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Luminescence. 2014 Aug;29(S1):55-106
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Abstract
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the involvement of a negative charge in the transition state, as also indicated by kinetic results from our group (unpublished work).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 nitr
ogen 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. Furthermore, 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 expe
rimental 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 potential 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;2012:391. 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 ×&thin
sp;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 molecul
ar 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 opsin 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
