entomol. 2001;46:471-510. 4. Abrahams MV, Townsend LD. Ecology. 1993;74(1);258-260. [Figure: see text] [Figure: see text] P0083 Identification of a Novel Luciferase Fold Monika G. Wood, Keith V. Wood Promega Corporation, Madison, Wisconsin, USA Many evolutionarily unrelated luciferases share coelenterazine as their photon-emitting substrate. One of these, the heterotetrameric Oplophorus luciferase, was cloned in 2000(1) . The small 19 kDa subunit (Oluc-19) is sufficient for light production, and does not share significant sequence similarity with known luciferases or other proteins. We successfully employed a bioinformatics approach to search for distant homologs of Oluc-19. Using a fold recognition meta-server(2) we identified a structural motif shared between Oluc-19 and intracellular lipid binding proteins (iLBPs). iLBPs are a family of small cytoplasmic carrier proteins that bind long-chain fatty acids and other hydrophobic ligands. They fold into a 10-strand β-barrel that carries conserved residues in its cavity for binding charged groups in the ligand. We created a homology model of Oluc-19 based on several iLBP crystal structure templates. The 3D model allowed us to predict fold-specific stabilizing mutations that were subsequently confirmed experimentally. Further engineering and codon-optimization of Oluc-19 yielded a new ultra-bright glow-type luciferase, NanoLuc®, that preferentially uses a novel imidazopyrazinone substrate, furimazine(3) . Our recently solved structure by X-ray diffraction(4) confirms that the Oplophorus luciferase 19 kDa subunit adopts the predicted iLBP-related 10-strand β-barrel fold. The evident homology to a broadly abundant family of binding proteins suggests that this luciferase evolved from non-enzyme origins. The new 3D structure is also expected to contribute toward further insights into the enzymatic mechanisms for light generation. References; 1. Inouye S, Watanabe K, Nakamura H, Shimomura O.. Secretional luciferase of the luminous shrimp Oplophorus gracilirostris; cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett. 2000;481:19-25. 2. (16) Kurowski M. A., Bujnicki J. M. GeneSilico protein structure prediction meta-server. Nucleic Acids Res. 2003;31:3305-3307. 3. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11);1848-57. 4. This project was supported by grants from the National Center for Research Resources (5P20RR017708-10) and the National Institute of General Medical Sciences (8P20GM103420-10) from the National Institutes of Health. [Figure: see text] P0084 Dual-color bioluminescence imaging using green- and red-emitting beetle luciferases at subcellular resolution Mayu Yasunaga(a) , Yoshihiro Ohmiya(b) , Yoshihiro Nakajima(a) (a) Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa, Japan (b) Biomedical Research Institute, DAILAB, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Bioluminescent reporters have become an essential tool for studying various aspects of biological functions, including gene expression, post-transcriptional modification, and protein-protein interactions, because the sensitivity and range of the linear response are superior to those of other reporters, including β-galactosidase, chloramphenicol acetyltransferase, and fluorescent proteins. In particular, luciferases are used as sensitive reporters to monitor gene expression noninvasively, quantitatively, and longitudinally in living cells, explant tissues, and in vivo. Recent advances in luciferase technology have enabled the quantitative visualization of gene expression at single-cell resolution by imaging its luminescence in real time using a highly sensitive charged-coupled device (CCD) camera. Bioluminescence imaging is widely used to monitor cellular events, including gene expression, in vivo, in cellulo, and at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase ELuc and the red-emitting luciferase SLR are used as reporters localized to the peroxisome and the nucleus, respectively. We have captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus with high spatiotemporal resolution. Furthermore, the activation of nuclear factor-κB (NF-κB) triggered by tumor necrosis factor alpha (TNFα) has been accurately tracked in real-time by using this system where nuclear-targeted SLR and peroxisome-targeted ELuc are used as the test reporter and the internal control reporter, respectively. The results demonstrate that this imaging system can visualize the subcellular localization of reporters and track two gene expressions simultaneously at subcellular resolution. P0085 Determination of trace ferrum in power plant water using a reaction of luminol and dissolved oxygen Li Ying Xi'an Thermal Power Research Institute Co. Ltd., No. 136 Xingqing Road, Xi'an 710032, China In power plants, in order to maintain high reliability and security operation of the units and to realize their expected lifetime and economic operation, high water quality in all the water systems within a power plant have to be achieved [1]. It is essential to monitor the presence and movement of ionic impurities in the various water streams used in the power generation process, including the feed water, boiler water, steam, condensate polisher water and cooling water. The presence of corrosive ionic species, such as sodium, chloride, magnesium and ferrum in the water-steam cycle, even at microgram level, are closely related to deposits on turbine blades, steam generator tubing and other plant components, and the deposits not only impair heat transfer, they also can propagate stress corrosion cracking and other corrosion mechanisms in the all segments of the wate r-steam cycle, that can eventually lead to component failures and plant shutdowns resulting in millions of dollars in lost revenue. Such damage can be minimized by maintaining impurity levels as low as possible and by taking an active role in identifying sources of the impurities. The ionic impurities are accurate measured throughout the power generation process can provide valuable information regarding the source of contamination, the likely rates of contaminant build-up and probable rates of corrosion, as well as extremely useful and timely data during the start-up and shutdown of power plants. The measurement of trace ferrum provides valuable information for preventing corrosive conditions, unacceptable contamination levels, and other depositing conditions. Ferrum is recognized as the ion which indicates the corrosion potential of the water and gives insight into the proper production and maintenance of water within the water-steam cycle. The monitoring of ferrum in the process water is very important for the control of corrosion processes. As the acceptable limits of ionic impurities in the power industry become more stringent, more demand is placed on the measurement technique to ensure that the water quality is within acceptable operating parameters. In recent years, various chemical analysis techniques have distinct advantages in sensitivity, reproducibility, simplicity, cost effectiveness, flexibility and rapidity. A range of analytical techniques have been used for analysis of ferrum in various samples including spectrophotometry [2], atomic absorption spectrometry [3], voltammetry [4], and mass spectrometry [5]. Spectrophotometric methods occupy special position due to their simplicity, less expensive instrumentation and high sensitivity. A number of chromogenic reagents, such as o-phenylenediamine with hydrogen peroxide, p-anisidine with N, N-dimethylaniline, ammonium thiocyanate and salicylate have been reported for the determination of ferrum. Chemiluminescence analysis has received much attention in chemical analysis due to the high sensitivity, rapidity and simplicity of this method. The chemiluminescence method with different chemiluminescence systems has been exploited for the determination of ferrum [6]. It is well know that the reaction between luminol and dissolved oxygen could emit chemiluminescence, which has been applied to different fields. In this work, it was observed that ferrum (III) could enhance the chemiluminescence reaction between luminol and dissolved oxygen. On the basis of this a simple, sensitive and rapid procedure was developed for the indirect determination of ferrum (III). The increment of intensity was linear with ferrum (III) concentration over the range from 1.0 ng mL(-1) to 100.0 ng mL(-1) , R(2)  = 0.9988, with the relative standard deviations (RSD) less than 3.0%, and the detection limit was 0.3 ng mL(-1) (3σnoise ). At a flow rate of 2.0 mL min(-1) , a complete determination of ferrum (III), including sampling and washing, could be completed in 0.5 min, offering the sampling efficiency of 120 h(-1) accordingly. The proposed procedure was applied successfully to the determination of ferrum (III) in water samples from power plant. Acknowledgments This work was financially supported by Xi'an Thermal Power Research Institute, and the author gratefully acknowledges Xi'an Thermal Power Research Institute. References 1. Lu Z, Liu Y, Barreto V, Pohl C, Avdalovic N, Joyce R, Newton B. Determination of anions at trace levels in power plant water samples by ion chromatography with electrolytic eluent generation and suppression. Journal of Chromatography A 2002;956:129-138. 2. Mohammed AK, Alaa SA. Spectrophotometric determination of iron in environmental and food samples using solid phase extraction. Food Chemistry 2013;141:1941-1946. 3. Mohammad RP, Ali AS, Masoud A, Seyed RY, Majid H H, Mohammad R. Column solid phase extraction and flame atomic absorption spectrometric determination of manganese(II) and iron(III) ions in water, food and biological samples using 3-(1-methyl-1H-pyrrol-2-yl)-1H-pyrazole-5-carboxylic acid on synthesized graphene oxide. Materials Science and Engineering; C 2014;35:370-378. 4. Rodrigo S, María I T, Verónica A. Determination of iron in water samples by adsorptive stripping voltammetry with a bismuth film electrode in the presence of 1-(2-piridylazo)-2-naphthol. Talanta 2008;75:973-977. 5. Tim MC, Angela D R, Jess FA, Seth G J. A new method for precise determination of iron, zinc and cadmium stable isotope ratios in seawater by double-spike mass spectrometry. Analytica Chimica Acta 2013;793:44-52. 6. Simon JU, Angela M, William ML, Kakar A, Marie JMS, Toby H, Eric PA, Abdul N, Paul JW. Investigation of iron(III) reduction and trace metal interferences in the determination of dissolved iron in seawater using flow injection with luminol chemiluminescence detection. Analytica Chimica Acta. 2009;652:259-265. P0086 Origin of chemiluminescence accompanying the reaction of acridinium esters with hydrogen peroxide Beata Zadykowicz, Karol Krzyminski University of Gdansk, Faculty of Chemistry, Gdansk, Poland Acridinium derivatives are readily oxidized with hydrogen peroxide, persulfates, and other oxidants in alkaline media leading to chemiluminescence (CL). The light-emitting species are electronically excited 9-acridinones formed as a result of the decomposition of the addition products of OOH() to acridinium cations. This feature of acridinium salts has prompted their use as chemiluminescent indicators or chemiluminogenic fragments of labels in medical, biochemical, chemical, and environmental analyses. The mechanism of oxidation of N-methyl-9-(phenoxycarbonyl)acridinium cations with hydrogen peroxide in alkaline media has been discussed by several authors;[1-3] we examined it [4] at the density functional theory (DFT, B3LYP functional) level using gradient techniques and 6-31G** basis set. The solvent effect was included in the single-point DFT calculations utilizing the polarized continuum model (PCM). Computations have shown that the commonly suggested reaction pathway,[1,2] involving the formation of dioxethanone as intermediate and the elimination of CO2 , is not the most probable means by which light is generated. We found [3,4], that such processes need to overcome quite high activation barriers. The barriers predicted for the latter process, are respectively (Δa,298 H(o) , Δa,298 G(o) (gaseous phase), Δa,298 G(o) (aqueous phase)) equal to (in kcal/mol) 14.3, 14.9, and 15.9). The results of our studies [4] suggest that light-emitting molecules of 10-methyl-9-acridinone are formed as a result of the elimination of the phenyl carbonate anion, from the cyclic intermediate that is formed after the initial addition of OOH() to the 10-methyl-9-(phenoxycarbonyl)acridinium cation and the subsequent abstraction of a proton by OH() . The predicted barriers for the latter process are moderate (28 kcal/mol in aqueous phase for the various substituted at the benzene moiety 9-(phenoxycarbonyl)acridinium cations), which means that elimination of phenyl carbonate anions and the formation of electronically excited 10-methyl-9-acridinone molecules is achieved under the experimental conditions applied. The formation of "pseudobase" initiates nonchemiluminescent pathways of acridinium cations depletion. According to our predictions, the energy predicting reaction of 9-(phenoxycarbonyl)acridinium cations to 10-methyl-9-acridinone is too small to electronically excite the above product. The activation barrier for the latter process are higher than that generated light emission (Δa,298 H(o) , Δa,298 G(o) (gaseous phase), Δa,298 G(o) (aqueous phase)) equal to (in kcal/mol) 28.4, 30.2, and 25.2). Acknowledgements This study was financed from the State Funds for Scientific Research though NSC grant 2011/03/D/ST4/02419 (contract no. UMO-2011/03/D/ST4/02419). B.Z. acknowledges financial support from the European Social Funds within the project "The development program of the University of Gdansk in Europe areas 2020 (UG 2020)". The calculations were done on computers of the Wroclaw Centre for Networking and Supercomputing using the GAUSSIAN 09 program package. 1. McCapra F. Chemiluminescence of organic compounds. Pure Appl Chem 1970;24:611-29. 2. Weeks I, Beheshti I, McCapra F, Campbell AK, Woodhead JS. Acridinium esters as high-specific-activity labels in immunoassay. Clin. Chem. 1983;29:1474-9. 3. Rak J, Skurski P, Blazejowski J. Toward an understanding of the chemiluminescence accompanying the reaction of 9-carboxy-10-methylacridinium phenyl ester with hydrogen peroxide. J. Org. Chem. 1999;64:3002-8. 4. Krzyminski K, Ozog A, Malecha P, Roshal AD, Wroblewska A, Zadykowicz B, Blazejowski J. Chemiluminogenic features of 10-methyl-9-(phenoxycarbonyl) acridinium trifluoromethanesulfonates alkyl substituted at the benzene ring in aqueous media. J. Org. Chem. 2011;76:1072-85. P0087 Simultaneous quantification of catecholamines in rat brain by high-performance liquid chromatography with on-line gold nanoparticle-catalyzed luminol chemiluminescence detection Chunlei Mu, Qunlin Zhang School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, China Catecholamines (CAs), such as norepinephrine (NE), epinephrine (EP), and dopamine (DA), play critical roles in the function of brain and the signal transduction of nervous system as neurotransmitters. Because of the significance of CAs in the clinical diagnosis and medical treatment, a highly sensitive method for the determination of these compounds in either pharmaceutical preparations or biological fluids is especially attractive. However, the quantification of CAs in some biological samples encounters low physiological concentrations and easy oxidations of the catechol groups of CAs. High performance liquid chromatography (HPLC) with chemiluminescence (CL) detection has become more and more attractive for the determination of CAs at trace levels. Up to date, for HPLC assay of CAs, several CL reactions including luminol, peroxyoxalate, and 6-aminomethylphthalhydrazide (6-AMP), have been reported. However, in peroxyoxalate- and 6-AMP-based methods, specific derivatization agents were needed, and derivative pretreatment steps led to complicated operation process. In this paper, a selective and sensitive method based on HPLC coupling with on-line gold nanoparticle-catalyzed luminol CL detection was developed for the simultaneous quantitation of CAs in rat brain. In the present CL system, gold nanoparticles were produced by the on-line reaction of H2 O2 , NaHCO3 -Na2 CO3 (buffer solution of luminol), and HAuCl4. NE, EP, and DA could strongly enhance the CL signal of on-line gold nanoparticle-catalyzed luminol system. The UV-visible absorption spectra and transmission electron microscopy studies were carried out, and the CL enhancement mechanism was supposed. CAs promoted the on-line formation of more gold nanoparticles, which better catalyzed the luminol-H2 O2 CL reaction. The good separation of NE, EP, and DA was achieved with isocratic elution using a mixture of methanol and 0.2% aqueous phosphoric acid (5;95, v/v) within 8.5 min. Under the optimized conditions, the detection limits defined as a signal-to-noise ratio of 3, were in the range of 1.32-1.90 ng/mL, corresponding to 26.4 - 38.0 pg for 20 μL sample injection. The recoveries of CAs added to rat brain sample were more than 94.6%, with the precisions of less than 5.5%. The validated HPLC - CL method was successfully applied to determine NE and DA in rat brain, with the advantages of short-time analysis, lower cost, simpler sample pretreatment, and much less waste of organic solvents. The measured concentrations of NE and DA were 0.55 ± 0.03 and 0.69 ± 0.07 ng/mg tissue (rat brain, n = 3). These values agree with the reported data. The application potential of this new method to monitor and diagnose some CA-related diseases is under further investigation. Acknowledgements The support of this research by the National Science Foundation of China (Grant No. 30973674) and Science and Technological Fund of Anhui Province for Outstanding Youth (Grant No. 1308085JGD10) are gratefully acknowledged. References 1. Lu C, Li J, Yang Y, Lin JM. Talanta 2010;82:1576-80. 2. Veretinskaya AG, Vekshina NL, Stanishevskaya AV, et al. Neurochem. J. 2013;7:135-8. Copyright © 2014 John Wiley & Sons, Ltd.

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