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Luminescence. 2014 Aug;29(S1):6-55
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Abstract
O0001 On the efficiency of the peroxyoxalate system using a simple experimental and theoretical approach Felipe A. Augusto, Carolina P. Frias, Wilhelm J. Baader Instituto de Química, São Paulo, SP, Brazil The peroxyoxalate system is the most efficient intermolecular chemiluminescent reaction with chemiluminescence quantum yields reaching up to three orders of magnitude higher than other similar systems.(1) It consists in the reaction of an oxalic ester with hydrogen peroxide, typically catalyzed by a base, forming a high-energy intermediate which interacts with a compound called activator, ACT.(2,3) This interaction leads to the ACT in its singlet excited state in a mechanism that involves a charge/electron transfer initially from the ACT to the peroxide and then back to the ACT.(2,3) Several important details have been experimentally determined about the mechanism, like the cyclization rate constant and the direct interaction between the high-energy intermediate and the ACT.(4,5) However the identity of the high-energy intermediate is still a matter of discussion, with only a few possible intermediate structures being properly studied or definitely discarded.(6) The study and characterization of the peroxyoxalate reaction with a relatively small ACT, like naphthalene, could allow a detailed theoretical study of the chemiexcitation step, contrarily to the case of the compounds normally used as ACTs which would involve prohibitive computational costs. Therefore, the reaction of bis(2,4-dinitrophenyl) oxalate (DNPO) with hydrogen peroxide catalyzed by imidazole (IMI-H) was studied using naphthalene as ACT. The observed rate constant (kobs ) showed linear dependence with the [H2 O2 ] (Fig. , kH2O2 = 23 ± 1 L mol(-1) s(-1) ) and [IMI-H] (Fig. , kIMI-H = 202 ± 7 L mol(-1) s(-1) ) and no dependence with [DNPO] or [ACT] (data not shown). [Figure: see text] The study of the peroxyoxalate system, employing naphthalene as electronically simple ACT, indicates the existence of a linear relationship between the kobs and the H2 O2 as well as IMI-H concentration, in general agreement with the mechanism proposed for the initial steps of the transformation. This fact indicates a normal behavior of the system in the conditions utilized. Therefore, the peroxyoxalate reaction with naphthalene as ACT will now be subject to theoretical studies in order to elucidate the exact mechanism of the chemiexcitation step, with the intention to understand the reason for the extremely high efficiency of the system even so it involves, apparently, intermolecular electron transfer steps. References 1. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. 2. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;2012:391. 3. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APF, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader W. J. Quim. Nova 2011;34:544. 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. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. O0002 Kinetic studies on the sodium salicylate catalyzed peroxyoxalate reaction Glalci A. Souza, Wilhelm J. Baader Instituto de Química da Universidade de São Paulo, São Paulo-SP, Brazil The peroxyoxalate reaction is the only chemiluminescence reaction which appears to involve the intermolecular Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism in its chemiexcitation step that possess highest emission quantum yields of up to 60%.(1-3) Detailed kinetic studies on this highly efficient CL system has been performed mainly using imidazole as base catalyst and the mechanistic elucidation of the complete reaction has shown that this compound is acting also as nucleophilic catalyst.(4-5) However, it has been shown also that the base and nucleophilic catalyst imidazole leads to a decrease in the CL emission quantum yield, apparently due to its interaction with the high-energy intermediate.(4-6) In order to further elucidate the mechanism of the peroxyoxalate system, the kinetics of the reaction were studied with sodium salicylate as base catalyst. The CL emission obtained in the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) (0.1 mM) with hydrogen peroxide, catalyzed by sodium salicylate, in the presence of 9,10-diphenylanthracene (DPA) (0.2 mM) as activator, in ethyl acetate at 25 °C, was measured in different conditions. When the reaction was performed varying the sodium salicylate concentration, the rate constants corresponding to the emission decay increased with the base concentration, showing a saturation curve like behavior. The decay rate constant also increased with increasing hydrogen peroxide concentrations and at low H2 O2 concentrations the rate constants show a linear dependence on the hydrogen peroxide concentration allowing the determination of a bimolecular rate constant (kbim ). These rate constants showed to depend also on the sodium salicylate concentration; kbim = (0.56 ± 0.06) 10(-3) ; (1.6 ± 0.2) 10(-3) and (2.04 ± 0.06) 10(-3) L mol(-1) s(-1) , for sodium salicylate concentrations of 0.3, 1.0 and 5.0 mmol L(-1) , respectively. Upon variation at higher hydrogen peroxide concentrations the rate constants showed saturation curves, indicating a change in the rate-limiting step. The rate constants corresponding to the initial rise in the emission intensity have been shown independent of the hydrogen peroxide concentration showing that this reagent does not participate in the reaction step measured in this part of the kinetic curve. Similarly to the behavior observed with imidazole, the CL emission quantum yield showed to decrease with an increase in the sodium salicylate concentration. The maximum quantum yields obtained with sodium salicylate were s (max) = (1.24 ± 0.06) 10(-3) E mol(-1) when the sodium salicylate concentration was 0.5 mmol L(-1) . These data indicate that, like imidazole, also sodium salicylate appears to interaction with the high-energy intermediate in the peroxyoxalate reaction, which diminishes the CL emission quantum yields. Financial support; FAPESP, Capes, CNPq. 1. Stevani CV, Silva SM, Baader WJ. Eur. J. Org. Chem. 2000;4037. 2. Augusto FA, Souza GA, Souza Jr., SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013, 89, 1299. 3. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Bastos EL, Albrecht S, Brandl H, Zimmermann T, Baader, WJ. Arkivoc;2012 (iii), 391. 4. Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFLM, Stevani CV, Baader WJ, Luminescence 2002;17:313. 5. Stevani CV, Lima DF, Toscano VG, Baader WJ, J. Chem. Soc. Perk. Trans. 2 1996;989. 6. Ciscato LFML., Bartoloni FH, Bastos EL., Baader WJ, J. Org. Chem. 2009;74:8974. O0003 How can quantitative bioluminescence and in-situ fluorescence of firefly oxyluciferin in luciferase be compared with theoretical calculations? Hidefumi Akiyama(a) , Yu Wang(a,b) , Miyabi Hiyama(a) , Toshimitsu Mochizuki(a) , Kanako Terakado(c) , Toru Nakatsu(c) (a) University of Tokyo, Kashiwa, Chiba 2778581, Japan (b) Institute of Genetics and Developmental Biology, Beijing 100101, Japan (c) Kyoto University, Sakyo-ku, Kyoto 6068501, China To investigate color-determination mechanisms from physicist points of view, we study quantitative spectra of firefly bioluminescence, and the in-situ absorption and fluorescence spectra of oxyluciferin contained in luciferase in a consumed reaction mixture, and intend to compare them with quantum-chemistry theoretical calculations. We have so far measured quantitative in-vitro firefly bioluminescence spectra influenced by pH, kinds of bivalent metal ions, temperatures, and mutant luciferase using our total-photon-flux spectrometer with our new light standards. We found that all the spectra were systematically and quantitatively decomposed into one environment-sensitive and two environment -insensitive Gaussian peaks, and that no intensity conv
ersion between yellow-green and red emissions but mere intensity variation of the pH-sensitive green peak at 2.2 eV causes the changes in apparent emission colors [1,2]. Therefore, answers for the color-determination problem need not only the assignment of the peaks, but also the explanation of the intensity change of the green peak. [Figure: see text] We next measured in-situ absorption and fluorescence characteristics of oxyluciferin, still combined with luciferase in a consumed reaction mixture [3]. The in-situ absorption spectra indicated that neutral oxyluciferin was dominant, which was in weak pH-dependent equilibrium with oxyluciferin mono-anions. The neutral oxyluciferin was more dominant in luciferase environments than in bare water environments. The in-situ fluorescence spectra shown in Fig. clarified that the neutral oxyluciferin is a blue emitter and the oxyluciferin mono-anion is a green emitter (Fig. ). Even in red-mutant luciferase environment, the in-situ fluorescence has shown strong blue and green fluorescent emissions (Fig. ). In short, the spectra of in-situ fluorescence of oxyluciferin in luciferase and those of bioluminescence have shown significant discrepancy. [Figure: see text] Therefore, the above-mentioned discrepancy between bioluminescence and in-situ fluorescence cast an important question, how they can be consistently compared with quantum-chemistry theoretical calculations, which are recently published in a large numbers. The above results may suggest that enzyme microenvironment affects the transition state in bioluminescence chemical reaction, but not the oxyluciferin states after the reaction. Physicists need to discuss this issue with biologists and chemists in the bioluminescence research community. 1. Ando Y, Niwa K, Yamada N, Irie T, Enomoto T, Kubota H, Ohmiya Y, Akiyama H. Nature Photonics 2008;2:44-47. 2. Wang Y, Akiyama H, Terakado K, Nakatsu T. Scientific Reports 2013;3:2490. 3. Wang Y, Hayamizu Y, Akiyama H. J. Phys. Chem. B 2014;118:2070-2076. O0004 Determination of the total phenolic / antioxidant content in honey samples using formaldehyde / potassium permanganate chemiluminiscence system in a novel microfluidics device Butheina A. M. Al Haddabi, Haider A. J. Al Lawati, FakhrEldin O. Suliman, Gouri B. Varma Sultan Qaboos University, Al-Khod, Oman Microfluidc device has been explored as a tool for the estimation of the total phenolic content/ antioxidant content in honey using acidic potassium permanganate chemiluminescence (KMnO4 -CL) detection system. Selected phenolic antioxidants including quercetin, catechin, gallic acid, caffeic acid and ferulic acid elicited analytically useful CL with detection limits ranging between 2.38 nmol L(-1) for galic acid and 33.9 nmol L(-1) O-coumaric acid for only 2 μL injection volume. The parameters that affect the CL signal intensity of each antioxidant were carefully optimized. . It was observed that formaldehyde can enhance the CL signal intensity of phenolic compounds up to 27 times (Fig. ). Additionally, it was observed that the chip volume and geometry both can play an important role in enhancing the CL signal intensity in this system. The CL signal intensity was enhanced five times when a spiral - flow split chip (SF) geometry was used, compared to the simple spiral chip (S) geometry commonly used (Fig. ). Other parameters were also optimized, including pH and concentration of reagents used and the flow rates. The effect of solvents and surfactants on CL signal intensities was also studied. The method was applied on Omani honey samples. Nine different honey samples resulted in total phenolic / antioxidant level range between 40 and 772 mg Kg(-1) with respect to gallic acid. Folin Coicalteu reagent (FCR) resulted in a good correlation with the developed method which was found to be a selective, rapid and sensitive method to estimate total phenolic / antioxidant level in a good agreement with reported results for honey samples. [Figure: see text] [Figure: see text] References 1. Costin JW, Barnett NW, Lewis SW, McGillivery DJ. Analytica Chimica Acta 2003;499:47-56. 2. Alvarez-Suarez JM, Gonzalez-Paramas AM, Santos-Buelga C, Battino M. J. Agric. Food Chem. 2010;58:9817-9824. O0005 Electrochemiluminescence sensor based on tris(2,2'bipyridyl)ruthenium(II)/poly(AHNSA) for chlorpheniramine maleate analysis Mohammed M. Alhinaai, Emad A. Khudaish Sultan Qaboos University, AlKhude, Muscat, Oman Since the discovery of impressive luminance property of tris(2,2'bipyridyl)ruthenium(II), [Ru(bpy)3 ](2+) , it becomes one of intensive used reagent for chemiluminescence and electrochemiluminescence (ECL) analysis with a wide range of coreactants. Immobilizing of the expensive Ru(II)-complex on the electrode surface began early 1980s using Nafion as an exchanger polymer [1] is still a hot and continuous topic by many research groups [2]. The main objective of the present work was to develop a solid-state ECL sensor based on immobilizing [Ru(bpy)3 ](2+) on a conducting polymer for chlorpheniramine maleate (CPM) determination. The sensor was fabricated by composite electropolymerization of 2.0 mM [Ru(bpy)3 ](2+) and 1.5 mM of 4-amino-3-hydroxy-naphthalene sulfonic acid (AHNSA) in acidic medium via potentiodynamic repetitive cycles between -0.8 and +2.0 V at 0.1 V s(-1) . The fabrication parameters were optimized carefully in order to produce a stable film and obtain an intense ECL signal. Figure shows the electrochemical characterization of the composite surface film where a redox peak of [Ru(bpy)3 ](2/3+) are well defined at 1180 mV (anodic) and 980 mV (cathodic), respectively. Impedance spectroscopy analysis showed that the charge transfer resistance of the composite film is greatly lowered by doping Ru(II)-complex on the moiety of the PAHNSA which suggests that Ru(II)-complex is acting as a charge transfer center [3]. [Figure: see text] This sensor exhibited excellent ECL behaviour toward CPM analysis as shown in Fig. with a good stability and reproducibility. The standard deviation of 16 measurements in flow stream was 2.35%. It also has a very good lifetime when stored in 5 °C for two weeks where the recovery measurement approached 94%. The sensor was also applied to estimate CPM in pharmaceutical preparations. The linear dynamic range was from 0.1 to 32 µg/mL (R(2) = 0.9956). The detection limit was 23 µg/L and the recovery of real sample analysis was from 102.0% to 98.50% for syrup and tablets respectively. [Figure: see text] Interference studies showed no effect of common compounds and the acceptable molar concentration ratios of foreign species to CPM were higher than1000-fold for Na(+) , K(+) , NO3 (-) , SO3 (2-) , 100-fold for Mg(2+) , Al(3+) , NH4 (+) , Cl(-) , lactose, sucrose, and glucose, and 10-fold for Fe(3+) and Co(2+) . The analytical parameters such as pH, flow rate, buffer concentration were systematically tested. In conclusion, a novel composite polymeric film was fabricated using simple electrochemical method and applied for determination of CPM in real samples. The sensor showed a good stability and sensitivity regardless the matrix of the pharmaceutical preparation. References 1. Rubinstein I, Bard A. J. J. Am. Chem. SOC. 1980;102;6641-6842. 2. Su M, Wei W, Liu S. Analytica Chimica Acta 2011;704:16-32. 3. Zhang B, Shi S, Shi W, Sun Z, Kong X, Wei M, Duan X. Electrochimica Acta 2012;67:133-139. O0006 Experimental Evidence of the Occurrence of an Intermolecular Electron Transfer in the Catalyzed Decomposition of spiro-Alkyl-1,2-Dioxetanones Fernando Heering Bartoloni(2,1) , Marcelo Almeida de Oliveira(1) , Luiz Francisco Monteiro Leite Ciscato(2,1) , Felipe Alberto Augusto(1) , Erick Leite Bastos(1) , Wilhelm Josef Baader(1) (1) Departamento de Química Fundamental do Instituto de Química da Universidade de São Paulo, Sao Paulo. SP, Brazil, (2) Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brazil Several chemical and biochemical reactions have light as a co-product and some of them can show high quantum efficiencies, include firefly biolumines
cence, the peroxyoxalate system, and the induced decomposition of 1,2-dioxetanes.(1) Cyclic peroxides have been frequently described as high-energy intermediates in the chemical formation of products in the electronic excited state because their decomposition fulfill both energetic and geometric criteria required for chemiexcitation. Nevertheless, the thermal decomposition of 1,2-dioxetanes and 1,2-dioxetanones results in inefficient chemiluminescence emission due to the preferential formation of products in the non-emissive triplet-excited state (ΦS < 10(-4) E mol(-1) vs. ΦT > 0.1 E mol(-1) ). ).(2,3) However, it has been reported that fluorescent polycyclic aromatic hydrocarbons with low oxidation potentials (referred to as activators, ACT) catalyze the decomposition of 3,3-dimethyl-1,2-dioxetanone (1), resulting in noticeable increase in light emission intensity, and high singlet chemiexcitation quantum yields (ΦS = 0.1 E mol(-1) ).(4) Contrarily, our group found recently that the ΦS for the catalyzed decomposition of 1,2-dioxetanones, the model intermediate in firefly bioluminescence, were overestimated in several orders of magnitude.(5) Consequently, the validity of this system as a model for excited states formation might be questioned. Therefore, we report here our results of a kinetic study on the catalyzed decomposition of the spiro-substituted 1,2-dioxetanone derivatives, spiro-adamantyl-1,2-dioxetanone (2) and spiro-cyclopentyl-1,2-dioxetanone (3) by several activators and confirmed the occurrence of an intermolecular electron or charge transfer in this transformation. The 1,2-dioxetanone derivatives 2 and 3 were prepared, purified and handled as described elsewhere;(6) kinetic assays and data treatment were performed as detailed before.(5) The kobs values for the decomposition of 2 and 3, determined in toluene in the absence and in the presence of different ACTs, do not depend on the nature and concentration of the ACT (kobs (2, 50 °C) = (6 ± 1) 10(-3) s(-1) and kobs (3, 25 °C) = (9 ± 3) × 10(-4) s(-1) ). Therefore, the bimolecular rate constant (kCAT ) cannot be determined directly; however, the kCAT /kD ratios and the chemiexcitation quantum yields at the infinite ACT concentration (ΦS (∞) ) can be calculated for each ACT from the double reciprocal plots of the singlet quantum yields (ΦS ) versus the ACT concentrations.(5) The kCAT /kD values show linear free-energy relation with the ACT's oxidation potential, indicating the importance of an intermolecular electron transfer from the ACT to the peroxide in the chemiexcitation step. The low efficiency in excited states formation in the catalyzed decomposition of these cyclic peroxides are rationalized by steric interactions between the activator and the bulky alkyl substituents on the peroxidic ring, thereby lowering the charge-transfer complex formation constant between the peroxide and the activator. Financial support; FAPESP, Capes, CNPq. 1. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. 2. Adam W, Baader WJ. J. Am. Chem. Soc. 1985;107:416. 3. Adam W, Baader WJ. Angew. Chem. Int. Ed. Engl. 1984;23:166. 4. Schuster GB, Schmidt SP. Adv. Phys. Org. Chem. 1982;18:187. 5. de Oliveira MA, Bartoloni FH., Augusto FA, Ciscato LFML, Bastos EL, Baader WJ. J. Org. Chem. 2012;77;10537. 6. Bartoloni FH, de Oliveira MA, Augusto FA, Ciscato LFML, Bastos EL, Baader JW. J. Braz. Chem. Soc. 2012;23:2093. O0007 Chemiluminescent detection of Nitric Oxide Martina Bancirova Palacký University, Olomouc, Czech Republic The chemistry of nitric oxide inside humans and other mammals is perhaps the most interesting aspect of this simple molecule's behaviour. NO is involved in controlling blood pressure; transmitting nerve signals and a variety of other signalling processes. When tissues in the body become inflamed for long periods of time, the concentration of nitric oxide within them increases and this can be used to diagnose disease. But also NO secreted by activated cells appears to be a complex "cocktail" of substances (see Fig. .)(1) [Figure: see text] So is necessary to take in account the presence of ROS during the determination of nitric oxide. One of the chemiluminescent detections is based upon the chemiluminescence reaction between NO and the luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-H2 O2 system. The luminol-H2 O2 system is specifically reactive to NO, so that other nitrogen-containing compounds (organic nitrite, organic nitrate, and thio-nitroso compounds do not interfere. (2) The light emission of luminol as detection of bloodstains is a complex process based on hydrogen peroxide decomposition catalyzed by haemoglobin. Three common known methods of bloodstains; detection according to Grodsky, Weber and by Bluestar® Forensic reagent(3), are based on the luminol chemiluminescence (complex process based on hydrogen peroxide decomposition catalyzed by haemoglobin). The Bluestar® Forensic Magnum was chosen because of its declared stability as a "new" detection system for nitric oxide. The different dilutions (up to three orders) of the Bluestar® Forensic Magnum were used. The experiments were done by using luminometer (type FB 12, Berthold Detection Systems, Germany) in the total volume of 1 mL. Sodium azide was used as a specific quencher of singlet oxygen to prove its presence (see Fig. .) [Figure: see text] 1. Kroncke KD, Fehsel K, Kolb-Bachofen V. Nitric Oxide; Cytotoxicity versus Cytoprotection-How, Why, When, and Where?, NITRIC OXIDE; Biology and Chemistry 1997;1:107-120. 2. Kikuchi K, Nagano T, Hayakawa H, Hirata Y, Hirobe M. Detection of Nitric Oxide Production from a Perfused Organ by a Luminol-H202 System. Analytical Chemistry 1993;65:1794-1799. 3. Blum LJ, Esperança P, Rocquefelte S. A new high-performance reagent and procedure for latent bloodstain detection based on luminol chemiluminescence. Canadian Society of Forensic Science Journal. 2006;39:81-100. Financial support from the Czech Science Foundation, project 301/11/0767. O0008 An In-depth study on Blue electrochemiluminescent Iridium(III) complexes Gregory Barbante(a) , Egan Doeven(a) , Paul Francis(a) , Timothy Connell(c) , Paul Donnelly(c) , Conor Hogan(b) , David Wilson(b) (a) Deakin University, Geelong, Victoria, Australia (b) La Trobe University, Melbourne, Victoria, Australia (c) Melbourne University, Melbourne, Victoria, Australia Electrogenerated chemiluminescence (ECL) is a form of luminescence produced by high-energy reactions between electrogenerated precursors,([1,2]) in which the electronically excited states responsible for the emission of light can be generated through the annihilation between oxidised and reduced forms of the same species, or by using a sacrificial co-reactant. The application of a co-reactant ECL as a highly sensitive mode of detection has been predominantly based on the use of tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3 ](2+) ), and related polyimine-ruthenium(II) complexes, with characteristic orange/red emissions (max ca. 590-700 nm).([1,2]) Over the last decade, however, numerous researchers have begun to explore chemiluminescence and ECL reactions with cyclometalated iridium(III) complexes exhibiting a wide range of electrochemical properties and emission maxima that can be tuned through subtle changes in the structure of one or more ligands.([3]) These complexes have created new possibilities for multiplexed ECL detection systems.([4]) However, in contrast to the vast range of orange/red-emitting metal complex electrochemiluminophores,([5,6]) relatively few blue emitters are available, and the most effective design of blue-emitting complexes for ECL detection is yet to be fully elucidated. We have therefore used electrochemical, spectroscopic and computational techniques to explore a series of blue-emitting iridium(III) complexes (see Fig. ) that exhibit various potentially attractive structural attributes for conventional and multiplexed ECL detection. Theoretical and experimental studies reveal the most effective strategies for the design of
blue-shifted iridium(III) complexes for efficient electrogenerated chemiluminescence. Stabilisation of the HOMO while only moderately stabilising the LUMO increases the energy gap, thus ensuring favourable thermodynamics and kinetics for the reaction leading to the excited state. Of the iridium(III) complexes examined, [Ir(df-ppy)2 (ptb)](+) was most attractive as a blue-emitter for ECL detection, featuring a large hypsochromic shift (max = 454 and 484 nm), superior co-reactant ECL intensity than the archetypal homoleptic green and blue emitters; [Ir(ppy)3 ] and [Ir(df-ppy)3 ] (by over 16-fold and threefold, respectively), and greater solubility in polar solvents. We have therefore used electrochemical, spectroscopic and computational techniques to explore a series of blue-emitting iridium(III) complexes (see Fig. ) that exhibit various potentially attractive structural attributes for conventional and multiplexed ECL detection. Theoretical and experimental studies reveal the most effective strategies for the design of blue-shifted iridium(III) complexes for efficient electrogenerated chemiluminescence. Stabilisation of the HOMO while only moderately stabilising the LUMO increases the energy gap, thus ensuring favourable thermodynamics and kinetics for the reaction leading to the excited state. Of the iridium(III) complexes examined, [Ir(df-ppy)2 (ptb)](+) was most attractive as a blue-emitter for ECL detection, featuring a large hypsochromic shift (max = 454 and 484 nm), superior co-reactant ECL intensity than the archetypal homoleptic green and blue emitters; [Ir(ppy)3 ] and [Ir(df-ppy)3 ] (by over 16-fold and threefold, respectively), and greater solubility in polar solvents. [Figure: see text] 1. Bard AJ. Electrogenerated Chemiluminescence. Marcel Dekker; New York, 2004 2. Forster RJ, Keyes TE. Neuromethods 2013;80:347-367 3. Zanarini S, Felici M, Valenti G, Marcaccio M, Prodi L, Bonacchi S, Contreras-Carballada P, Williams RM, Feiters MC, Nolte RJM, De Cola L, Paolucci F. Chem. Eur. J. 2011;17:4640-4647 4. Doeven EH, Zammit EM, Barbante GJ, Hogan CF, Barnett NW, Francis PS. Angew. Chem. 2012;124:4430-4433 5. Barbante GJ, Hogan CF, Wilson DJD, Lewcenko NA, Pfeffer FM, Barnett NW, Francis PS. Analyst 2011;136:1329-1338 6. Gorman BA, Francis PS, Barnett NW. Analyst 2006;131:616-639 O0009 Chaetopterus variopedatus tissue autofluorescence spectral characteristics Anna Belousova(a) , Fyodor Kondrashov(b) , Maria Plyuscheva(b) (a) Moscow State University, Biological Faculty, Invertebrate Zoology Department, Moscow, Russia (b) Centre de Regulació Genòmica (CRG), Barcelona, Spain Chaetopterus variopedatus is a sedentary marine polychaete that lives in a parchment U-shaped tube. It has a body with three distinct regions, each region contains different morphologically developed segments. [3] The polychaete is famous for being capable of emitting light with its epithelium and producing blue luminous mucous. Studies on anatomy and morphology of phenomenon of its epithelial bioluminescence have pointed out some special areas of intensity, such as notopodial structures of middle and posterior regions. [2] Referring to the chemical approach, previous research on the chemical compounds of Chaetopterus bioluminescent system have proved that the photoprotein takes part in the reaction. [1] As for the whole biochemical pathway of the Chaetopterus bioluminescence - it is still a question to solve. Photoproteins that are involved in the luminescence reaction are known for becoming fluorescent after the reaction of luminescence takes place. [1] Therefore, the distribution of the products of the bioluminescence reaction can be studied using the confocal light microscopy methods. The autofluorescence itself is usually relatively stable either continious, or intensive. The distribution of fluorescence of different wavelengths was observed with a confocal microscope on several cross-sections of the worm's body and on epithelium of various parts of the body. The specimens of the worms tissue exhibit fluorescence of some ranges of wavelengths - excited by lasers from 405 to 633 nm. Confocal observations have shown that the UV excitation of a tissue results most efficiently in the strong fast-bleaching fluorescence in far-red (630 nm) spectrum area. Purified far-red autofluorescent component which is supposed to be a part of bioluminescence reaction [4] is stored in the vesicles which can be visualised with the confocal microscopy. Though having a strong intensity, the far-red fluorescence is bleaching very fast which makes it more challenging to observe. References 1. Shimomura O. Bioluminescence; chemical principles and methods. - World Scientific, 2012. 2. Anctil M. The epithelial luminescent system of Chaetopterus variopedatus //Canadian Journal of Zoology. - Т. 57. - №. 6. - С, 1979;1290-1310. 3. Harvey EN. Bioluminescence. - Academic Press, 1952. 4. Branchini BR, et al. Chemical analysis of the luminous slime secreted by the marine worm Chaetopterus (Annelida, Polychaeta) //Photochemistry and photobiology. - Т. 90. - №.1. - С, 2014;247-251. [Figure: see text] [Figure: see text] O0010 Construction of lux operon of the ancestor of bioluminescent bacteria and proposal of evolutionary hypothetical theories on the origin and propagation of luminescent bacterial species Ramesh CH, Mohanraju R Pondicherry University, Pondicherry, India Using the processes, "Horizontal gene transfer (HGT) or Chromosomal exchange mechanism (CEM) or Acquisition, Plasmid DNA exchange, genetical processes like Interchromosomal rearrangements (ICR), and geological processes" we assembled lux genes together and constructed the luminescent bacterial ancestor that might have not yet been isolated or extincted in the biological evolution during the geological processes. The construction of lux operon of this luminescent ancestor was carried out with different lux genes such as the regulatory and structural genes, and with other genes which are flanking towards upstream and downstream of the lux operon of different luminescent bacterial species. The lux operon of this ancestor was constructed based on the approximate base pairs of different lux genes. The approximate order of lux operon is as follows luxZYLOPUMNQRSTICDABFEGH-ribEBHA. Where rib genes of rib operon are linked to downstream of the lux operon. Hypothetically it is possible to construct this luminescent bacteria ancestor, while we expect possibilities of finding of this luminescent ancestor. The processes "Insertion, Deletion, Horizontal gene transfer (HGT) or Chromosomal exchange mechanism (CEM) and Interchromosomal rearrangements (ICR), geological processes and Plasmid DNA exchange (PDE) might have given origin for modern luminescent bacteria. These processes provides base for this ancestor construction and supports for the presence or possibility in constructing the ancestor of luminescent bacteria. We also propose new strong hypothetical theories which speak about the existence of luminescent ancestor and origin of modern luminescent bacterial species. O0011 Ecological functions of shark luminescence Julien Claes, Jérôme Mallefet Laboratoire de Biologie Marine, Earth and Life Institute, Université catholique de Louvain, Louvain-la-Neuve, Belgium Introduction; Sharks from the Etmopteridae and Dalatiidae families are among the most enigmatic bioluminescent organisms. Although they encompass about 12% of current shark diversity, with over 50 described species, their luminescence is rarely observed [1]. Moreover, contrary to the situation encountered in other animals, their intrinsic light organs (photophores) are primarily controlled by hormones rather than by nerves [2,3] and form a diversity of patterns whose adaptive advantage has long remained obscure. This work aims to synthetize recent advances made in the field of shark luminescence ecology as well as to present novel experimental data in order to inspire future research. It involves various techniques such as in vivo luminescence measurements [via an optic fibre coupled to a luminometer (Berthold FB12)], spectrophotometry [with a mi
ni-spectrometer (Hamamatsu Photonics C10083CA)], stereology and visual modelling. Results and discussion; In the last five years, we observed and characterized the spontaneous luminescence of one dalatiid and three etmopterid shark species. At 0.5 prepelvic length, their luminescence [downward emission; λmax at 457-488 nm; ventral intensity = 0.34-130.78 Mq s(-1) mm(-2) (n = 31)] appears physically similar to the residual downwelling light present in their environment, supporting a function of camouflage by counterillumination [1,4]. However, digital photography revealed that etmopterid luminescence (i) was not homogeneous on the ventral side (Fig. A), a result confirmed by stereological analysis of photophore distribution; and (ii) was also present dorsally, as dim glows underlying several structures including fin spines, eyes and nostrils (Fig. B). This leads to a variable angular distribution pattern along the etmopterid body, as it can be seen in E. spinax, whose luminescence adopts caudally (at the level of clade-specific lateral photophore markings) a more lateral distribution well suited for intraspecific communication (Fig. ). Visual modelling demonstrated that spine-associated glow signal the presence of the defensive fin spines to predators at several meters and hence might be used for aposematism [5]. Finally, the association of photophores with photoreceptive tissues likely provides a reference for counterillumination while nostril luminescence might be used as a torch to improve prey detection. We suggest the luminescence versatility of etmopterid sharks to have powered their rapid radiation in the deep-sea. Acknowledgments Julien M. Claes and Jérôme Mallefet are respectively postdoctoral researcher and research fellow of the Fonds National de la Recherche Scientifique (FNRS, Belgium). This is a contribution to the Biodiversity Research Center (BDIV) and to the Centre Interuniversitaire de Biologie Marine (CIBIM). References 1. Claes JM, Nilsson DE, Straube N, Collin SP, Mallefet JM. Sci. Rep. 2014;4:4328 2. Claes JM, Mallefet J. J. Exp. Biol. 2009;212:3684-3692. 3. Claes JM, Ho HC, Mallefet J. J. Exp. Biol. 2011;215:1691-1699. 4. Claes JM, Aksnes DL, Mallefet J. J. Exp. Mar. Biol. Ecol. 2010;388:28-32. 5. Claes JM, Dean MN, Nilsson DE, Hart NS, Mallefet J. Sci. Rep. 2013;3:1308. [Figure: see text] [Figure: see text] O0012 Prolonging Light Emission in Enhanced Chemiluminescence (ECL) Leopoldo Della Ciana(a) , Michele Zucchelli(a) , Luca Covello(a) , Dario Foglietta(a) , Ivan Yu Sakharov(b) (a) Cyanagen, Bologna, Italy (b) Department of Chemistry of Lomonosov Moscow State University, Moscow, Russia The chemiluminescent oxidation of luminol catalyzed by peroxidase finds wide application in the detection and quantitation of antigens, haptens, and nucleic acids and, in particular blotting tests, i.e., Western (proteins), Southern (DNA), Northern (RNA) blots, and ELISA. Because peroxidases are poor catalysts in luminol oxidation, certain compounds known as enhancers are added to the substrate mixture to increase chemiluminescent (CL) intensity. Although a number of compounds were successfully used in the enhancement of peroxidase-induced CL [1,2], currently the most effective enhancer is 3-(10-phenothiazinyl)propane-1-sulfonate (SPTZ) [3]. This compound can increase CL induced by horseradish peroxidase (HRP) and soybean peroxidase (SbP), by an order of magnitude when compared to previously known enhancers, such as p-iodophenol, p-coumaric acid or p-iodophenylboronic acid [3]. Furthermore, it was shown that the introduction of some 4-dialkylaminopyridines such as 4-morpholinopyridine (MORP) a reaction mixture containing luminol, hydrogen peroxide, and SPTZ resulted in a further 10-fold increase of CL intensity. Because 4-dialkylaminopyridines enhanced CL only in the presence of a primary enhancer (SPTZ) and did not act as enhancers in the absence of SPTZ, these compounds were named " secondary enhancers" . A recent study [4] suggests that " for an implementation of its enhancing ability, 4-dialkylaminopyridines should get bound to a protein fragment of peroxidase located near the entrance in the canal of the active site, where adsorption of peroxidase substrates commonly occurs The existence of such a complex near the active site may help in binding SPTZ to the peroxidase due to the formation of some charge transfer and ionic bonds between 4-dialkylaminopyridines and SPTZ and, consequently, may improve the efficiency of the enzymatic oxidation of SPTZ with the formation of SPTZ(•+) , that, reacting with luminol, results in the increase of CL intensity" . No significant effect was observed when secondary enhancers were included in the formulation containing primary enhancers other than SPTZ. Apart from chemiluminescent signal intensity, another important feature of ECL systems is light output duration. A prolonged light emission is highly desirable, especially in the blotting techniques, where esposure parameters may need adjustement, without the need to repeat the experiment. In addition, to increase detection it may be useful to prolong exposures to many hours. Again, when considering formulations with only primary enhancers, SPTZ substrates are by far the best in terms of prolonged light emission. The addition of secondary enhancers such as MORP causes a gradual decrease in light output duration, reaching its minimum at the highest initial signal level. Thus, the aim of this study is the search for additives and/or reaction conditions which could prolong light output in these systems. In particular, we have extended our screening to chelators, free-radical scavengers, electron/energy transfer mediators. As a result, we discovered some formulations with significantly improved light duration. These favorable properties were also observed in model dot-blot assays and ELISA. 1. Thorpe GHG, Kricka LJ, Moseley SR, Whitehead TP. Clin. Chem. 1985;31:1335. 2. Kricka LJ, Cooper M, Ji X. Anal. Biochem. 1996;240:119-125. 3. Marzocchi E, Grilli S, Della Ciana L, Prodi L, Mirasoli M, Roda A. Anal. Biochem. 2008;377:189. 4. Yu I, Sakharov MM. Vdovenko Analytical Biochemistry 2013;434:12-14. O0013 Marine luciferases; are they really taxon-specific? A putative luciferase evolved by co-option in an echinoderm lineage Jérôme Delroisse(a) , Patrick Flammang(a) , Jérôme Mallefet(b) (a) Biology of Marine Organisms and Biomimetics, University of Mons, Mons, Belgium (b) Laboratory of Marine Biology, Catholic University of Louvain, Louvain-La-Neuve, Belgium The bioluminescence reaction can be generalized as the oxidation of a luciferin substrate catalysed by a luciferase enzyme [1]. Although some luciferins are shared by phylogenetically distant organisms, it is commonly admitted that luciferases are clade-specific [2]. The European brittle star Amphiura filiformis emits a blue light using a coelenterazine-luciferase system [3, 4]. However, brittle star luciferases (and echinoderm luciferases in general) have not been characterized so far. Using genomic and transcriptomic data, we highlighted the presence of several putative coelenterazine-specific luciferase sequences in A. filiformis. Sequence comparisons revealed that these enzymes are similar to the luciferase of the luminous sea pansy Renilla sp (up to 47% of identical amino acids, up to 69% of general similarity) despite the large phylogenetic distance between these two species. Luciferase-like genes are also predicted in the purple sea urchin genome and surprisingly, mRNAs were also specifically identified in different transcriptomes from non-luminous echinoderms. Luciferase-like protein expression in non-luminous organisms raises the question of whether luciferin could be the limitative parameter of the bioluminescence reaction. A physiological approach, performed on tube feet of the common sea star (organs expressing luciferase-like mRNA) demonstrated that coelenterazine supplementation did not induce light emission in crude extracts from tube feet. Therefore, this luciferase-like enzyme must have a different function in sea stars, as it was previously suggested fo
r the purple sea urchin [5]. Assuming Renilla luciferase derived from haloalkane dehalogenases [6], we can hypothesize that haloalkane dehalogenases were presumably independently co-opted in luciferases in both Renilla sp and A. filiformis. Using anti-Renilla luciferase antibody, immunodetections were performed on the arm of A. filiformis. Specific immunolabeling was observed in the stroma of the spines, organs that we previously described as the unique photogenic areas (Fig. ). Our results confirm the probable implication of an enzyme similar to Renilla luciferase in the bioluminescence of the brittle star A. filiformis. Two luminous systems using the same luciferin and homologous luciferases seem to have emerged in a convergent manner in two phylogenetically distant species. The similar way of life of these benthic suspension-feeding species could constitute a strong selective pressure for the emergence of bioluminescence. [Figure: see text] Acknowledgements Jérôme Delroisse, Patrick Flammang and Jérôme Mallefet are respectively research fellow, research director and research associate of F.R.S.-FNRS (Fonds de la Recherche Scientifique). Thanks to Olga Ortega-Martinez, Sam Dupont and Magnus Rosenblad (University of Gothenburg) for the access to Amphiura genomic database. Contribution to the "Centre interuniversitaire de la Biologie Marine". Work supported in part by a FRFC Grant n° 2.4590.11. References 1. Henry JP, Michelson AM. Bioluminescence. Photochemistry and Photobiology 1978;27(6);855-858. 2. Haddock SH, Moline MA, Case JF. Bioluminescence in the sea. Marine Science 2010;2. 3. Shimomura O. Bioluminescence; chemical principles and methods. World Scientific Publishing Company, 2012. 4. Mallefet J, Parmentier B, Mulliez X, Shimomura O, Morsomme P. Characterisation of Amphiura filiformis luciferase (Ophiuroidea, Echinodermata). In Echinoderms in a Changing World - Johnson (ed) CRC Press The Netherlands. 293, 2013. 5. Fortova A, Sebestova E, Stepankova V, Koudelakova T, Palkova L, Damborsky J, Chaloupkova R. DspA from Strongylocentrotus purpuratus; The first biochemically characterized haloalkane dehalogenase of non-microbial origin. Biochimie 2013;95(11);2091-2096. 6. Loening AM, Fenn TD, Wu AM, Gambhir SS. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Engineering Design and Selection 2006;19(9);391-400. O0014 Ultrasensitive bioanalytical application using silica nanoparticles doped with new thermochemiluminescent 1,2-dioxetane derivatives Massimo Di Fusco(a) , Massimo Guardigli(b) , Mara Mirasoli(b) , Arianna Quintavalla(b) , Marco Lombardo(b) , Claudio Trombini(b) , Aldo Roda(b) (a) CIRI-MAM, Alma Mater Studiorum, University of Bologna, Bologna, Italy (b) Department of Chemistry "G. Ciamician", Alma Mater Studiorum, University of Bologna, Bologna, Italy Thermochemiluminescence (TCL), i.e., the light emission originating from the thermolysis of a suitable molecule, was proposed in the late '80s as a detection technique for immunoassays [1]. Being TCL emission simply triggered by heat, this technique would allow for reagentless luminescence-based detection, thus simplifying the microfluidic network in miniaturized analytical devices and biosensors. However, TCL detection was abandoned due to methodological problems, such as the high operating temperature (200-250 °C) and the poorer detectability in comparison with other labels. Despite these advantages, TCL detection remains very attractive because it potentially offers the same advantages of other chemiluminescent techniques. Recently, we tried to overcome the problems related to the reported TCL studies and in particular we described the synthesis of a library of TCL acridine-based 1,2-dioxetane derivatives (1-11 in Fig. ) proposed as new TCL labels [2,3]. Suitable structural modifications were introduced to decrease the emission triggering temperature down to 80-100 °C and to produce highly efficient fluorophores in the singlet excited state. [Figure: see text] In the first stage of the work we evaluated the photophysical properties of the acridanone derivatives and the TCL properties of 1,2-dioxetane derivatives using an ITO-coated glass slide as heating element placed directly in contact with a thermoelectrically cooled CCD sensor through a fiber optic taper. Such a lensless contact imaging configuration combined adequate spatial resolution and high light collection efficiency within a small size portable device. We showed that the 10-ethylacetate-9-acridanone derivative moieties produced in the singlet excited state were the main responsible for luminescence emission with fluorescence quantum yields (ϕF ) in the range 0.1-0.5. In addition, with the more efficient 1,2-dioxetane derivative 10 we obtained a limit of detection 17 times lower than 1. Herein, we described the encapsulation of these 1,2-dioxetane derivatives in silica nanoparticles (SiNPs), both alone or together with fluorescent energy acceptors, to obtain amplification of the TCL signal and their superficial modification with biotin for biosensing applications. The amino-functionalized SiNPs loaded with TCL compounds and fluorescent energy acceptor dipyridamole (DP) or 9,10-bis(phenylethynyl)anthracene (BPEA) thanks to the signal amplification due to the large number of 1,2-dioxetane molecules (about 104) in each SiNP and the increased emission efficiency due to the energy transfer to the fluorescent acceptor, could be revealed by TCL imaging with a detectability close to that of the CL enzyme label horseradish peroxidase [2]. In conclusion, the new TCL compounds showed emission triggering temperatures much lower (i.e., < 100 °C) than the compounds used in the past and higher emission yield. In addition, the entrapment of this compound in functionalized SiNPs, used as probes to amplify the TCL signal exploiting the strong biotin-avidin interaction, demonstrated its suitability for the development of TCL-based immune or nucleic acid biosensors. 1. Hummelen JC, Luider TM, Wynberg H. Complementary immunoassays. Chapter 14, Ed. W.P. Collins, 1988;191-208. 2. Roda A, Di Fusco M, Quintavalla A, Guardigli M, Mirasoli M, Lombardo M, Trombini Anal C. Chem. 2012;84:9913-9919. 3. Di Fusco M, Quintavalla A, Trombini C, Lombardo M, Roda A, Guardigli M, Mirasoli M. J. Org. Chem. 2013;78:11238-11246. O0015 Strategies towards Multi-colour Electrochemiluminescence Sensors Egan Doeven, Gregory Barbante, Emily Kerr, Paul Francis Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria, Australia In recent times electrochemiluminescence (ECL) has emerged as an important analytical technique, its selectivity and sensitivity making it suitable for the detection of a wide range of compounds. ECL is exploited routinely in commercial applications for rapid, sensitive detection and quantification of biomarkers, food borne pathogens and biowarfare agents. New applications of ECL as a sensing technique continue to appear in a wide range of fields, with a range of novel ECL-active luminophores having diverse properties being developed. Generally a single luminophore is excited in an ECL experiment without wavelength discrimination, for example tris(2,2'-bipyridyl)ruthenium(II) ([Ru(bpy)3 ](2+) ) emits strong co-reactant ECL centred at 620 nm. This work explores the use of multiple, selectively excited ECL luminophores, as well as the recently discovered inhibition of Ir(ppy)3 ECL(1) (under certain conditions) in order to selectively detect three emitting species in a single solution.(2) The emitting species of interest have been developed to have complimentary photophysical and electrochemical properties, and can thus be selectively excited via application of different electrode potentials. Quantification of the emission from the luminophores is investigated using two different approaches. Simultaneously collecting electrochemical and spectral data using a potentiostat and typical wavelength-sensitive detector such as a CCD can be exploited
to generate a 3D map of the emission vs. applied potential. Alternatively, the use of a low cost consumer-level digital camera and image analysis algorithms to isolate and quantify the contribution of each complex has been explored. Using this approach we demonstrate simultaneous detection of three emitting species at the low micro-molar level. This low cost multiplexed ECL detection system has potential applications in the emerging fields of mobile phone based telemedicine, as well as expanding the utility of current ECL based assays. 1. Doeven EH, Zammit EM, Barbante GJ, Francis PS, Barnett NW, Hogan CF. A potential-controlled switch on/off mechanism for selective excitation in mixed electrochemiluminescent systems. Chemical Science 2013;4(3);977-982. 2. Doeven EH, Barbante GJ, Kerr E, Hogan CF, Endler JA, Francis PS. Red-green-blue electrogenerated chemiluminescence utilizing a digital camera as detector. Analytical Chemistry 2014. DOI; 10.1021/ac404135f. O0017 Application of quinone as a selective chemiluminescent reagent for determination of biothiols in biological fluids Mohamed Elgawish(a,b) , Naoya Kishikawa(a) , Kaname Ohyama(a) , Naotaka Kuroda(a) (a) Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki, Japan (b) Pharmaceutical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt Background The physiological significance of low molecular weight thiols is well recognised with the levels of these compounds within biological fluids such as plasma and urine serving as valuable biomarkers in a number of clinical situations(1) . While there is a clear need to monitor these important analytes and indeed many procedures proffered, considerable scope remains for the development of fast protocols that require minimal sample pre-treatment. The biological important of quinones can be assign to their electrophilic and versatile oxidative properties, which are capable of promoting Michael-addition with cellular thiols, such as free glutathione, cysteine, and cysteine residues of proteins and electron transfer in living system through redox cycling of quinone/semiquinone/quinol triad system. Michael-addition-type probes have been actively developed in recent years and exploited for the design of chromo-and fluorogenic probes for thiol sensing(1) . In this context, we applied for the first time the Michael-addition reaction for chemiluminescence (CL) determination of biothiols. The principal of the proposed method relies on the application of quinone as Michael acceptor to react rapidly and specifically with biothiols. The liberated adducts retain the redox cycling capability of parent quinones to react with reductant, dithiothreitol (DTT), releasing reactive oxygen species (ROS) which can be measured by luminol-CL assay.(2) Materials and Methods Sample preparation One hundred microliters of human plasma, diluted with 500 mmol/L HEPES buffer, pH 8.5 to approximately 300 μL, was mixed with 10 μL of tris (2-carboxyethyl) phosphine (TCEP) solution (100 mmol/L in HEPES buffer, pH 8.5) and allowed to react at room temperature for 15 min. 10 μL of menadione (MQ) solution (100 mmol/L in acetonitrile (ACN)) was added and the sample was spin for 15 min at room temperature. Oasis HLB 1 cm(3) /30 mg cartridges were used to isolate the resulting adducts from each biological sample. The cartridges were conditioned with 0.5 mL of methanol and equilibrated with 0.5 mL of purified water. The samples were passed through individual cartridges, after which the cartridges were washed two times with 250 μL of purified water. The target analytes were eluted with 150 μL of 40% ACN, followed by 150 μL of neat ACN. Each mixture was vortex mixed, diluted ten times, and 20 μL was then inject ed into the HPLC-CL system (Fig. ) [Figure: see text] Results Four of the most important thiols in our body, cysteine (CYS), homocysteine (HCY), glutathione (GSH), and N-acetylcysteine (NAC), were selected under our investigation. MQ, the highly reactive and selective compound of studied quinones, overcame the problems of commonly utilized probe and reacted with thiol group specifically and rapidly at lowest possible temperature. All studied thiols reacted with MQ in 0.5 M HEPES buffer, pH 8.5, at room temperature. The reaction was carried out for 5 min at MQ to thiol molar ratio of about ten. The reaction was specific to aminothiol compared with other aminoacids which shown no reactivity. The calibration curves of MQ-thiol adducts showed excellent linearity over the range 0.0025-2 µmol/L with excellent r values and the detection limits at a signal-to-noise ratio of 3 were 0.0002-0.0008 µmol/L for all analytes. The derivatization of thiols was occurred before solid phase extraction technique to prevail the high polarity which makes their extraction from biological matrices very difficult. The proposed method could successfully quantify the studied thiols in human plasma samples with reasonable accuracy and precision. The protocol shown here clearly provides a sound footing from which further studies can be advanced to the measurement of other sulfhydryl thiols and matrices. References 1. Chen X, Zhou Y, Peng X, Yoon J et al Chem. Soc. Rev. 2010;39:2120. 2. Elgawish MS, Shimomai C, Kishikawa N, Ohyama K, Wada M, Kuroda N et al Chem. Res. Toxicol. 2013;26:1409. O0018 CASPT2//CASSCF Study Of the Ring-opening Mechanism of Dewar Dioxetane Pooria Farahani(a,b) , Marcus Lundberg(a) , Roland Lindh(a) , Daniel Roca-Sanjuán(b) (a) Uppsala University, Uppsala, Sweden (b) Universitat de València, Valencia, Spain Light emission from the heating of Dewar benzene was reported by McCapra.(1) Since the process was observed to be dependent on the presence of oxygen and most of the chemiluminescence reactions occur through an O-O cleavage,(1) the light observed was suggested to be produced after the ring opening of an intermediate structure, named Dewar dioxetane (see Fig. ).(2) The oxidation of Dewar benzene might lead to Dewar dioxetane and, after O-O and C-C cleavage,to the 2,4-hexadiendial product. The thermally activated decomposition mechanism of the Dewar dioxetane has been studied here by the multiconfigurational CASPT2//CASSCF approach,(3,4,5) and accurate reaction path strategies based on minimum energy path and intrinsic reaction coordinate computations. A two-steps biradical mechanism is determined for the process. It involves asynchronous O-O' and C-C' bond cleavage as in the related system 1,2-dioxetane.(6) Moreover, a radiationless decay path to the ground-state potential energy surface has been determined for the molecule along the manifold of the excited triplet state, while in the excited singlet state the system evolves toward an equilibrium structure that might be responsible of the light emission. This findings provide clues for rationalizing the observed light and point to a higher efficiency of fluorescence than phosphorescence. [Figure: see text] References 1. McCapra F. QUARTERLY Rev. 1966;20:485. 2. Koo J-Y, Schmidt S, Schuster G. Proc. Natl. Acad. Sci. USA 1978;75:30-33. 3. Roca-Sanjuán D, Aquilante F, Lindh R. WIREs Comput. Mol. Sci. 2012;2:585-603. 4. Andersson P, Malmqvist P.-Å, Roos B.O. J. Chem. Phys. 1992;96:1218. 5. Andersson P, Malmqvist P.-Å, Roos B.O, Sadlej A, Wolinski K. J. Chem. Phys. 1992;94:5483. 6. Farahani P, Roca-Sanjuán D, Zapata F, Lindh R. J. Chem. Theory Comput. 2013;9:5404-5411. O0019 Bioluminescence of Obelin; identification of the light emitters using QM/MM models Shufeng Chen(a,b) , Isabelle Navizet(c,e) , Roland Lindh(d) , Yajun Liu(b) , Nicolas Ferré(a) (a) Aix-Marseille Université, Marseille, France (b) Beijing Normal University, Beijing, China (c) Université Paris-Est, Marne-la-Vallée, France (d) Uppsala University, Uppsala, Sweden (e) University of Witwatersrand, Johannesburg, South Africa The chemiluminescent compound coelenterazine is related to the bioluminescence of a wide range of marine organisms, eg the Obelia Longissima hydrozoan. While the corresponding photochemical reaction (an oxidative decarboxylation
of oxo-coelenterazine in which the coelenteramide product is in an excited electronic state) is commonly used as a luminescent probe,(1) the details of its mechanism are still unknown. In particular, the chemical nature of the light emitters responsible for the multi-modal bioluminescence and fluorescence emission spectra is still matter of debate. Up to now, the neutral coelenteramide molecule and its phenolate anion (obtained through a proton transfer towards the close His22 residue, hence forming an ion-pair) are the two most serious candidates.(2) Using hybrid QM/MM calculations,(3) we confirm the implication of the neutral coelenteramide in its first excited state as the primary light emitter (the computed TDDFT/MM vertical emission is 339 nm). However our results demonstrate that the postulated ion-pair is not a stable light emitter. Actually, an electron transfers together with the proton to form a diradical state (Fig. ) and the corresponding system ultimately evolves towards a point of degeneracy between the ground and first excited states. Hence a non-radiative decay path is suggested to compete with the light emission process. [Figure: see text] Alternatively, the phenolate coelenteramide is found to be a light emitter, as long as His22 looses another proton at the same time its accepts the one coming from coelenteramide, hence keeping its electric neutrality (the computed TDDFT/MM vertical emission is about 500 nm, in excellent agreement with the experimental λ(max) ). Our calculations show that the final location of the proton is not of primary importance. Finally, using the unique modeling capabilities of QM/MM calculations, and comparing our results with previous computations of coelenteramide in gas phase or in a solvent(4) , we assess the different contributions responsible for the color of the emitted light. Besides the protonation state of the luminophore, the steric constraints induced to the tight cavity in which coelenteramide is bound is the most important factor, far more than the electrostatic interaction with the protein. References 1. Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES. Violet and Greenish Photoprotein Obelin Mutants for Reporter Applications in Dual-color Assay. Anal. Bioanal. Chem. 2008;391:2891-2896. 2. Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG. Spectral Components of Bioluminescence of Aequorin and Obelin. J. Photochem. Photobiol., B 2008;92:117-122. 3. Chen S-F, Navizet I, Lindh R, Liu Y-J, Ferré N. Hybrid QM/MM Simulations of the Obelin Bioluminescence and Fluorescence Reveal an Unexpected Light Emitter. J. Phys. Chem. B 2014 (in press, dx.doi.org/10.1021/jp412198w). 4. Chen S-F, Navizet, I, Roca-Sanjuá n D, Lindh R, Liu Y-J, Ferré N. Chemiluminescence of Coelenterazine and Fluorescence of Coelenteramide; A Systematic Theoretical Study. J. Chem. Theory Comput. 2012, 8, 2796-2807. O0020 Identification of a fluorescent compound from the bioluminescent polychaete Tomopteris Warren Francis(a,b) , Meghan Powers(a,b) , Steve Haddock(a) (a) Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA (b) University of California, Santa Cruz, Santa Cruz, CA, USA Tomopteris is a cosmopolitan genus of luminous polychaetes that release bright yellow particles from the parapodia when stimulated. Although the yellow bioluminescence of this genus has been the subject of a few investigations, the chemistry is essentially unstudied. All that is known is that the reaction does not involve any of the known molecules, like coelenterazine. The connection between fluorescence and bioluminescence has been a topic of great discussion. A brief report half a century ago described the yellow fluorescence of the parapodia with an identical similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here we report the isolation of an abundant, fluorescent yellow-orange compound found in the luminous exudate and in the body of the animals. LCMS revealed the mass to be 270 m/z with a molecular formula of C15 H10 O5 , which ultimately was shown to be aloe-emodin, an anthraquinone previously found in various Aloe plant species. From known redox properties and chemiluminescence from other anthraquinones, we hypothesize that aloe-emodin is the oxyluciferin for Tomopteris bioluminescence. O0021 Chemiluminescent methods for explosives (TNT, TATP, HMTD) detection Stefano Girotti(a) , Elida Ferri(a) , Marcello D'Elia(b) , Mara Mirasoli(c) , Aldo Roda(c) , Luigi Ripani(d) , Giuseppe Peluso(d) , Roberta Risoluti(e) , Elisabetta Maiolini(a) , Francesco Saverio Romolo(f,g) (a) Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Bologna,, Italy (b) Gabinetto Regionale di Polizia Scientifica per l'Emilia Romagna, Bologna, Italy (c) Dipartimento di Chimica, Università di Bologna, Bologna, Italy (d) Reparto Investigazioni Scientifiche (RIS) Carabinieri, Roma, Italy (e) Dipartimento di Chimica, Università "La Sapienza", Roma, Italy (f) Institut de Police Scientifique, Université de Lausanne, Lausanne, Switzerland (g) Legal Medicine Section, Università "La Sapienza", Roma, Jamaica The terroristic attacks performed in the last ten years have focused the attention on the protection and security of the citizen and the detection of various types of explosives is included in this purpose. Our work was finalised to develop chemiluminescent methods that permit the detection of TNT (2,4,6-trinitrotoluene), TATP (Triacetone triperoxide) and HMTD (Hexamethylene triperoxide diamine), displaying higher detectabilityand ease of use with respect to currently available methods (1-2). TNT is one of the most employed explosives in the 20th century and at the same time one possible well known environmental pollutant for its toxicity (3). For these reasons its detection could permit the prevention of terrorism acts (or the identification of the explosive used for these purposes) and for an early sign of environmental pollution. TATP (Triacetone triperoxide) and HMTD (Hexamethylene triperoxide diamine) are compounds extremely instable because they contain peroxide groups (4). Due to their simple synthesis, which requires, as reagents, compounds easily available at any supermarket, and that can be performed at home, they are frequently used in terrorist attacks. For TATP and HMTD we developed a chemiluminescent method that permits their indirect identification. Upon treatment with acidic solutions, explosive residues are decomposed into radical peroxides, which are then quantified as substrates of the horseradish peroxidase (HRP) enzyme in the light-emitting oxidation of Luminol. Both assays shown good sensitivity (values of Limit of Detection, LOD, and IC50 in the ppb range) and reproducibility (CV value less than 10%), and when applied to real samples they showed a great specificity. In the field of detection of nitroaromatic based explosives, we developed three indirect competitive immunochemical assays specific for TNT; an ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label and a chemiluminescent-LFIA (CL-LFIA). Both LFIA methods showed good performances in terms of qualitative and semiquantitative capability, resulting especially practical and easy to use on-site. The colorimetric LFIA showed to be very selective in cross reactivity studies and displayed a LOD (MDA) of 1 µg mL(-1) . Using optimised working conditions the CL-LFIA method showed a limit of detection of 0.05 µg mL(-1) and additionally offered the possibility of a semi-quantitative screening in situ. The total analysis time was 15 minutes. ELISA showed the best sensitivity (LOD of 0.4 ng mL(-1) ) and a good reproducibility (CV value about 7%) but needed more intensive activities by qualified laboratory unit. The developed immunoassays were applied to real samples of various materials involved in controlled explosions of Improvised Explosive Devices (IEDs), based on massive military grade charges of 100 g, 1000 g and 2000 g of TNT, and finally to samples from hand
ling tests. Samples analysed after explosions were, soil sampled in the explosion area and target-surfaces made of six different materials; metal, plastic, cardboard, moquette, wood and adhesive tape fixed at the top of 180 cm-high wood poles. 1. Schulte-Ladbeck R, Kolla P, Karst U. A field test for the detection of peroxide-based explosives. The Analyst. 2002;127:1152 2. Vorbeck C, Lenke H, Fischer P, Spain JC, Knackmuss H-J. Appl. Environ. Microbiol. 1998;64:246 3. Kaplan DL, Kaplan AM. 2,4,6-Trinitrotoluene-surfactant complexes; decomposition, mutagenicity and soil leaching studies. Environ. Sci. Technol. 1982;16:566 4. Keinan E, Itzhaky H. Method and kit for the detection of explosives. US Patent 6767717, 2004 O0022 Characterization of photogenic sites in deep-sea planktonic worms; a comparative approach within Tomopteridae Anaïd Gouveneaux, Jérôme Mallefet Catholic University of Louvain, Louvain-la-Neuve, Belgium From the intracellular microsources to the most sophisticated photophores, the characterization of photogenic structures may, in some cases, require the development and combination of original approaches and criteria. While the majority of bioluminescent pelagic organisms are blue-emitters, some tomopterid polychaetes are able to produce yellow light emissions. ([1]) After Greeff described in 1880 the parapodial rosette glands containing fluorescent yellow-pigmented cells as the specific photogenic organs ([2]) , some species have been reported as bioluminescent referring on this morphological criterion only ([3]) . However, although it seems consistent hence being commonly admitted, the structure-function coupling has never been clearly demonstrated. Moreover, how to explain that both intracellular and secreted bioluminescence have been reported? ([1][2]) Here we compare the bioluminescent signals and structures from T. helgolandica Greeff, 1879, T. nisseni Rosa, 1908 and T. carpenteri Quatrefages, 1866 which are East Atlantic, Pacific and Antarctic species respectively. Through combined optic, fluorescent, confocal microscopy techniques, we provide the first experimental data for the clarification of the ambiguous status of the rosette glands in Tomopteridae. Thus, the bioluminescent activity and fluorescence reactivity have been co-localized in the yellow pigmented cells of T. helgolandica, confirming the close link between these cells and the photogenic events. Moreover, we observed that the fluorescence tissue regions extend during the light emission. Finally, we have recently demonstrated by immunohistochemical staining a dense network of tubules largely developed in the pinnae. Some similar structures have been described in 1964 by Terio in T. nationalis as the excretion pathway of the yellow fluorescent substance ([4]) . However, these channels partially converge to form the chromophile glands - another glandular type of unknown function - whereas the morpho-anatomical continuity with the rosette glands remains uncertain. We are currently performing additional analysis using confocal and transmission electronic microscopy in order to improve the dynamic morphological description of these structures. References 1. Dales RP. Bioluminescence in pelagic polychaetes. Journal of the Fisheries Board of Canada 1971;28(10);1487-1489. 2. Harvey EN. Bioluminescence. New York; Academic Press, 1952;649. 3. Bonhomme C. La Bioluminescence de quelques annélides méditerranéennes. (Etude histologique et hystophysiologique). Naturalia Monspeliensa 1952;2:7-137. 4. Terio B. Possibli interrelazioni tra bioluminescenza e fluorescenza di materiali fotosensibili presenti nelle pinne e sui parapodi dei Tomopteridi. Atti della Società Peloritana di Scienze Fisiche. Matematiche e Naturali 1964(10);127-138. O0023 Oxygen Binding Sites in Obelin; A Computational Study into the Formation of Active Photoproteins. Thomas M. Griffiths, Haibo Yu University Of Wollongong, Wollongong, NSW, Australia Incorporation of molecular oxygen is key to function of Ca(2+) regulated photoproteins. The photoprotein obelin contains a non-covalently bound 2-hydroperoxycoelenterazine substrate in the centre of the enzyme. Formation of the active form of obelin involved binding coelenterazine, [1] then the formation of the C2-hydroperoxyl derivative of coelenterazine. Before this can occur oxygen must bind to the protein. To date, the location of these oxygen binding sites are unknown, as well as the effect of the protonation state of the ligand on the energetics of oxygen binding. Here we use computational methods to identify oxygen-binding sites within obelin, and examine the effect of coelenterazine protonation on the position and thermodynamics of oxygen binding. All atom classical molecular dynamics (MD) simulations of obelin (PDB 1SL9), complexed with protonated and deprotonated forms of coelenterazine. A potential of mean force (PMF) map for the inclusion of molecular oxygen was generated from these trajectories by performing implicit ligand sampling (ILS), [2] to identify potential oxygen binding sites within the enzyme, shown in the figure below. We ran free energy perturbation (FEP) calculations with explicit oxygen molecules in the identified binding sites to more rigorously characterize the binding free energy of oxygen in the obelin coelenterazine complex. Further molecular mechanical (MM) and hybrid quantum mechanical/molecular mechanical (QM/MM) MD FEP calculations were performed to examine the deprotonation of coelenterazine in the binding pocket of obelin. Potential oxygen-binding sites were identified in each case. A shift of the binding site towards the C2 carbon of coelenterazine is seen when moving from the protonated, to the deprotonated form of the ligand, and energetically we see a decrease in the binding energy of oxygen upon deprotonation, which we hypothesize as due to a change in the structure of the binding site, and the electrostatics of the binding pocket upon deprotonation. [Figure: see text] 1. Shimomura O. Bioluminescence; Chemical Principles and Methods. World Scientific, 2008 2. Cohen J, Arkhipov A, Braun R, Schulten K. Imaging the Migration Pathways for O2, CO, NO, and Xe Inside Myoglobin. Biophysical Journal 2006;91:1844-1857. O0024 Recent discoveries in marine bioluminescence diversity and function Steven Haddock MBARI, Moss Landing, CA, USA Since Peter Herring's 1987 catalog of bioluminescent species, there have been many discoveries of bioluminescence capability in a variety of species and even phyla. Examples include sea anemones, arrow-worms, polychaetes, fish, and other species yet to be described. Many of these have arisen from exploration of the deep sea. In addition, new functions have been proposed to explain the presence of bioluminescence across such a broad diversity of taxa. Here we will review the present the discoveries of luminescent organisms and their behaviors. Special focus will be on the evolutionary origins of this diversity; from the species perspective this will include the potential for convergence, evolutionary precursors, and co-opting luminous substrates. From the functional perspective, this will highlight the many roles that luminescence plays, with even multiple roles in a single organism. O0025 Alternative approaches to increase stability of firefly luciferase through its flexibility switch Saman Hosseinkhani, Mehdi Imani, Zahra Amini-Bayat, Maryam Moradi Tarbiat Modares University, Tehran, Iran The thermal sensitivity of firefly luciferase has hampered its application in a range of fields. It is proposed that the stability of a protein can be increased by several procedures including site-directed mutagenesis and also solvent engineering. In this study, different approaches including addition of disulfide bridges, saturation of surface charges, decrease of segmental flexibility and removal of unusual residues have been used to increase thermostability of firefly luciferase. Introduction of disulfide bridge decreases the configurational entropy of unfolding and thereby make a more thermostable protein. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two s
eparate mutant enzymes. One of the designed mutant ( A103C/S121C mutant) showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was kept at higher temperatures (37 ◦C). B-factor analysis of firefly luciferase residues shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins show that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on argument. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type while D476N doesn't have any substantial difference. Protein engineering studies have shown that thermostable proteins from thermophilic organisms have a higher frequency of Arg, especially in exposed states. To further elucidate the arginine overload effect on thermostability of firefly luciferase, some of hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase are transformed to arginine. All of these residues are located at the external loops of L. turkestanicus luciferase. Introduction of double mutation (-Q35R/I232R) and triple mutation (-Q35R/I232R/I182R) were kept specific activity of firefly luciferase while its thermostability was enlarged and its flexibility was declined. On the other hand, in order to better understand the practical role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Residue Thr346 in a linking loop (341-348) of firefly luciferase is located in a forbidden region of Ramachandran plot. In this study, we have replaced this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were investigated using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability. O0026 Luciferase complementary assay implication in detection of early stages of apoptosis Saman Hosseinkhani, Masoud Torkzadeh-Mahani, Farangis Ataei, Maryam Nikkhah, Shiva Akbari-Birgani Tarbiat Modares University, Tehran, Iran Protein-protein interaction analyses have been used in many investigation including drug screening, biosensors and basic molecular studies. Firefly luciferase with high quantum yield, the ability of different color emissions and suitable structural properties is widely used for design of cell-based biosensors for monitoring pathways in nano-systems biology. To develop intracellular reporter, we have employed split firefly reporter strategy wherein a firefly luciferase is genetically split into two non-functional fragments so that luciferase activity is effectively stopped and then these fragments are fused to possibly interacting protein partners and upon induction if the target proteins interact, the fragments of the reporter protein are brought within proximity leading to signal generation that can be measured. For studying a specific signaling pathway, various optional interacting proteins can be taken into consideration in signaling cascades. Based on this strategy, novel whole-cell recombinant biosensors are designed to detect early-stages of apoptosis and release of IP3. Apoptosome formation (the main step in progress of intrinsic pathway of apoptosis) is triggered by release of cytochrome c from mitochondria followed by oligomerization of Apaf-1 monomers. In spite of momentous investigational support for apoptosome formation, but its detail structure within living cells is not clearly known. In order to direct confirmation of this model and also earlier detection of apoptosis, a novel method using a split luciferase biosensor is designed based on oligomerization of N-luc-Apaf-1 and C-luc-Apaf-1 monomers. Inositol-1,4,5-trisphosphate (IP3) is a vital second messenger that regulates complex signaling processes in various physiological events. According to the result, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed biosensor was able to monitor release of IP3 upon induction by different inducers like Bradykinin and ATP. The current biosensor not only delivers a specific IP3 detector in vitro but also facilitates monitoring of the response of IP3 in living organisms. Both current biosensors are being used to discriminate between apoptosis and differentiation of mouse embryonic stem cells into cardiomyocytes. O0027 Development of a flow cytometry-based BRET assay utilising genetically-encoded biosensors for mixed cell populations; Application to cannabinoid CB1 receptors. Morag Rose Hunter, Stephen G Edgar, Michelle Glass University of Auckland, Auckland, New Zealand Genetically-encoded bioluminescent resonance energy transfer (BRET)-based biosensors have been invaluable tools in the quantitative and qualitative analysis of intracellular signalling processes. BRET biosensors are typically transiently transfected into cultured cells, and the resulting signal detected using a plate reader. These plate readers integrate the results from large samples of cells - typically 30,000-100,000 cells per 96-well. Here, we report the development process of a high throughput assay for BRET biosensors in single cells with the primary aim of developing a method to differentiate and selectively analyse subpopulations of cultured cells. Specifically, we are developing a flow cytometry (FCM) method for single-cell cAMP measurement, using the genetically-encoded BRET-based biosensor CAMYEL (1). FRET by FCM has previously been demonstrated (e.g. (2)), but to date there have been no published examples of FCM BRET detection, although this approach would help overcome the spectral overlap of FRET-pair fluorophores. The BRET-based biosensor assay was optimised using a Becton Dickinson LSRII flow cytometer, with the 488 nm laser occluded to prevent laser-excitation of the BRET acceptor. As the 488 nm laser typically provides the forward and side-scatter data used to identify cells, an antibody-based cell-surface marker was utilised to identify and selectively gate for cells. We found that, while the Rluc and YFP BRET pair had insufficient energy for detection, with a sufficiently slow flow rate, the high emission of the variants Rluc8 and Venus provided satisfactory signal for detection of both wavelengths. Site directed mutagenesis was therefore employed to modify CAMYEL to optimise detection, and results with the optimised biosensor will be presented. In conclusion, while it is technically challenging to establish a suitable single cell detection method, such a high throughput approach would be a valuable to analysing cell signalling biology and for drug discovery. 1. Jiang LI, Collins J, Davis R, Lin K-M, DeCamp D, Roach T, et al. JBC. 2007;282(14);10576-84. 2. Banning C, Votteler J, Hoffmann D, Koppensteiner H, Warmer M, Reimer R, et al. PLoS ONE. 2010;5(2);e9344. O0028 Biosynthetic Components and Decarboxylation from L-Cysteine in Firefly Luciferin Biosynthesis Shusei Kanie(a) , Yuichi Oba(a) , Naoki Yoshida(a) , Makoto Ojika(a) , Satoshi Inouye(b) (a) Nagoya University, Nagoya, Japan (b) JNC Corporation, Yokohama, Japan The aim of this work is to demonstrate the biosynthetic components of firefly luc
iferin in vivo. The luminescence properties and the crystal structure of firefly luciferases have been investigated, and its cDNA has been used as a reporter in various assay systems. However, there has been no conclusion in the biosynthetic components of firefly luciferin for over 30 years. Clarifying the biosynthetic components of firefly luciferin is beneficial to elucidate the biosynthetic pathway, including the biosynthetic enzyme. We performed incorporation studies by injecting stable isotope-labeled compounds, including 1,4-[D6 ]-hydroquinone, L-[U-(13) C3 ]-cysteine, L-[1-(13) C]-cysteine, L-[3-(13) C]-cysteine, into the adult lantern of the living Japanese firefly Luciola lateralis. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was determined by LC/ESI-TOF-MS as follows. Firstly, the incorporation experiment with 1,4-[D6 ]-hydroquinone showed that 1,4-hydroquinone was incorporated into the benzothiazole unit of firefly luciferin. Secondly, the incorporation experiment with L-[U-(13) C3 ]-cysteine showed that L-cysteine was incorporated into both the benzothiazole and thiazoline units of firefly luciferin. Interestingly, firefly luciferin biosynthesized from L-[U-(13) C3 ]-cysteine was not only L-luciferin but also D-luciferin. Further, in the incorporation experiments with L-[1-(13) C]-cysteine and L-[3-(13) C]-cysteine, the positions of the stable isotope atoms in firefly luciferin were identified by the mass fragmentation of firefly luciferin. These results showed that only the carboxyl group of L-[1-(13) C]-cysteine was eliminated during the benzothiazole ring formation of firefly luciferin. In addition, we examined the presence of free 1,4-hydroquinone in fireflies using HPLC because the presence of it has not been reported. Although free 1,4-hydroquinone was not detected in our conditions, we successfully detected arbutin, a glycoside derivative of 1,4-hydroquinone. Our present works demonstrate for the first time that D-firefly luciferin is biosynthesized in the lantern of the adult firefly from two L-cysteine molecules with 1,4-hydroquinone, accompanied by the decarboxylation from L-cysteine. Moreover, this work suggests that 1,4-hydroquinone is stored as a glycoside derivative such as arbutin in fireflies. O0029 First experimental evidence for an enzyme-generated chemiluminescence-induced trans-cis isomerization of chip-immobilized zearalenone in a microfluidic cell of a biosensor Susanna Oswald, Reinhard Niessner, Dietmar Knopp Chair of Analytical Chemistry, Technische Universität München, München, Germany In a recent research project, which was focused on the development of a microarray-based flow-through chemiluminescence immunoassay (CL-ELISA) for multiplex determination of mycotoxins in cereals, a stand-alone platform was used and reusable biochips developed (1). Surprisingly, the method failed for ZEA. After performing a set of additional experiments, the photochemical trans-cis isomerization of biochip-immobilized ZEA by chemiluminescence (CL) light generated by horseradish peroxidase (HRP) catalyzed oxidation of luminol revealed as the ultimate cause. To our knowledge, this is the first observation of a photochemical isomerization by enzyme-generated CL light in a microfluidic cell of a biosensor. The principle of the multiplex flow-through assay is based on the indirect competitive ELISA and use of mycotoxin derivatives covalently immobilized on DAPEG-functionalyzed glass chips (Fig. ). In the final step, the enzyme substrate (luminol/H2 O2 ) was pumped over the chip and an image was taken for 60 s by the CCD camera. After recording, the chip was exhaustively regenerated with glycine buffer (pH 3.0) with addition of surfactant (0.1% SDS), i.e., all bond primary and secondary antibodies were removed. While for AFB1, FB1, OTA and DON almost constant maximal CL signals for up to 50 measurement cycles for blank samples were obtained, a significant loss (~45%) of signal intensity was observed for ZEA already in the second measurement. To elucidate the reason for this abnormal behavior of ZEA, several experiments were conducted. Beside others, we speculated about possible structural changes of the chip-immobilized ZEA molecule which could lead to a lowered binding of primary ZEA antibody. Already in 1972, Peters (2) reported on the photochemical trans-cis stereoisomerization of ZEA using a 450-W medium pressure mercury lamp. Later, it was proved that this reaction could take place under real-life conditions by storage of methanolic solution (3) or edible oils (4) in glass bottles, and even in wet maize (5). With the traditional microtiter plate ELISA, the used ZEA-antibodies (obtained from Dr. R. Dietrich and Prof. Dr. E. Märtlbauer, LMU, München) showed only 5% cross-reactivity of cis-ZEA (graciously donated by Dr. M. Koch, BAM, Berlin) compared to the trans-isomer. However, it was hardly conceivable that stereoisomerization of ZEA could happen with HRP-generated CL light (emission maximum at 425 nm) in the used microfluidic cell. Nonetheless, light intensity per spot area (~250 µm) might be adequate, possibly. In another experiment, two ZEA-spotted biochips were run in the chip reader to investigate the effect of CL light on the signal intensity along several measurements. As the main outcome, five incomplete cycles (in the last step, luminol/H2 O2 was replaced by buffer) did not lead to a reduction of CL signal intensity in the first subsequent complete measurement. This result revealed a strong evidence for a CL light-induced molecular transformation of the immobilized ZEA. This was further supported by an investigation using a different detection principle, i.e., surface plasmon resonance (Biacore X100) and ZEA-BSA conjugate-loaded CM5 Sensor Chip (Fig. ). Over five sequential cycles of ZEA-antibody binding/regeneration (same regeneration buffer was used as with the chip reader) no change of signal (measured as refractive units) was observed, as supposed. To conclude; This finding should be important for bio(immuno)analytical determinations of analytes which show trans/cis-isomerization and make use of competitive assay formats with solid-phase immobilized target analyte and CL as the detection principle. Acknowledgements This research was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V. Bonn); project AiF 381 ZN. [Figure: see text] [Figure: see text] References 1. Oswald S, Karsunke XYZ, Dietrich R, Märtlbauer E, Niessner R, Knopp D. Anal. Bioanal. Chem. 2013;405:6405-6415. 2. Peters CA. J. Med. Chem. 1972;15:867-868. 3. Miles CO, Erasmuson AF, Wilkins AL, Towers NR, Smith BL, Garthwaite I, Scahill BG, Hansen RP. J. Agric. Food Chem. 1996;44:3244-3250. 4. Köppen R, Riedel J, Proske M, Drzymala S, Rasenko T, Durmaz V, Weber M, Koch M. J. Agric. Food Chem. 2012;60:11733-11740. 5. Brezina U, Kersten S, Valenta H, Sperfeld P, Riedel J, Dänicke S. Mycotoxin Res. 2013;29:221-227. O0030 A novel method of synthesis of peptide-6-amino-D-luciferin conjugates for detection of peptidase activity Anita Kármen Kovács(a,b) , Péter Hegyes(b) , László G. Puskás(b) , Gábor K. Tóth(a) (a) University of Szeged, Szeged, Hungary (b) Avidin Biotechnology Ltd., Szeged, Hungary Aminoluciferin (aLuc) is a luciferin with its 6-position hydroxyl group substituted with an amino group. This modification allows aminoluciferin to form amide bonds with a peptide, while retaining the transport and bioluminescent properties of luciferin, resulting in a good substrate for different important proteases(1) , which can be used for the determination of the enzymic activity in different therapeutically highlighted cases. The synthesis of several peptide-aLuc conjugates and their precursors have been published(2) and some of them are commercially available. However, due to their high price and difficulties with their synthesis the application of these conjugates is very limited. Our aim was to extend the utilization of bioluminescent determination for the fib
roblast activation protein (FAP) and prolyl oligopeptidase (POP) enzymes, which has therapeutic potential for metastatic epithelial cancers. For the aforementioned purposes the appropriately protected aminoluciferin derivatives would be necessary which in the required amount have an enormously high price. In addition, the elongation of the peptide chain at the amino function is very difficult due to its deactivation. Therefore, an entirely new synthesis strategy has been worked out for the preparation of the desired peptide-aLuc conjugates. Briefly, the appropriately protected amino acid was coupled to 6-amino-2-cyano-benzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was attached then to different types of functionalised resins. The resulted loaded resins can be used for the solid-phase synthesis of any desired peptide-aLuc conjugates without difficulty. For example, we successfully prepared the following derivatives; Z-Arg-Arg-Pro-aLuc, Z-Gly-Pro-aLuc, Z-Phe-Phe-Pro-aLuc, which were used as bioluminescent probes for fundamental biochemical study of cancer stroma and high-throughput inhibitor screening. 1. Hickson J, Ackler S, Klaubert D, Bouska J, Ellis P, Foster K, Oleksijew A, Rodriguez L, Schlessinger S, Wang B, Frost D. C. Death and Diff. 2010;17:1003-1010. 2. O'Brien MA, Daily WJ, Hesselberth PE, Moravec RA, Scurria MA, Klaubert DH, Bulleit RF, Wodd KV. J. of Biomol. Scr. 2005;10:137-148. O0031 Application of enzyme bioluminescence in ecology Valentina Kratasyuk(a,b) , Elena Esimbekova(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics, Siberian Branch of RAS, Krasnoyarsk, Russia The general principles of bioluminescent enzymatic toxicity bioassays were examined in this review. This review describes the applications of these methods and their implementation for commercial biosensors. Bioluminescent Enzyme System Technology (BEST) has been proposed, where the bacterial couple enzyme system; NADH;FMN-oxidoreductase-luciferase substitutes for living organisms [1-6]. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications [1-6]. To BEST wide-spread use the multi-component reagent "Enzymolum" has been developed, which contains the bacterial luciferase, NADH;FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel [1 ]. "Enzymolum" is the central part of Portable Laboratory for Toxicity Detection (PLTD), which consists of a biodetector module, a sampling module, a sample preparation module, and a reagent module. PLTD immediately signals chemical-biological hazards and allows us to detect a wide range of toxic substances. "Enzymolum" can be integrated as a biological module into the portable biodetector-biosensor of original construction for personal use. By the example of " Enzymolum" and the algorithm of creation of new enzyme biotests with tailored characteristics a new approach was demonstrated - biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. The possible applications of enzyme bioassays are seen in the examples of their use in medical diagnostics, assessment of the effect of physical load on sportsmen, analysis of food additives and in new practical courses for higher educational institutions and schools [2-6 ]. The advantages of enzymatic assays are their rapidity (the time of analysis does not exceed 3-5 minutes), high sensitivity, simple measuring procedure, possibility of automation of ecological monitoring procedure, availability and safety of reagents, and a wide market of bioluminometers. 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. Esimbekova EN, Kratasyuk VA, Torgashina IG. Disk-shaped immobilized multicomponent reagent for bioluminescent analyses; correlation between activity and composition/ Enzyme and microbial technology. 2007;40(2);343-346. 2. Vetrova EV, Kudryasheva NS, Kratasyuk VA. Redox compounds influence on the NAD(P)H;FMN-oxidoreductase - luciferase bioluminescent system. Photochem. Photobiol. Sci. 2007;6:35-40. 3. Vetrova E, Esimbekova E, Remmel N, Kotova S, Beloskov N, Kratasyuk V, Gitelson I. A bioluminescent signal system; detection of chemical toxicants in water. Luminescence 2007;22(N3);206-214. 4. Kratasyuk V, Esimbekova E, Correll M, Bucklin R. Bioluminescent enzyme assay for the indication of plant stress in enclosed life support systems. Luminescence 2011;26(6);543-546. 5. Rimatskaya NV, Nemtseva EV, Kratasyuk VA. Bioluminescent assays for monitoring of air pollution. Luminescence 2012;27(2);154. 6. Esimbekova E, Kondik A, Kratasyuk V. Bioluminescent enzymatic rapid assay of water integral toxicity. Environmental Monitoring and Assessment 2013;185(7);5909-5916. O0032 Structure-property relationships disclosed in the family of acridinium esters - chemiluminogenic molecules of great practical importance Karol Krzyminski(a) , Lucyna Holec-Gasior(b) , Justyna Czechowska-Kryszk(a) (a) University of Gdansk, Faculty of Chemistry, Wita Stwosza 63 Str., 80-308 Gdansk, Poland (b) Gdansk Univeristy of Technology, Microbiology Chair, Narutowicza Street 11/12, 80-233 Gdansk, Poland Introduction Acridinium esters (AE), since their introduction to the analytics 30 years ago, make one of the most important groups of chemiluminogenic (CL) systems, that have been employed in medical diagnostics and environmental analysis today [1]. So far, however, no systematic studies on the structure-property relationships were carried out in this family of compounds. The latter features likely determine their practical utility and facilitate rational designing of the new labels and indicators with their use. Investigations, that we were conducting in recent years over those molecular systems enabled us to determine some crucial parameters, influencing luminogenic properties of AEs [2-6]. The latter problems, together with the examples of our original analytical solutions with the use of selected AEs will be addressed during the presentation [7]. Methods The study involved a wide group of specially developed AEs (Fig. A), their precursors (Fig. B) and the products of their luminogenic transformations in solutions (Fig. C, D). [Figure: see text] Several methods have been employed, including liquid, solid and gaseous phases of AEs. To determine the effect of substituents on the geometry of acridine derivatives, the stuctural rentgenography of monocrystals was employed. This approach also enabled us to characterize the intermolecular interactions occurring in AEs in the crystalline phase. Thermal stability, solid phase thermodynamics, phase transitions, as well as the crystal lattice energies of AEs and their precursors were assessed, applying thermoanalytical methods (DSC, TG). Electron spectroscopy, IR, MS and NMR techniques, supported by the findings of theory (DFT level), enabled us to determine the types of electronic transitions, quantum efficiencies and charge distributions, associated with the chemical reactivity in the family of AEs. In order to determine CL quantum yields, hydrolytic stability, kinetics of emission and influence of the environment on the luminometric properties of AEs, a plate luminometry techniques were employed. HPLC chromatography (fluorescence and absorption detection) allowed us to study the population of CL reaction products and the kinetics of hydrolytic side processes. Results In the crystal lattices of AEs, in addition to the prevailing Coulomb inter-ionic interactions, there are a number of hydrogen (C - H(…) O) as well as short-range interactions (C - F(…) π a S - O(…) π, C-H(…) π), involving both the acridinium cations and the anions (TfO(-) ) . The flat aromatic fragments additiona
lly form a dense network of weak π(…) π - type contacts. AEs melt in the range 470-540 K; the enthalpies of this phase transition fall in the range of 30-50 kJ mol(-1) ; several tens degrees above the melting starts thermal decomposition of the salts. The crystal lattice energies of AEs fall in the range of 560-605 kJ mol(-1) , expressing typical values for the systems build of complex monovalent ions, the enthalpy of formation are similar for variously substituted AEs, attaining strong negative values at the level of -1000 kJ mol(-1) [2]. A number of structure-property relationships were disclosed in the family of AEs, among which a significant is the linear dependence of (1) H chemical shifts of nuclei neighboring to the ester group and the values of the LCAO coefficient of pz LUMO orbital of the acridinium C9 atom. Analysis of these factors indicated, that C9 atom is the site of primary nucleophilic attack, resulting in the formation acridane non-planar adduct. One of such an adducts was obtained in the crystalline form and its structure fully confirmed the theoretical predictions [3]. Luminescence studies revealed that the AE's emission quantum yields decrease linearly with the hydrodynamic volume of phenoxycarbonate anions, presumably the leaving groups in CL process. Proposed by us parameter, called utility (U) (the product of CL quantum yield and the factor determining the hydrolytic stability of AEs in solutions), differentiates the cations in terms of their usefulness. It was revealed, that U varies non-linearly with the volume of PhOCO3 (-) , with the maximum around 220 Å(3) for alkyl-substituted AEs [4]. Substitution of the acridinium nucleus with OCH3 group is not only manifested by the batochromic shift of emission range, but is also accompanied by the increase in the emission efficiency - as it was clearly observed in non-aqueous systems [5]. Taking into account conclusions drawn from the basic research, a new analytical solutions were developed by us. The tests enable to perform quantitative assays of the antioxidant activity of exogenous substances (such as food additives, dietary supplements, etc.) as well as biological samples (e.g. blood plasma) [6]. Currently, our research activity is focused on the design and testing of new acridinium luminogenic labels, designed to optimize emission efficiency and stability in aqueous solutions. Some preliminary results will be disclosed during the presentation. Conclusions Short-range interactions determine the unique crystal structures of AEs. AE are thermodynamically stable and durable below 500 K. They can be stored for any length of time at room temperature, if their analytical use is planned. The efficiency of AEs emission correlate with a variety of physicochemical parameters, among them with the volume is of the hydration layer of the of leaving group during CL process. Original analytical tests were developed with the use of new indicators and markers based on AE. Acknowledgments The studies were financed by the Polish State National Science Center through the grants No. N N204 375740 (contract No. 3757/B/H03/2011/40) for the period 2011-2014 and No. UMO-2012/05/B/ST5/01680 for the period 2013-2015. References 1. Kricka LJ. Anal. Chim. Acta 2003;500:279-286. 2. Zadykowicz B, Krzymiński K, Storoniak P, Błażejowski J. J. Therm. Anal. Calorim. 2010;101:429-437. 3. Niziołek A, Zadykowicz B, Trzybiński D, Sikorski A, Krzymiński K, Błażejowski J. J. Mol. Struct. 2009;920:231-237. 4. Krzymiński K, Ożóg A, Malecha P, Roshal AD, Wróblewska A, Zadykowicz B, Błażejowski J. J. Org. Chem. 2011;76:1072 - 1085. 5. Krzymiński K, Roshal AD, Zadykowicz B, Białk-Bielińska A, Sieradzan A, J. Phys. Chem. A 2010;114:10550-10562. 6. Kozin YI, Dyubko TS, Sokolyk OA, Roshal AD, Krzymiński K. ArchivEuromedica, 1(st) & 2(nd) Edition, 2011;91-93. O0033 Bioluminescence as a tool for study Mechanisms of Radiation Hormesis and Radiation Toxicity Nadezhda Kudryasheva(a,b) , Maria Selivanova(a) , Alena Petrova(b) , Tatiana Rozhko(b) , Anna Tugarova(c) , Alexander Kamnev(c) , Anna Devyatlovskaya(d) (a) Institute of Biophysics Sb Ras, Krasnoyarsk, Russia (b) Siberian Federal University, Krasnoyarsk, Russia (c) Institute Of Biochemistry and Physiology of Plants and Microorganisms Ras, Saratov, Russia (d) State Technological University Lb, Lesosibirsk, Krasnoyarsk Region, Russia Marine luminous bacteria are widely used as bioassays for monitoring the environmental toxicity for more than forty years [1]. The effects of toxic compounds on luminous bacteria and their enzyme reactions were intensively studied; this is why the bioluminescence (BL) assay systems are convenient tools for study mechanisms of toxic effects of exogenous compounds, including radioactive ones. In this work, the BL intensity was monitored in solutions of the alpha-emitting radionuclide of high specific activity Am-241 (Am(NO3 )3 , 0.4-6.7 kBq/L) [2,3] and the beta-emitting radionuclide H-3 (HTO, 10-10(5) kBq/L) [4,5] under the conditions of low-dose irradiation. Three BL assay systems were used to study the effects of the radionuclides; the marine bacteria Photobacterium phosphoreum (intact and lyophilized) and coupled enzymatic reactions. Three stages of the bacterial BL response to the radionuclides were found in the BL kinetics; (1) the absence of any effect, (2) activation, and (3) inhibition. The bacterial response was interpreted in terms of the standard reaction of organisms to a stress factor; it includes the following successive stages; (1) stress recognition, (2) adaptive response/syndrome, and (3) suppression of the physiological function. Stage (1) demonstrates the "threshold effect" of ionizing radiation, stage (2) - radiation hormesis, and stage (3) - radiation toxicity. The duration of stages (1) and (2) were compared in solutions of Am-241 and HTO for the lyophilized and intact bacteria. It was found that the damage of the bacterial cells by the lyophilization procedure reduced the stages. Additionally, stages (1) and (2) were shorter in Am-241 solutions than in HTO, revealing higher toxicity of the alpha-irradiation (as compared to the beta-irradiation) under similar doses delivered to the bacteria. In the enzyme system, in contrast to the bacterial ones, the kinetic stages mentioned above were not revealed. Additionally, the role of radiolysis products in biological effects of alpha- and beta-emitting radionuclides was studied. The increase of the peroxide concentrations and the rates of NADH (i.e. endogenous reducer, the component of bacterial bioluminescent enzyme system) oxidation in (241) Am aquatic solutions were demonstrated; these were not found in HTO. The result attributes the biological effects of (241) Am to reactive oxygen species generated in water solutions as secondary products of the radioactive decay. An increase in the content of beta-structured proteins in bacterial cells exposed to HTO was demonstrated by diffuse reflectance FTIR spectroscopic studies. The changes involving the secondary structure components of cellular proteins were interpreted in terms of a stress response of the bacterial cells to the low-dose chronic radioactivity. Similar changes were not found in bacterial cells exposed to Am-241 [6]. Accumulation of Am-241 and H-3 in bacterial cells and in DNA was determined. References 1. Girotti S, Ferri EN, Fumo MG, Maiolini E. Anal Chim Acta 2008 2008;608:2-29 2. Rozhko TV, Kudryasheva NS, Kuznetsov AM, Vydryakova GA, Bondarev, LG, Bolsunovsky AY, Photochem Photobiol. Sci. 2007;6:67-70 3. Alexandrova M, Rozhko T, Vydryakova G, Kudryasheva N. J Environ Radioact 2011;102:407-411. 4. Selivanova MA, Mogilnaya OA, Badun GA, Vydryakova GA, Kuznetsov AM, Kudryasheva NS. J Environ Radioact 2013;130:19-25. 5. Alexandrova MA, Badun GA, Kudryasheva NS. Luminescence 2012;27:95. 6. Kamnev AA, Tugarova AV, Selivanova MA, Tarantilis PA, Polissiou MG, Kudryasheva NS. Spectrochim Acta A; Mol Biomol Spectrosc 2013;100:171-175. O0034 A theoretical study on bacterial bioluminescence Cong Hou(a) , Nicolas Ferré(b) , Ya-Jun Liu(a) (a) College of Chemistry, Beijing Normal Un
iversity, Beijing, China (b) Aix-Marseille Université, Institut de Chimie Radicalaire, Marseille, France Bacterial bioluminescence (BL) has been successfully applied in water-quality monitoring and in vivo imaging. The attention of researchers has been attracted for several decades, but the mechanism of bacterial BL is still largely unknown due to the complexity of the multistep reaction process. Debates mainly focus on three key questions; How is the bioluminophore be produced? What is the exact chemical form of the bioluminophore? How does the protein environment affect the light emission? Via quantum mechanics (QM), combined QM and molecular mechanics (QM/MM) and molecular dynamic (MD) calculations in gas-phase, solvent and protein environments, the current theoretical study investigated the entire process of bacterial BL, from flavin reduction to light emission. This investigation revealed that (1) the chemiluminescent decomposition of flavin peroxyhemiacetal does not occur via intramolecular chemical initiated electron exchange luminescence (CIEEL) or the 'dioxirane' mechanism, as suggested in the literatures. Instead, the decomposition occurs according to our previously proposed mechanism of gradually reversible charge-transfer-initiated luminescence (GRCTIL) for the thermolysis of dioxetanone. (2) The first excited state of 4a-hydroxy-4a,5-dihydroFMN (HFOH) was affirmed to be the bioluminophore of bacterial BL. This study provides details regarding the mechanism by which bacterial BL is produced and is helpful in understanding bacterial BL in general. References 1. Yue L, Liu Y-J, Fang W-H. J. Am. Chem. Soc. 2012;134:11632-11639. 2. McCapra F. Methods Enzymol. s;305:3-47. 3. Hastings JW, Balny C, Peuch CL, Douzou P. Proc. Natl Acad. Sci. 1973;70;3468-3472. 4. Aquilante F, De Vico L, Ferré N, Ghigo G, Malmqvist P-å, Neogrády P, Pedersen TB, Pitoňák M, Reiher M, Roos BO, Serrano-Andrés L, Urban M, Veryazov V, Lindh R. J. Comput. Chem. 2010;31:224-247. 5. Chen S-F, Ferré N, Liu Y-J. Chem. Eur. J. 2013;19:8466-8472. O0035 Comparison between different ATP-related measures of hospital hygiene Arne Lundin, Nadia Touma BioThema AB, Handen, Sweden Introduction Hospital-acquired infections affect more than 10 % of indoor patients in most hospitals and are spread within the patient and between patients, personnel and visitors, directly or indirectly via surfaces and medical devices. This results in huge costs for the society as well as human suffering, especially as multiresistant bacteria are becoming more and more frequent. Clearly tools are required for discovering routes of transmission of infections. Materials and Methods We have developed a new method for the estimation of living bacteria on surfaces by measuring bacterial ATP after degradation of non-bacterial ATP as shown in Fig. . In-house reagents (BioThema, Sweden) were used. Total ATP was measured in the same way but obviating the degradation step. The measurement of ATP + AMP (1) was done using LuciPac Pen and Lumitester PD-20 (Kikkoman, Japan). Each individual test of total ATP and bacterial ATP was calibrated by measuring the light before and after adding a known amount of ATP standard. This compensates for all analytical interferences and allows calculation of ATP in moles (2). Calibration of each individual test is not possible with LuciPac Pen. Instead we added a know amount of ATP standard to unused swabs and measured the light. This does not compensate for analytical interference from e.g. inhibitory detergents on the sampling surfaces, but it allows calculating at least approximate results in moles. Results and Discussion Measurements were done with three ATP related hygiene methods (bacterial ATP, total ATP and ATP + AMP) in five hospitals wards at the Karolinska Hospital at five times. The three methods measure entirely different aspects of hygiene and, as expected, there is no correlation between the three parameters. Median values were calculated for all surfaces sampled over time and over wards. When corrected for the 4 times larger sampling area with ATP + AMP, values for total ATP and bacterial ATP were 120 and 26 %, respectively, of the ATP + AMP value. With ATP + AMP the swab is rounded and wetted with water rather than extractant. This results in a rather poor collection of ATP + AMP from the surface. With total ATP and bacterial ATP we used a swab, which is flat and flexible. Furthermore the extractant is added directly on the surface where it releases all the ATP. This makes it much easier to cover the entire surface and to get a reproducible result. This explains why total ATP gives higher values than ATP + AMP. Bacterial ATP specifically measures living bacterial cells. Total ATP and ATP + AMP differentiate neither between bacterial and human cells nor between living and dead cells. During cleaning strong detergents may kill the cells stabilising the released ATP on the surface. In food industry it is often argued that ATP and ATP + AMP are good indicators of biological contamination. Even if the food contact surface is sterile after cleaning, microbes, e.g. from the air, will start to grow in the time period between cleaning and start of production. This argument is undoubtedly correct in many situations, although there is a trend in food production to use more or less clean-room facilities. In a hospital environment the situation is more complex as one wants to trace the routes of transmission of bacteria. The assay of bacterial ATP should be a complementary tool in these efforts. The method is obviously not limited to hospital environments, but can be used wherever one is interested in hygiene at a bacteriological level. References 1. Sakakibara T, Murakami S, Eisaki N, Nakajima M, Imai K. An Enzymatic Cycling Method Using Pyruvate Orthophosphate Dikinase and Firefly Luciferase for the Simultaneous Determination of ATP and AMP (RNA). Analytical Biochemistry 1999;268:94-101. 2. Lundin A. Use of Firefly Luciferase in ATP-Related Assays of Biomass, Enzymes, and Metabolites. Methods in Enzymology 2000;305:346-370. [Figure: see text] O0036 Bioluminescent properties of hydromedusan Ca(2+) - regulated photoproteins and their semi-synthetic derivatives Natalia Malikova(a,b) , Eugene Vysotski(a,b) (a) Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia (b) Siberian Federal University, Krasnoyarsk, Russia Calcium ion is a ubiquitous intracellular messenger carrying out this function in many eukaryotic signal transduction pathways. To understand the regulation mechanisms of Ca(2+) and how their disturbances are associated with diseases, it is necessary to measure [Ca(2+) ]i . Ca(2+) -regulated photoproteins are successfully used for this purpose for more than four decades. Here, in identical set of in vitro experiments, we characterize five wild-type recombinant photoproteins; aequorin from Aequorea victoria, obelins from Obelia geniculata and Obelia longissima, mitrocomin from Mitrocoma cellularia, and clytin from Clytia gregaria regarding their suitability for the use as a tool for detection of [Ca(2+) ]i . Although photoproteins reveal a high degree of sequences identity and spatial structures [1], and have, apparently, a common mechanism of bioluminescence reaction [2], they differ in affinity to Ca(2+) , sensitivity of bioluminescence to physiological concentration of Mg(2+) , and rates of rise of luminescence signal at a sudden change of [Ca(2+) ], i.e. in properties which are very important at their use for [Ca(2+) ]i measurements. For instance, aequorin shows the highest sensitivity to calcium, whereas clytin is the most insensitive; its affinity is almost one and half order lower than that of aequorin. In contrast to other photoproteins, aequorin affinity to calcium goes down in the presence of 1 mM Mg(2+) , what is more, the effect of Mg(2+) is more pronounced at low [Ca(2+) ], i.e. concentrations corresponding to those within many types of living cells. The rate of rise of light intensity after rapid mixing with Ca(2+) is what limits the ability of the light signal to f
ollow rapid changes in [Ca(2+) ]. For that matter, photoproteins also differ from each other; obelins from O. geniculata and O. longissima show the highest rates of rise of luminescence signal (607 ± 4.6 s(-1) and 510 ± 5.0 s(-1) , respectively) whereas aequorin reveals rate constant equal to 123 ± 0.5 s(-1) . It is obvious that photoprotein with a higher speed of rise will have the advantage in tracking rapid changes in [Ca(2+) ]i . It should be noted that 1 mM Mg(2+) has stronger effect on aequorin as well, the krise constant value decreases almost twice. As it is well known that coelenterazine analogues affect bioluminescence characteristics of photoproteins, we also studied some properties of recombinant obelin from O. longissima and aequorin charged by several novel coelenterazine analogues [3, 4]. Analogues having electron-donating groups on the C6 phenol moiety or an extended resonance system at the C8 position showed a significant red shift of light emission but did not change the affinity to calcium. Thus, currently a suitable photoprotein among available wild-type recombinant photoproteins and coelenterazine analogues depending on the task to be solved can be selected. 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, Ding W, Eremeeva EV, Markova SV, Lee J, Vysotski ES, Liu ZJ. Crystal structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support a catalytic function of a water molecule in the reaction. Acta Crystallogr D Biol Crystallogr 2014; D70; [doi;10.1107/S1399004713032434]. 3. Gealageas R, Malikova NP, Picaud S, Borgdorff AJ, Burakova LP, Brûlet P, Vysotski ES, Dodd RH. Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues. Anal Bioanal Chem 2014; [doi;10.1007/s00216-014-7656-4]. 4. Prolume Ltd. (http;//www.nanolight.com/69_NanoFuels/Luciferase_substrates.html). O0037 First luminescence survey of Okinawa brittle stars Jerome Mallefet(a) , Toshihiko Fujita(b) (a) Marine biology laboratory, Catholic University of Louvain, Louvain-la-Neuve, Belgium (b) National Museum of Nature and Science, Tsukuba-shi, Ibaraki, Japan For the last 10 years, a series of field trips allowed to discover and describe the luminous capabilities of numerous brittle stars [1]. A multidisciplinary approach permitted to obtain physiological, morphological, ethological and finally biochemical data for some brittle stars species mainly because luminous representatives of this class can be found from the intertidal zone downwards [2]. The present project aim to extend the number of brittle stars species tested for their luminous capabilities, in order to understand why so many critters glow in the dark. This work represents the first survey of brittle stars fauna in Okinawan waters (Japan). One hundred twenty specimens were collected mainly by SCUBA (down to 30m depth with the help of researchers and students from Ryukyus University) but we also gain access to deeper zone thanks to the use of a remoted operated vehicle from Churaumi aquarium. A total of ten dives in various biotopes were performed and collected animals were transferred alive to the laboratory to be tested for their luminous capabilities before preservation for further identification (figure ). After a first species screening, a total of 47 different species were identified and out of these (i) 14 + 3 (not yet determined at specific level) responded positively to potassium chloride (KCl 200mM) depolarization by either brief flash or strong long lasting glows; (ii) 9 species produced dubious responses i.e. very brief flash during KCl injection or unrepeatable luminescence, and finally (iii) 21 species showed not sign of luminescence after whole depolarization. Some biochemical data using coelenterazine and vargula luciferin for cross reactions were also performed, results suggest the presence of one luciferin/luciferase reaction based on coelenterazine in Amphiura luetkeni. Since the cosmopolitan species, Amphipholis squamata was present in our sample; this field study allows us to discover at least 13 (+ possibly 3) new luminous brittle stars species to add to the list of luminescent echinoderms [3,4]. . This first survey in Okinawa indicated that many luminous brittle stars species remains unknown and new field works should be planned to complete our knowledge about the fascinating phenomenon of the living light in echinoderms. [Figure: see text] Acknowledgements Special thanks to Prof Hirose who hosted us and provided support at Ryukyus University. JM is research associate of F.R.S.-FNRS (Fonds de la Recherche Scientifique). Contribution to the Biodiversity research center (UCL) and Centre interuniversitaire de la Biologie Marine (Cibim). This work was partially supported by one FNRS grant FC 28302 - 1.5.278.08 F (Belgium) and funding sources from National Museum of Nature and Science (Japan). References 1. Mallefet J. News in Echinoderm's luminescence. Luminescence 2012;27:138. 2. Mallefet J. Echinoderm bioluminescence. In Bioluminescence in Focus - A Collection of Illuminating Essays (Eds. VB Meyer-Rochow), Research Signpost; India, 2009;67-83. 3. Herring PJ. Systematic distribution of bioluminescence in living organisms. J Biolumin Chemilumin. 1987;1(3);147-63. 4. Herring PJ. Bioluminescent echinoderms; Unity of function in diversity of expression? In; Emson RH, Smith AB, Campbell AC, eds. Echinoderm Research. Rotterdam; Balkema AA, 1995;9-17. O0038 Protective effect of deferoxamine, as a potential anti-inflammatory chemical, on ROS-mediated cell and DNA damage in chemiluminescence systems Jalil Mehrzad(a) , Mohammad Javad Chaichi(b) , O. Nazari(b) , A. Ehtesham(b) , M Parvar(b) , S. Hosseinkhani(c) , H. Golchoubian(b) , H.J. Schuberth(d) (a) Ferdowsi University of Mashhad, Mashhad, Iran (b) University of Mazandaran, Babolsar, Iran (c) Tarbiat Modares University, Tehran, Iran (d) University of Veterinary Medicine, Hannover, Germany Inhibition of iron-mediated generation of reactive oxygen species (ROS) by a synthetic chemical might help treat many (non)infectious diseases in humans and animals. Deferoxamine (DFO) would be one of the chemicals of choice for that purpose; as a naturally occurring sideramine, the DFO is specifically used to treat patients with iron-mediated disorders and inflammatory diseases; medical application of DFO is undoubtedly increasing. In the first study, ROS scavenging effect of DFO against superoxide radical (O2 (־•) ), hydrogen peroxide (H2 O2 ) and hydroxyl radical (OH(•) ) was examined by chemiluminescence (CL) assays; effect of DFO on the kinetics of some CL systems was also studied. In the second study, the protective effect of DFO on DNA and neutrophils was investigated using agarose gel electrophoresis and CL systems, respectively; using cellular and acellular CL assays the effects of DFO was assessed on; CL from added OCl(-) , flow cytometry assays of necrosis, apoptosis, CL-based bactericidal capacity of neutrophils and phagocytosis and killing of Escherichia (E.) coli and Staphylococcus (S.) aureus by neutrophils. Briefly, isolated bovine blood neutrophils were exposed with 0 and 0.1 mM of DFO for 1 and 18 h depending on the assay. Further, blood neutrophils were exposed with the DFO for 3 h and phagocytosis and killing activities against S. aureus and E. coli were examined. Our study strongly confirms the scavenging effects of DFO on O2 (־•) , H2 O2 and OH(•) in the luminol and orthophenanthroline CL systems. Maximal quenching capacity of DFO was observed in Fenton's reaction where participation of catalyst, iron ions, to the CL system is central. DFO also strongly protected DNA from ROS-mediated damages. Mechanistically, the observed quenching effect of DFO on ROS clearly revealed the static part of quenching properties of DFO for ROS generation systems. Though the effect of DFO on extracellular ROS greatly
diminished, the DFO-treated neutrophils showed a remarkably increased phagocytosis-depended luminal CL. No neutrophils necrosis and apoptosis was induced by DFO. Phagocytosis rates and killing of E. coli and S. aureus by DFO-treated neutrophils were significantly higher than those of non-treated ones. Our results show the extracellularily anti-oxidant and pro-phagocytic properties of pharmacologically relevant level of DFO for bovine neutrophils. Application of bovine neutrophils as a cellular model for CL system revealed that DFO behaved differently in cellular and acellular CL. The scope of the enhancing effects of the in vitro DFO on neutrophil functions should be considered in mammals as a pharmacologically relevant chemical for clinical implications. O0039 Bioluminescent Magnetotactic Bacteria as powerful Bioanalytical tool for Lab-On-A-Chip analysis Elisa Michelini(a) , Luca Cevenini(a) , Maria Maddalena Calabretta(a) , Sarah Borg(b) , Dirk Schüler(b) , Aldo Roda(a) Elisa Michelini(a) , Luca Cevenini(a) , Maria Maddalena Calabretta(a) , Sarah Borg(b) , Dirk Schüler(b) , Aldo Roda(a) (a) Dept of Chemistry, University of Bologna, Bologna, Italy (b) Ludwig-Maximilians-Universität München, München, Germany The increasing need for rapid, robust and cost-effective toxicity screening systems led to the development of miniaturized analytical devices exploiting the potentiality of genetically engineered living cells for biosensing. The incorporation of living cells within a miniaturized system offers the advantage of small reagent and sample volume requirements combined with the possibility to move the cells in different areas of the chip designed to carry out specific functions. However several critical issues still need to be addressed such as limit of detection, sensitivity and reproducibility [1,2]. In an effort to obtain bioreporters with enhanced analytical performance suitable to chip integration, we produced smart whole-cell biosensors using genetically engineered bioluminescent magnetotactic bacteria (BL-MTB). MTB, which have the ability to produce magnetosome chain, (i.e. magnetite nanoparticles enveloped in a phospholipid membrane) to orient according to an external magnetic field [3], have been genetically engineered to express bioluminescent reporter proteins and used as a whole cell biosensors based on reported gene technology. As first proof of concept Magnetospirillum gryphiswaldense strain was genetically engineered to constitutively express the red-emitting click beetle luciferase (CBR, λmax = 610 nm) and used as general toxicity sensor. A simple microfluidic chip has been fabricated by using multilayered polydimethylsiloxane (PDMS) constituted by three parallel diamond shape incubation chambers connected via microchannels to separate detection areas. BL-MTB were incubated with different cell-toxic compounds (e.g., bile acids, DMSO) and then transferred and magnetically trapped in the detection area which is placed in contact with a CCD sensor [4]. BL images were acquired upon addition of the D-luciferin substrate and light emission inhibition as a result of cell toxicity was evaluated with ImageJ software to obtain quantitative measurements. Despite the analytical performance of BL-MTB is not yet competitive with BL microbial biosensors, our preliminary results represent the first step toward the obtainment of a new class of biosensors based on magnetic bioluminescent bacteria that can be integrated into microfluidic platforms and controlled by external magnetic fields [5]. 1. Michelini E, Cevenini L, Calabretta MM, Spinozzi S, Camborata C, Roda A. Field-deployable whole-cell bioluminescent biosensors; so near and yet so far. Anal Bioanal Chem. 2013 Jul;405(19);6155-63. 2. Roda A, Roda B, Cevenini L, Michelini E, Mezzanotte L, Reschiglian P, Hakkila K, Virta M. Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters. Anal Bioanal Chem. 2011 Jul;401(1);201-11. 3. Schüler D. Int Microbiol. 2002 Dec;5(4);209-14. 4. Roda A, Cevenini L, Michelini E, Branchini B.R. Biosens Bioelectron. 2011,26:3647-53. Bioengineered bioluminescent magnetotactic bacteria as a powerful tool for chip-based whole-cell biosensors. 5. Roda A, Cevenini L, Borg S, Michelini E, Calabretta MM, Schüler D. Lab Chip. 2013 Dec 21;13(24);4881-9. O0040 Performances of on-chip integrated amorphous silicon photodiodes for chemiluminescence and bioluminescence-based assays. Mara Mirasoli(a) , Augusto Nascetti(b) , Martina Zangheri(a) , Domenico Caputo(c) , Riccardo Scipinotti(c) , Giampiero De Cesare(c) , Aldo Roda(a) (a) Laboratory of Analytical and Bioanalytical Chemistry, Alma Mater Studiorum - University of Bologna, Bologna, Italy (b) Department of Astronautics, Electrical and Energy, Sapienza University of Rome, Rome, Italy (c) Department of Information, Electronics and Communication Engineering, Sapienza Univerity of Rome, Rome, Italy Chemiluminescence (CL) and bioluminescence (BL)-based microfluidic chips are being increasingly proposed for clinical, agrofood and environmental analyses [1,2]. Although offering high detectability and specificity, CL reactions generally involve the emission of low light levels, thus requiring highly sensitive detectors for their sensitive measurement. To enable point-of-care (POC) applications, full integration of all the system components in a low-cost microfluidic platform is required. Herein, we describe a microfluidic-based analytical device integrating an array of 16 hydrogenated amorphous silicon (a-Si;H) photodiodes [3] for on-chip detection of CL signals and the evaluation of its analytical performance, as compared with a state-of-the-art cooled slow-scan charge coupled device (CCD)-based acquisition system (Night Owl LB 981, Berthold Technologies). The a-Si;H photodiodes, which are p-doped/intrinsic/n-type stacked structures, were deposited by Plasma Enhanced Chemical Vapour Deposition (PECVD) on a glass substrate covered with an indium tin oxide (ITO) layer. An array of polydimethylsiloxane (PDMS) microwells were bonded to the opposite side of the glass for CL measurements. Device performances were characterized at photosensor level and at analytical level in terms of noise, sensitivity, detectability with different CL and BL systems. Thanks to the optimal design of the system, limits of detection obtained were comparable with those obtained in parallel with the reference luminograph, in particular, 130 amol for alkaline phosphatase (ALP), 30 amol for horseradish peroxidase (HRP), and 3 fmol for firefly luciferase (Luc). In addition, high inter-sensor reproducibility in the array (maximum variability of the measured signal between photosensors was 3%) and low cross talk between photosensors (around 1%) was detected, demonstrating that the geometry design of the array is optimal. Finally, applicability for bioanalytical assays was shown by employing a model CL assay, namely a HRP-based assay for measuring the total antioxidant activity of red grapes must extracts and a cytotoxicity assay employing genetically engineered HEK293 cells expressing Luc. Preliminary experiments performed in microfluidic regimen for a model immunoassay employing HRP proved the ability to implement on-chip assays opening the route toward practical applications outside the lab environment. References 1. Mirasoli M, Guardigli M, Michelini E, Roda A. Recent advancements in chemical luminescence-based lab-on-chip and microfluidic platforms for bioanalysis. J Pharm. Biomed. Anal. 2014;87:36-52. 2. Roda A, Mirasoli M, Dolci LS, Buragina A, Bonvicini F, Simoni P, Guardigli M. Portable device based on chemiluminescence lensless imaging for personalized diagnostics through multiplex bioanalysis. Anal. Chem. 2011;83:3178-3185. 3. Caputo D, de Cesare G, Nascetti A, Negri R, Scipinotti R. Amorphous silicon sensors for single and multicolor detection of biomolecules. IEEE Sens. J. 2007;7:1274-1280 O0041 Chemiluminescence-based biosensor for fumonisins and aflatoxins quantitative detection in maize samples Mara Mirasoli(a) , Martina Zangheri(a) , Laura Anfossi(b) , Fabio Di Nardo(b) ,
Donato Calabria(a) , Cristina Giovannoli(b) , Claudio Baggiani(b) , Aldo Roda(a) (a) Department of Chemistry "G. Ciamician", Alma Mater Studiorum - University of Bologna, Bologna, Italy (b) Department of Chemistry, University of Turin, Turin, Italy Aflatoxins and fumonisins, two mycotoxins mainly found in corn and derived products represent a potential hazard for human health [1], thus maximum residue limits (MRLs) of these mycotoxins in corn-derived foodstuff have been established by the European Union. To enable on-field screening, portable devices providing satisfactory analytical performance are required. In this work a multiplex chemiluminescent (CL) biosensor for simple, rapid and ultrasensititive on-site quantification of Aflatoxin B1 (AfB1) and type B-Fumonisins in maize flour samples is presented. The biosensor integrates an indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed CL detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration [2]. The ready-to-use LFIA strips contain immunoreagents immobilized in specific areas and additional dry reagents that are activated by applying the fluid sample. The use of CL detection improves performances with respect to conventional LFIA, providing quantitative measurements and low limits of detection (LOD) [2, 3, 4]. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30-min assay time. The use of CL detection allowed accurate and objective analytes quantification, enabling to detect simultaneously type B-Fumonisins and AfB1 down to 6 g Kg(-1) and 1.5 g Kg(-1) , respectively, thus fulfilling the standard imposed by the legislation of European Union. Maize flour samples spiked with both Fumonisin B1 and AfB1 were subjected to analysis obtaining recoveries ranging from 79 to 119% and coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 89-120%. The selectivity of the assay was evaluated towards several mycotoxins. Both anti-aflatoxin and anti-fumonisin antibodies showed cross-reactivities below 2% for Zearalenone (ZEA), Deoxynilvalenol (DON), Ochratoxin A (OTA). In addition, the cross-reactivity of anti-aflatoxin B1 antibody towards other aflatoxins was investigated; 38% cross-reactivity was found for aflatoxin G1, while values below 2% were found for aflatoxins B2 and G2. In the future, the portable device will enable the analysis directly on field and, by simultaneously detecting several mycotoxins in one sample, provide multiple information through a single analysis. Furthermore the proposed CL-LFIA protocol is suitable for identifying, within the regulatory limits, samples that require further confirmatory analysis, therefore reducing the overall number of samples subjected to analysis by reference chromatographic assays and thus costs. This allows performing frequent analyses monitoring the entire production chain (e.g., on field, at harvest, during storage and transportation) according with the HACCP procedures. 1. International Agency for Research on Cancer, IARC, 1993;301-366. 2. Mirasoli M, Buragina A, Dolci LS, Simoni P, Anfossi L, Giraudi G, Roda A. Chemiluminescence based biosensor for fumonisins quantitative detection in maize samples. Biosens. Bioelectron. 2012;32:283-287. 3. Cho I-H, Paek E-H, Kim Y-K, Kim J-H, Paek S-H. Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography. Anal. Chim. Acta 2009;632:247-255. 4. Mirasoli M, Buragina A, Dolci LS, Guardigli M, Simoni P, Montoya A, Maiolini E, Girotti S, Roda A. Development of a chemiluminescence based quantitative lateral flow immunoassay for on field detection of 2,4,6trinitrotoluene. Anal. Chim. Acta 2012;721:167-172. O0042 On the role of the C-domain in maintaining the structure of the green emitter in firefly luciferase catalytic reaction Yulia Modestova, Mikhail I. Koksharov, Natalia N. Ugarova Department of Chemistry, Moscow, Russia Firefly luciferases catalyse the bioluminescent reaction of firefly luciferin oxidation. The mechanism of color modulation in this reaction remains unclear. The studies concerning the correlation between the structure of luciferase and the resulting bioluminescent spectra are mostly focused on the N-domain of the enzyme, in which many strong color-shifting mutations were discovered. The role of the C-domain in color modulation is rarely discussed, and very few mutations in this region are known to affect the color of bioluminescence. Recently we reported a mutation of the absolutely conserved C-domain residue E457K (L. mingrelica luciferase) [1]; the mutation reduced the enzymatic activity to ~40% and resulted in a strongly red-shifted bioluminescent spectrum. The possible mechanism behind this effect is of interest because it can reveal the role of the C-domain in maintaining the bioluminescence color. By means of site-directed mutagenesis, we obtained the following set of mutants; E457K, E457V, E457Q, E457D, A534R, and E457V/A534R. The set was design to vary the properties of the side-chain at the position 457 and to introduce an additional hydrogen bond between the residues 457 and 534 (A534R mutant). The TS form of L. mingrelica luciferase with a thermostabilized N-domain was used as a parent enzyme in order to increase the enzyme tolerance for mutagenesis [2]. The enzymes were purified to homogeneity by Ni(2+) -affinity chromatography. Their catalytic properties, thermal stability and bioluminescence spectra at three different temperatures were studied. The properties of the mutants are summarized in the Table. In most cases, the effect of the mutations on the catalytic parameters of the TS enzyme was rather mild. However, the bioluminescent spectra were significantly altered (see Figure O0042;1). The bioluminescent spectra of TS enzyme were bimodal in the temperature range 10-42°C; at 10°C, the emission maximum (λmax ) is in the green region; at 42°C, in the red. The spectra obtained for the E457D and A534R mutants were similar to that of TS, yet the shoulder in the green region is less pronounced. The spectra obtained for the E457Q, E457V and E457V/A534R mutants were strongly red-shifted even at low temperatures; the spectra of the E457K mutant were red-shifted, monomodal and temperature independent. At 42°C the bioluminescent spectra are monomodal for all the mutants; their λmax values were equal within the error limits. We managed to represent the experimental spectra as the superposition of two theoretical spectra, where the λmax(1) of the red spectra was equal to that of the monomodal experimental spectra of the mutants at 42°C; the λmax(2) of the green spectra was also equal for all the mutants within the error limits. The difference between the values of λmax for these spectra was λmax(2) - λmax(1) ~ 30 nm. It implies that there are two forms of the emitter in the system. Our data correspond to the previously obtained fluorescence parameters of keto- and enol/enolate forms of oxyluciferin [3] supporting the theory of keto-enol tautomerism as a possible mechanism for the color modulation in luciferases. The structures of the emitters should be stabilized by two conformations of the enzyme active center that differ by rigidity and polarity. Our data shows that the relative stability of these conformations is controlled by the mutations in the C-domain. The analysis of the computer models of mutated enzymes demonstrate that most of the mutation cause steric hindrances in the enzyme structure, weakening the interactions between the elements of the C-domain. Thus, the intact structure of the C-domain is essential for maintaining the green color of bioluminescence. The results of this study reveal certain aspects of the role of the C-domain in luciferase structure and supports the theory of the oxyluciferin keto-enol tautomerism as one of the color-tu
ning mechanisms in luciferase reaction. This work was supported by the Russian Foundation for Basic Research (grants 08-04-00624 and 11-04-00698). 1. Koksharov MI, Ugarova NN. Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence. Photochem. Photobiol. Sci. 2013;12:2016-27. 2. Koksharov MI, Ugarova NN. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng. Des. Sel. 2011;24:835-44. 3. Leont'eva OV, Vlasova TN, Ugarova NN. Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase. Biochemistry (Moscow) 2006;71:51-55. [Table: see text] [Figure: see text] O0043 Bioluminescence and fluorescence in Obelia species Valerie Jane Morse(a,b) , Kenneth T Wann(a) , Anthony K Campbell(a,b) (a) Cardiff University, Cardiff, UK (b) Darwin Centre for Biology and Medicine, Pembrokeshire, UK Bioluminescent hydroids from the genus Obelia are found commonly in coastal waters worldwide. Light emission is triggered naturally by touch, or artificially by depolarisation of the nerve net or photocytes directly using K(+) , isosmotic with sea water. These cause Ca(2+) to enter the photocyte, which triggers the Ca(2+) -activated photoprotein obelin (1). Obelin emits blue light. However, the intact hydroids emit green light, because the photocytes also contain a green fluorescent protein, which acts as an energy transfer acceptor, and is the actual light emitter. There are four clear species of Obelia. However, in the past, there has been a problem in distinguishing them accurately (2). In fact, there are publications that have misidentified Obelia geniculata for Laomeda flexuosa, which is not bioluminescent, and has no GFP. We have established that the problems of misidentification can be solved by utilising the different fluorescent photocyte patterns of Obelia longissima, Obelia geniculata and Obelia dichotoma. For example, the fluorescent photocytes in Obelia geniculata are distributed randomly along the stem of the coenosarc, and are endodermal. However in Obelia longissima the photocytes are solely at the base of the hydranths, the feeding organ with tentacles. The patterns were found to be different in specimens from the Milford Haven waterway in Pembrokeshire, UK, and the same differences were found in specimens obtained from Plymouth Sound, UK. The fourth species Obelia bidentata was not found in Pembrokeshire or Plymouth (it is known to prefer warmer waters). But we predict that it will have a different photocyte pattern. GFP in live specimens of Obelia was found to photobleach far slower than GFP in EGFP expressed in E. coli (Table ). This supports our hypothesis that Obelia has a molecular mechanism which protects its GFP from photobleaching. The pattern of light emission from photocytes in Obelia geniculata, recorded using the Photek imaging system, was different from that of Obelia longissima (3). This suggests that the mode of entry of Ca(2+) into the photocytes of these two species is different. Morin and Cooke have previously established that K(+) depolarises the membrane of Obelia, leading to a bioluminescent response (4). To investigate the exact pathway by which initial stimulation of Obelia caused Ca(2+) to enter the Obelia photocytes, experiments were conducted using K(+) channel blockers. The potassium ion channel blockers tetraethyl ammonium chloride and 4-aminopyridine both produced a bioluminescent response in Obelia geniculata. This suggested that the pathway included K(+) channels and that some of those channels are "A" type. Fluorescence was recorded in the hydrothecae and tentacles of Obelia dichotoma. FLIM analysis suggested it was GFP. The hydranths of Obelia do not produce bioluminescence; therefore we suggest that GFP in this position might act as a UV protectant (3). [Table: see text] [Figure: see text] References 1. Campbell AK. Extraction, partial purification and properties of obelin, the calcium- activated luminescent protein from the hydroid Obelia geniculata. Biochem. J. 1974;143:411-418. 2 Crowell S. The regression-replacement cycle of hydranths of Obelia and campanularia. Physiol. Zool. 1953;26:319-27. 3. Morse VJ. The regulation and origin of bioluminescence in the hydroid Obelia. Thesis Cardiff University, 2013. 4. Morin J, Cooke MI. Behavioural physiology of the colonial hydroid Obelia;geniculata; stimulus-initiated electrical activity and bioluminescence. J. Exp. Biol. 1971(a);54:707-721. O0044 Bioluminescence lights up science for schools and the public Valerie Morse(a,b) , Anthony Campbell(a,b) (a) Cardiff University, Cardiff, UK (b) Darwin centre for biology and medicine, pembrokeshire, UK Working through the Darwin Centre for Biology and Medicine (1), we have utilised the 'wow' factor revealed by the bioluminescence and fluorescence of local marine organisms, to excite a wide range of age groups to participate in marine science workshops, and carry out investigative projects. Projects have included the circadian rhythm in dinoflagellates, the bioluminescence in brittle stars, and the flashing of hydroids from the genus Obelia. The latter revealed the amazing green fluorescent protein, which has had such an impact on biomedical sciences. Project students learnt a variety of key scientific principles, experimental methods, and developed key skills in STEM subjects (STEM = Science, Technology, Engineering and Maths). By studying the medical applications of bioluminescence and fluorescence, they also gained an insight into cutting edge science. Since 2000, 1228 CREST (Creative Research in Engineering Science and Technology) science awards have been gained by individual students for their project work. CREST is a national school project scheme in the UK. A number of the projects gained European Science week awards (2). Workshop participants also improved their skills in taxonomy and microscopy. A research project on bioluminescent hydroids in Pembrokeshire was linked with Ireland. This included a cross border research seminar. Students assisted with the bioluminescent research, and included it in their personal statements for University. One student has progressed to achieve first class honours in biochemistry, and is now studying bioluminescent bacteria for a PhD. Several students were also captivated by the Photek photon imaging system, and a high-powered fluorescent microscope. Such systems involving ICCD cameras are increasingly featuring in science syllabuses. The investigations were also linked to key skill qualifications. Junior school students carried out projects allowing them to cover the key stage areas of interdependence of organisms, life cycles, binomial naming system and food chains. The projects also contributed towards the movement for 'education outdoors', allowing students to develop fieldwork skills from an early age. Most projects had scientific experts from the University or Further Education sectors acting as mentors. We have also developed a bioluminescent roadshow with 'hands on demonstrations' in a black tent. This has toured Welsh junior schools, and included work on ocean food chains. A senior version, 'How to genetically engineer a rainbow', was presented at the Royal Society summer science exhibition, and the Edinburgh Science Festival. With the support of Milford Haven Port Authority, we have set up a marine research laboratory close to the Milford Haven waterway. Marine ply boards have been installed in the Milford Haven marina, so that samples of bioluminescent Obelia hydroids were easily obtained for use in the laboratory. We have found that marine bioluminescence is an exciting vehicle to stimulate curiosity in studying a range of scientific topics. It can also be used to enhance student's skills, and introduce them to fieldwork. Members of the public, with their families, have also been taken on glow-worm hunts during the season in July. These were preceded by a lecture, showing how curiosity about the wonderful phenomenon of bioluminescence has transf
ormed biomedical research, led to one Nobel Prize, and quite surprisingly created three individual billion dollar markets. [Figure: see text] [Figure: see text] References 1. www.darwincentre.com 2. Pazzagli M, Gelmini S, et al. The Da Vinci-Darwin- Linnaeus Initiative towards a new renaissance for science in Europe. Bioluminescence and Chemiluminescence progress and current applications. Edit Stanley PE, Kricka LJ. World scientific, 2002. O0045 Control of peroxyoxalate chemiluminescence by ligand-metal ion host-guest interaction Jiro Motoyoshiya Shinshu University, Ueda, Japan Due to its practical simplicity and high efficiency, the peroxyoxalate chemiluminescence (PO-CL) is not only used as a chemical light source, but also comprehensively applied to analytical chemistry by means of the signals of the light emission. Since a CIEEL mechanism (chemically induced electron exchange luminescence) was applied to the PO-CL [1], there have been several documents that support its validity. We reported [2] that the addition of the strongly electron-donating nitrogen containing ligands with the dipicolylamino groups effectively quenched the light emission of the PO-CL, whereas the CL turned on in the presence of the metal ions well incorporating with the ligands. Applying the CIEEL to this observation, the ligands can interact electronically with the high-energy intermediates (HEI) instead of the fluorophore to quench the CL, but chelation of the suitable metal ions to the ligands permits the CIEEL between the HEI and the fluorophores (Scheme ). Based on this finding, the PO-CL involving the host-guest chemistry can be applied to a reporter of such the interaction, for example, the metal ion-ligand interaction. [Figure: see text] The structures of the nitrogen-containing ligands used in this study are shown in Fig. 32, which are characterized by the dipicolylamino groups or a monoazacrown ether. The PO-CL using bis(4-chlorophenyl) oxalate, alkaline hydrogen peroxide, and perylene as a fluorophore was effectively quenched, when these ligands (500 eq. to the oxalate) were added to this PO-CL system except for 2b and 3 with the poor electron-donating ability (vide infra). [Figure: see text] Contrary, the distinct CL was observed when the CL reactions were carried out in the presence of the suitable metal ions. Namely, the combination of Zn(2+) and 1a, 4, or 5 caused the CL, but no significant emission was observed in the presence of other metal ions (Li(+) , Na(+) , K(+) , Mg(2+) , Ca(2+) , Ni(2+) , Fe(2+) , Fe(3+) , Pd(2+) , Ag(+) , and Al(3+) ). 1a was also found to form the complex with Cu(2+) by CL turning on. The marked effect was conducted for the combination of 5 and Ag(+) , which was known by the other method [3], but 5 was newly found to capture Zn(2+) by this chemiluminescent method. These results are dependent on compatibility of the ligands and metal ions, i.e., ionic radius, charge on the metal ion, etc. It is worthy to note that the CL quenching effect of the selected ligands is closely related to their oxidation potentials, and the ligands behave like the quenchers by the electronic interaction. Thus, the formation of the complex prevents the electronic interaction between the HEI and the ligands but enables the CIEEL between the HEI and the fluorophores leading to CL, which we would like to call the CIEEL switching. Thus, chelation of these ligands with the suitable metal ions was readily detected by this method, the PO-CL involving the host-guest chemistry. Consequently, this chemiluminescent method based on the CIEEL of the PO-CL will be useful as a reporter of the host-guest interaction of unknown combination. References 1. Stevani CV, Silva SM, Baader WJ. Eur. J. Org. Chem. 2000;24:4037-4046. 2. Maruyama T, Fujie Y, Oya N, Hosaka E, Kanazawa A, Tanaka D, Hattori Y, Motoyoshiya J. Tetrahedron 2011;67:6927-6933. 3. Thaler A, Bergter R, Ossowski T, Cox BG, Schneider H. Inorg. Chim. Acta. 1999;285:1-9. O0046 Three-dimensional structures of mutants of Ca(2+) -regulated photoprotein obelin provide insight into molecular mechanisms underlying changes in bioluminescent properties Pavel Natashin(a,c) , Eugene Vysotski(a,c) , Zhi-Jie Liu(b,d) (a) Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia (b) National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China (c) Laboratory of Bioluminescence Biotechnology, Chair of Biophysics, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, Russia (d) iHuman Institute, Shanghai Tech University, Shanghai, China The substitutions of amino acid residues situated in a substrate-binding cavity of Ca(2+) -regulated photoproteins in hydrogen-bond distances from atoms of 2-hydroperoxycoelenterazine significantly affect the bioluminescent properties. To elucidate the molecular mechanism underlying these effects, the crystal structures of two obelin mutants with substitutions of Tyr138 to Phe and Phe88 to Tyr in two conformational states before bioluminescent reaction, i.e. bound with 2-hydroperoxycoelenterazine, and after reaction, i.e. bound with coelenteramide and Ca(2+) , were determined. The comparison of the determined spatial structures with those of the wild-type obelin in corresponding conformation states clearly evidences that mutations have no influence on the overall structures but lead to local changes affecting bioluminescence properties. For instance, from three-dimensional structures of Y138F obelin mutant we conclude that the slowdown of bioluminescence kinetics of this mutant is caused by the lack of water molecule in the internal cavity near Phe138. This water molecule appears in the cavity of obelin Y138F through the solvent exposed opening, and the bioluminescence reaction takes place, but with a slow rate.(1) The substitution of Phe88 to Tyr renders the bioluminescence spectrum of F88Y obelin towards the spectral properties of aequorin. We compare the hydrogen-bond network formed by 2-hydroperoxycoelenterazine and coelenteramide with the amino acid residues facing into the ligand-binding cavity in obelin F88Y with those of wild-type obelin and aequorin and conclude that the main reason of different bioluminescence colors of obelin and aequorin is a different arrangement of the hydrogen-bond network near the 6-(p-hydroxyphenyl) group of coelenterazine due to the presence of Phe or Tyr residue.(2) This work was supported by RFBR grants 12-04-91153, 12-04-00131 and the China-Russia International Collaboration grant from the Chinese Academy of Sciences and NSFC, by the Programs of the Government of the Russian Federation 'Measures to Attract Leading Scientists to Russian Educational Institutions' (grant 11.G34.31.0058) and 'Molecular and Cellular Biology' of the RAS, the President of the Russian Federation 'Leading Science School' (grant 3951.2012.4). PVN was supported by RFBR grant 14-04-31092. References 1. Natashin PV, Ding W, Eremeeva EV, Markova SV, Lee J, Vysotski ES, Liu ZJ. Crystal structures of the Ca(2+) -regulated photoprotein obelin Y138F mutant before and after bioluminescence support a catalytic function of a water molecule in the reaction. Acta Crystallogr D. 2014;D70:720-732. 2. Natashin PV, Markova SV, Lee J, Vysotski ES, Liu ZJ. Crystal structures of F88Y obelin mutant before and after the bioluminescence reveal molecular insight into spectral tuning among hydromedusan photoproteins. FEBS J. 2014;281:1432-1445. O0047 Keto or Enol-That is the Question; Recent Progress in Spectra-Structure Correlations of Firefly Oxyluciferin and its Derivatives Pance Naumov(a) , Kyril M. Solntsev(b) , Sergey Laptenok(c,d) , Mateusz Rebarz(e) , Boris-Marko Kukovec(a) , Oleg V. Maltsev(f) , Cyril Ruckebusch(e) , Michel Sliwa(e) , Lukas Hintermann(f) (a) New York University Abu Dhabi, Abu Dhabi, United Arab Emirates (b) Georgia Institute of Technology, Atlanta, USA (c) CNRS, Ecole Polytechnique, Palaiseau, France (d) INSERM U696, Palaiseau, France (e) Universite Lille 1, Lille, France (f) Technische Universität München, Munich, Germa
ny Despite being of critical relevance for the bioanalytical performance, two key aspects of the firefly bioluminescence (BL) have long remained a subject of speculation and dispute. First, the exact chemical identity of oxyluciferin (OxyLH2 ) complexed with the luciferase (Luc) at various stages of the BL reaction sequence and at various conditions has not been determined yet. A second thoroughly debated issue is the molecular origin of natural or artificially (point-mutation) induced tuning of the emission energy. [Figure: see text] In this presentation, the most recent results from our steady-state([1-5]) and time-resolved([6]) spectroscopic and structural studies of OxyLH2 and its derivatives will be described. These results provide direct insight into the structure and fate of different forms of the emitter in solvated form and when complexed with Luc. By studying the photochemical behavior of several synthetic derivatives (Figure), we have assessed the effects of its functional groups and support the assignments by two non-natural fluorophores. The natural emitter OxyLH2 has been studied intensively, and it behaves as a typical photoacid with an estimated pKa * of -0.5. Despite the common belief that the hydroxyl of the thiazoline-substituted phenolic group serves as a source of photogenerated protons, we were very surprised to observe excited-state proton transfer (ESPT) from HOxy, which has only one enolic hydroxyl group. Moreover, the photoacidity of HOxy was stronger than of the parent OxyLH2 . Usually the HO-photoacids are based on the phenolic compounds, and the examples of non-traditional photoacids are extremely rare. We are tempted to conclude that the ESPT reactivity associated with the phenol photodissociation was misinterpreted. [Figure: see text] Based on these observations, we interpreted the photoiduced behavior of OxyLH2 in water and in the complex with Luc as a complex ESPT cascade. Depending on pH, the emitting species included keto- and enol tautomers in various protonation steps. Our spectral assignments could be directly utilized in the analysis of the multiband Luc chemiluminescence spectra. O0048 DFT study of chemiluminescence and fluorescence of coumaranone derivatives Isabelle Navizet Laboratoire MSME UMR 8208 CNRS Université Paris-Est Marne-la-Vallée, 5 bd Descartes, 77454 Marne-la-Vallée Cedex 2, France Experimental study of chemi and fluorescence of coumaranone derivatives has shown that the colours emitted in the two processes are different [1]. The chemiluminescence results from the leaving of CO2 leading to a carbonyl group (see figure). This reaction can be put in parallel of the last reaction in the bioluminescence systems. Theoretical studies in bioluminescent model have already shown that chemi and fluorescence can results to emission of different wavelength [2]. These two results presented in the last ISBC in Guelf have lead to a theoretical study in order to understand the reason of this discrepancy between chemiluminescence and fluorescence in the case of derivatives of coumaranone. In the present study, DFT calculations of the different conformation isomers of the reactants and the products can rationalize this difference in the derivatives of coumaranone. In order to take into account the solvent, calculations has been done in vacuo and in THF. Linear response has been used to optimize the first excited state in PCM and the electronic transitions have been calculated with a state specific treatment. The conformations due to the values of the dihedral angle between the different part of the molecules explain the difference of the wavelength in chemi and fluorescence experiments. [Figure: see text] 1. Schramm Stefan, Weiss Dieter, Navizet Isabelle, Roca-Sanjuán Daniel, Brandl Herbert, Beckert Rainer, Goerls Helmar. Investigations on the Synthesis and Chemiluminescence of novel 2-Coumaranones. ARKIVOC 2013;(iii);174-188. 2. Roca-Sanjuán Daniel, Delcey Mickael, Navizet Isabelle, Ferré Nicolas, Liu Ya-Jun, Lindh Roland. Chemiluminescence and fluorescence states of a small model for coelenteramide and Cypridina oxyluciferin; A CASSCF/CASPT2 study Journal of Chemical Theory and Computation 2011;7(12);4060-4069 O0049 Chemical scission of albumins, peptides and amino acids and accompanying chemiluminescence after peroxidation with hydrogen peroxide Osamu Nozaki(a) , Motohiro Shizuma(b) (a) Kinki University School of Medicine, Osaka-Sayama, Osaka, Japan (b) Osaka municipal technical research institute, Osaka, Osaka, Japan [Introduction] A novel method of cleaving albumins, peptides, amino acids was investigated in this study. Albumins, peptides and amino acids are important constituents of human body, are altered in disease, and also can be used as therapeutic agents. Therefore, methods for rapid assay and cleaving albumins, peptides and amino acids are required. A method using gamma ray and singlet oxygen for peroxidation of albumins, peptides and amino acids has been reported(1,2)) , but is not popular. The methods of cleaving proteins employing heating with acid and digesting enzymes(3)) do not reveal the course of the process. In this study, a method of chemical scission of albumin, peptide, and amino acid and the accompanying chemiluminescence was investigated. For this purpose, a novel method of peroxidation of albumins, peptides, and amino acids with hydrogen peroxide at ambient temperature was developed. [Materials and Methods] Materials used were proteins (human serum albumin (HSA), bovine serum albumin (BSA), peptides (insulin, alanyl-histidine, alanyl-glutamine, alanyl-proline), amino acids (alanine, N-acetyl-alanine, aspartic acid, lysine, tryptophan, histidine, et. al.). Peroxidation of albumins, peptides, and amino acids were carried out as follows; 0.1 mg/ mL of albumins, peptides and amino acids were mixed with hydrogen peroxide (H2 O2 ), and incubated for two min. at room temperature. Then, 66 % ethanol in the Tricine solution (pH 12.5) was added, and incubated for 10 min at ambient temperature. For removing of H2 O2 from the peroxidation reaction solution, potassium iodide were added and incubated for 5 min at ambient temperature.The peroxy albumin, peptide, and amino acids were determined by chemiluminescence. The peroxy albumin (HSA, BSA), insulin, amino acids were oxidized with potassium ferricyanide (Fe(III)) to emit light. The light intensity and kinetics of light emission were monitored for 5 min at ambient temperature with a chemiluminometer. Peroxy insulin and its fragments were determined by MALDI-TOF MS in the positive ion mode. The peroxy insulin solution and its reactants from the chemiluminescence reaction were extracted with hydrophobic gels, washed, and eluted to desalt. The eluants were used as specimens for mass spectrometry. [Results] Different kinds of solvent for dissolving BSA were investigated. The mixture of dimethyl sulfoxide (DMSO) and ethyl alcohol (9;1, v/v) and 66.7 % ethanol in the Tricine solution (pH 12.5) were examined for producing peroxy BSA. Based on the the results, a mixture of DMSO and ethanol was superior to the 66.7 % ethanol in the Tricine solution for producing both signal and noise. On the other hand, the ethanol-Tricine solution was superior to the mixture of DMSO and ethanol for the ratio of signal to noise. Different kinds of solvents for dissolving H2 O2 were investigated for stability by means of a time course study. Water, ethanol and 1-propanol showed good results for stability of H2 O2 activity. Ferricyanide (Fe(III)), hemin, hematin, hemoglobin (Hb), horseradish peroxidase (HRP) were investigated as oxidants for peroxy BSA. The order of difference of light intensity of peroxy BSA and H2 O2 with the oxidants was HRP > Hb > hematin > hemin > Fe(III). The order of signal to noise ratio was Fe(III) > hematin > hematin = Hb = HRP. The optimal concentration of ethanol in the Tricine solution (pH 12.5) was investigated for producing peroxidized albumin and found to be 66.7 %. This provided maximum light intensity from both HSA and BSA. The reaction time of BSA and H2 O2 for pro
ducing maximum amount of peroxy BSA was 10 min after start of the reaction. The conditions for producing peroxy albumins were investigated. The peroxidization reaction was started by addition of the 66.7 % ethanol-Tricine solution to the mixture of BSA and H2 O2 . The method of removing free H2 O2 from the reaction solution after finishing the peroxidizing reaction was studied. Addition of potassium iodide (KI) was effective to keep light intensity of peroxy BSA constant for one hour. When no KI was added, the light intensity of the reaction solution decreased over time. Addition of potassium ferrocyanide(Fe(II)) showed poor result of lower light intensity than the blank signal. The peroxy albumins, peptides, and amino acids were investigated. The light intensities from the peroxy HSA, BSA, insulin, alanine, aspartic acid, lysine, histidine of the same mass (weight), showed the same light intensity. Those modified with o-phthalaldehyde (OPA) emitted higher light intensity than the light from the peroxides with no OPA modification. Correlation between concentrations of HSA, BSA, alanine, glutamic acid, lysine, histidine and the light intensities was investigated. The light intensity changed according to the concentrations of the specimens. When the concentrations were between 0.001 and 0.1 mg/mL, light intensity increased in relation to the concentrations. When the concentration was over 0.1 mg/mL, the light intensity of each specimen decreased. The calibration curve of the albumins was investigated. BSA and HSA were quantified when BSA was in concentration of between 1 ug/mL to 1 mg/mL, and HSA in concentration of 200 ng/mL to 0.2 mg/mL. Certification of chemical scission of insulin was investigated. Mass spectrometry of the peroxy insulin solution showed the peak of molecular weight of 5816. The peaks of smaller seized fragments (1700 - 3500 molecular weight) appeared as shown by MALDI-TOF MS of the solution obtained after the chemiluminescent reaction of the peroxy insulin. [Discussion] This study developed a novel method for peroxidation of albumins, peptides, amino acids with hydrogen peroxide instead of singlet oxygen. It enabled a 10-minute peroxidation reaction at room temperature. Production of peroxide was identified by chemiluminescence with different oxidants - Fe(III), hemin, hematin, Hb, HRP. The site of peroxidation of albumins, peptides, amino acids was speculated to be the alpha carbon. The reason was as follows. light emission occurred from not only histidine, tryptophan (imidazole base), but also other amino acids. This speculation coincided to the report on peroxidation with singlet oxygen. The chemical scission of insulin and light emission from insulin occurred simultaneously. This method allowed more rapid destruction of albumins with more mild conditions in comparison with the other methods of digesting proteins. This method, also, enabled observation of the process of protein destruction by monitoring its chemiluminescence. [Conclusion] This study developed a novel method of chemical scission of albumin, peptide, and amino acids that was accompanied by light emission. Albumins, peptides, amino acids were peroxided with hydrogen peroxide. The organic peroxides decomposed with light emission. [References] 1) Morgan P. E., Pattison D. I., Hawkins C. L., et al. Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins. Free Radic. Biol. Med. 2008; 45; 1279 - 89. 2) Alarcon E., Henriquez C., Aspee A., et. al. Chemiluminescence associated with singlet oxygen reactions with amino acids, peptides and proteins. Photochem. Photobiol. 2007; 83; 475-80. 3) Chowdhury SM1, Munske GR, Yang J, Zhukova D, Nguyen H, Bruce JE..Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry. Rapid Commun Mass Spectrom. 2014 Mar 30;28(6);635-44. O0050 Luciferase isotype involving in the dim glow of pupae and eggs in the Japanese firefly, Luciola lateralis Yuichi Oba(a) , Mana Furuhashi(a) , Manabu Bessho(a) , Shingo Sagawa(a) , Haruyoshi Ikeya(b) , Satoshi Inouye(c) (a) Nagoya University, Nagoya, Japan (b) Toin Gakuen High School, Yokohama, Japan (c) JNC Corporation, Yokohama, Japan We isolated cDNA for an isotype of luciferase, named LlLuc2, from the Japanese firefly, Luciola lateralis. The gene product showed approximately 60% amino acid identity to LlLuc1, which was isolated from adult lantern of L. lateralis, previously. The light emission maxima of recombinant LlLuc1 and LlLuc2 were 550 and 539 nm, respectively. RT-PCR analyses indicated that LlLuc1 was expressed predominantly in larval and adult lanterns, in contrast, LlLuc2 was expressed in eggs and pupae. The luminescence spectrum of recombinant LlLuc1 was in good agreement with in vivo luminescence of larval and adult lantern, and that of LlLuc2 was also in good agreement with in vivo luminescence of eggs and pupae (Figure). In addition, a homologous gene of LlLuc2, named LcLuc2, was isolated from the Japanese firefly, Luciola cruciata, and was also expressed in eggs and pupae. These results suggested that two functional luciferases are present and the gene duplication of luciferase occurred before divergence of the family Lampyridae. Further, one luciferase is used for the luminescence reaction in eggs and pupae and the other is used for the luminescence reaction in larval and adult lanterns. O0051 Bioluminescent cell multiplex assay for chemical library screening Guillaume Octobre, Ophélie Berthuy, Christophe Marquette, Loïc Blum Université Claude Bernard Lyon1 - UMR5246 - GEMBAS, Villeurbanne, France Analyzing multiple cell lines in parallel for screening purposes is a promising approach in which different cell lines are studied in the exact same conditions of induction, nutrition and kinetic. The present study aims to develop a cell biochip providing a tool for various therapeutic applications, particularly the screening of chemical compound libraries for potentially active molecules in cancer therapy. The system will exhibit the following features; Engineered surfaces allowing the localized and controlled culture of multiple cell populations (up to 64 different cell lines); real multiplex cell culture, Engineered multiple cell lines incorporating different promoters regulating the expression of a common bioluminescent gene; an activity screening tool, Encapsulation of these cell populations in biocompatible hydrogel beads (alginate), providing a low analysis volume (10-20 nL) of controlled, stable and homogenous composition; enhanced induction signal, Optical coupling between a dedicated silicon photomultiplier (SiPM) array (8x8 pixels) and the cell array allowing the continuous detection of secreted bioluminescent reporter molecules activity; real time kinetic, Imaging system for cell observation and control; cell viability control. In this framework, the cells will incorporate a reporter vector coding for the Gaussia princeps luciferase gene under the regulation of a promoter of interest. The induction will be performed by a chemical or a biochemical compound. The intensity of the promoter response will then be proportional to the intensity of the luciferase bioluminescent signal detected by the sensitive layer composed of an 8x8 array of single photon sensitive devices (silicon photomultipliers). [Figure: see text] [Figure: see text] O0052 Study on the periodicity of fungal bioluminescence Anderson Oliveira(a) , Jillian Emerson(d) , Chandru Mallappa(d) , Vadim Viviani(c) , Cassius Stevani(b) , Jennifer Loros(d) , Jay Dunlap(d) (a) Federal University of Sao Paulo, Sao Jose dos Campos, Sao Paulo, Brazil (b) University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil (c) Federal University of Sao Carlos, Sorocaba, Sao Paulo, Brazil (d) Dartmouth College, Hanover, New Hampshire, USA Circadian clocks are biological oscillators responsible for maintaining the internal rhythm of animals, plants, fungi and even some bacteria to the alternation of external stimuli as light and temperature (1). Interestingly, bioluminescence an
d chronobiology share some historical events. Light emission in Lingulodinium polyedrum (formerly Gonyaulax polyedra), for instance, is regulated by a cellular circadian biological clock, making the light intensity higher at night (2). Nevertheless, the regulation of light emission in fungi by circadian rhythm, whose study initiated at the same time as L. polyedra, has never been formerly proved. We present here experimental results that support the regulation of fungal bioluminescence by a circadian rhythm, using the bioluminescent fungus Neonothopanus gardneri as model (3,4). In addition, we hypothesize that the observed clock-controlled luminescence would confer some biological and ecological advantages to bioluminescent fungi. Freshly inoculated agar plates with N. gardneri mycelium were grown over 48h in a 12h/12h dark/light cycle under constant temperature (25 ºC) in an incubator equipped with a CCD camera. After the initial induction period of 48h, the light/dark cycle was interrupted and the cultures were maintained at total darkness during 6 days. The development of N. gardneri cultures during this interval was photographed every hour. Picture analyses by imaging software indicate that the light emission from the bioluminescent mycelium oscillates ca. 22h at 25ºC, thus suggesting a circadian rhythm. The value obtained is in agreement with those reported for Neurospora crassa, a non-luminescent ascomycete. N. crassa has a long history as genetic model and presents periodicity of ca. 22.5h in constant conditions (1). Moreover, the fungal circadian clock seems to be very consistent, as the cycles will also persist once a day in the absence of any periodic stimulus. Fungi, unlike fireflies, cannot neurologically control their own light emission. Hence, they could save a great amount of energy during the day by either lowering or even turning off the emission of light. It is important to mention that fungi emit a dim glow that is not visually perceptible under the daylight. Over the night the light intensity could be reestablished making them brighter, helping these fungi to be more attractive to fungivores or to the predators of these latter, and consequently increasing spore dispersal or predation of fungivores, hypotheses postulated to explain the ecological functions of fungal bioluminescence in Nature (5,6). References 1. Baker CL, Loros JJ, Dunlap JC. The circadian clock of Neurospora crassa. FEMS Microbiol. Rev. 2012;36:95-110. 2. Morse D, Fritz L, Hastings JW. What is the clock? Translational regulation of circadian bioluminescence. Trends in biochem. 1990;15:262-265. 3. Desjardin DE, Oliveira AG, Stevani CV. Fungi bioluminescence revisited. Photochem. Photobiol. Sci. 2008;7:170-182. 4. Berliner MD. Diurnal periodicity of luminescence in three basidiomycetes. Science. 1961;134:740. 5. Waldenmaier HE, Oliveira AG, Stevani CV. Thoughts on the diversity of convergent evolution of bioluminescence on earth. Int. J. Astrobiol. 2012;1:1-9. 6. Sivinski J. Arthropods attracted to luminous fungi. Psyche.1981;88:383-390. O0053 Effect of DMSO on fluorescence properties of Ca(2+) -discharged photoprotein obelin Alena Petrova(a) , Nadezhda Belogurova(b) , Rosa Alieva(a) , Nadezhda Kudryasheva(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Calcium-regulated bioluminescent reactions catalyzed by photoproteins are responsible for bioluminescence of marine coelenterates. Photoprotein is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen "pre-activated" substrate, 2-hydroperoxycoelenterazine which is tightly but noncovalently bound within a hydrophobic cavity inside the protein. Addition of calcium ions to photoprotein triggers a bioluminescent reaction resulting in light emission with λmax = 485 nm. The product of the bioluminescent reaction (enzyme-bound chromophore, coelenteramide) is a fluorescent protein; It is called 'discharged' photoprotein. Obelin isolated from hydroid Obelia longissima is one of the most studied among photoproteins. Obelin is stable and nontoxic natural complex; its spectra, and hence, color of luminescence, are variable. This is why it is considered as perspective bioluminescent and fluorescent marker [1] for biological and medical investigations in vitro and in vivo. Therefore, it is important to study effects of different physical and chemical factors on the spectral properties and stability of the photoprotein. We showed previously [2, 3] that combination of high-temperature exposure and variation of excitation wavelength can change emission spectra of Ca(2+) -independent discharged obelin. In papers [4] and [5] we demonstrated the dependence of obelin spectral characteristics on concentration of calcium ions and alcohols (glycerol and ethanol). DMSO is widely used as biomedical agent in cryobiology and medicine. In this work, we studied the effect of DMSO (C = 0.002 - 2.65 М) on intensity and color of light-induced fluorescence of Ca(2+) -discharged obelin. The spectra were measured at 280 and 350 nm excitation wavelengths, corresponding to tryptophan and polypeptide-bound coelenteramide absorption maxima, respectively. It was found, that the Increase of DMSO concentration causes a decrease of obelin luminescence intensity. All emission spectra were deconvolved into components using Gauss-based distribution and method of second derivative. It was found that the emission spectra (λexc = 350 nm) of the discharged obelin were a superposition of three components. The spectral components were attributed to different fluorescent forms of coelenteramide; protonated, partly protonated and deprotonated forms with maxima at 417, 504 and 566 nm, respectively. Addition of DMSO increases the contribution of protonated form and reduces the contribution of partly protonated form. Therefore, the increase of DMSO concentration affects the process of proton transfer from coelenteramide to a proton-acceptor environment. Emission spectra under 280 nm excitation involved the additional ultraviolet (λmax = 345 nm) and red (λmax = 660 nm) peaks, corresponding to fluorescence of tryptophan and hypothetical indole-coelenteramide exciplex, respectively. Fluorescent intensities and contributions of the components to experimental obelin spectra at λexc = 280 nm changed under addition of the DMSO; the DMSO increased contributions of ultraviolet, violet, and red components, while blue and green components decreased. This fact should be taken into account using discharged obelin as a fluorescent marker in medical and biological investigations. References 1. Frank LA. Sensors 2010;10:11287-11300. 2. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem 2013;405:3351-3358. 3. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. LUMINESCENCE 2012;27:96. 4. Belogurova NV, Kudryasheva NS. J Photochem Photobiol B 2010;101:103-108. 5. sAlieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem DOI; 10.1007/s00216-014-7685-z. O0054 The Photoacidity of Oxyluciferin; Excited State Processes Behind Firefly Bioluminescence Luís Pinto da Silva, Joaquim C.G. Esteves da Silva Centro de Investigação em Química (CIQ-UP), Faculty of Sciences of University of Porto, Porto, Portugal The firefly oxyluciferin family of fluorophores has been receiving a great deal of attention from the research community, due to the role played in the firefly bioluminescence system. Given the significant potential of this latter system for use in many practical applications, several systematic studies have been performed regarding the photophysical properties and photoprotolytic cycles of these fluorophores. Such studies have revealed that these molecules are strong photoacids.(1) Typical photoacids are weak acids in the ground state with pKa of 5-10. Upon photoexcitation, the pKa constant decrease by ~7 pH values, due to the ability of these molecules to donate a proton to a solvent molecule in the excited state.(1) Study of firefly oxyluciferin, dehydroluciferin and luciferin revealed many interesting, c
omplex and unexpected photoinduced phenomena.(1-6) Thus, in this communication we intend to present the photoacidity characterization of these molecules, performed by using a combination of experimental and computational methodologies. Steady-state and time-resolved fluorescence measurements have revealed that upon photoexcitation this family of photoacids transfer a proton to a solvent molecule, via Excited State Proton Transfer (ESPT).(1) The ESPT rate of oxyluciferin in water was found to be 2.2x10(10) s(-1) , which is lower than that found for luciferin (3.2x10(10) s(-1) ) but higher than the one found for dehydroluciferin (1.1x10(10) s(-1) ).(1,3,4) Further time-resolved analysis revealed a very efficient fluorescence quenching process for these molecules, due to irreversible proton geminate recombination.(1,3,4) Theoretical calculations indicate that the quenching is mainly due to excited state protonation of the nitrogen heteroatom of the benzothiazole moiety, which leads to a singlet to triplet intersystem crossing.(5) Studies of firefly oxyluciferin in solution showed that this molecule present two specific excited state phenomena; pH-sensitive fluorescence and photoinduced base-catalyzed tautomerism reaction.(2,6) In water, light is emitted from the enolate species at all pH values. The pH-sensitivity arises from the formation of ground state π-π stacking enolate complexes.(6) Upon photoexcitation, these complexes present different conformations at acidic and basic pH, thus accounting for the different emission wavelength.(6) It was also found that the keto form of oxyluciferin can be converted into an enol species via base-catalyzed ESPT.(2) With the obtained data we are now able to present a model for the photoprotolytic cycle of the oxyluciferin family of photoacids (Figure 38). Moreover, we intend to correlate the obtained information with the mechanisms behind firefly bioluminescence, as possible excited state decay paths. More important is the finding of the strong photoacidity of the enol group of oxyluciferin and its photoinduced tautomerism. These features once again indicate that the keto-enol tautomerism of oxyluciferin may be of importance for the firefly bioluminescence system. [Figure: see text] References 1. Pinto da Silva L, Simkovitch R, Huppert D, Esteves da Silva JCG. ChemPhysChem 2013;14:3441. 2. Solntsev KM, Laptenok SP, Naumov P. J. Am. Chem. Soc. 2012;134:16452. 3. Erez Y, Presiado I, Gepshtein R, Pinto da Silva L, Esteves da Silva JCG. J. Phys. Chem. A 2012;116:7452. 4. Presiado I, Erez Y, Simkovitch R, Shomer S, Gepshtein R, Pinto da Silva L, Esteves da Silva JCG. J. Phys. Chem. A 2012;116:10770. 5. Pinto da Silva L, Simkovitch R, Huppert D, Esteves da Silva JCG. J. Photochem. Photobiol. A 2013;266:47. 6. Pinto da Silva L, Simkovitch R, Huppert D, Esteves da Silva JCG. ChemPhysChem 2013;14:2711. O0055 Ultrastructure and morphology of the bioluminescent system in "bioluminescent" and "non-bioluminescent" scale-worms (Polychaeta, Polynoidae). N.B. Aneli(a) , A.A. Belousova(b) , V.B Saprunova(c) , V.N. Petushkov(d) , F.A. Kondrashov(e) , M.V. Plyuscheva(e) (1) Experimental Factory for Scientific Engineering of the Russian Academy of Sciences (EZAN), Chernogolovka, Moscow region, Russia, (2) Moscow State University, Biological Faculty, Invertebrate Zoology Departement, Moscow, Russia, (3) A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia, (4) Laboratory of Photobiology, Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk, Russia, (5) Centre de Regulació Genòmica (CRG), Barcelona, Spain For the first time Quatrefages described bioluminescence of scale worms in 1843, but the best and most full description has been given by Panceri (1875) who studied Malmgreniella lunulata (Delle Chiaje, 1830) (old name Polynoe lunulata), and Subadyte pellucida (Ehlers, 1864) (old name Pholoe brevicornis Panceri, 1875). Bioluminescence is restricted to the scales, a pair to a segment, which cower the dorsal surface of the worm in two imbricated rows, like shingles on a roof. Panceri noted that the luminous region might cover the entire scale (except for point of attachment) or a half-moon-shaped area of light, depending on the species. The initial idea about the morphology of bioluminescent system and photogenic cells must be attributed to Quatrefages (1843) who erroneously thought muscles were the source of light. Second idea came from Panceri (1875), who mentioned superabundance of nerve fibres in the elytra and decided that nerve endings might be the source of light. Jourdan in 1885 prepared serial sections of scales and suggested the role of photogenic cells to modified ventral epithelial cells that was looking like secretary cells. In 1909 Kutschera, working on Acholoe astericola (Delle Chiaje, 1841) in which the light beginning in the half-moon-shaped region and spreading over all the scale, concluded that light organs are presented by the cuticular papillae and gland cells, which are supposed to secret a luminous material into the sea water through a canal and pore. In addition to the luminous papillae there were tooth-like projections with few underlying cells on the anterior margin of the scale. Their function was not determined but presumably was sensory. Dalhgren (1916) figured the tooth-like projections and the papillae on Lepidonotus squamatus (Linnaeus, 1758) scales and was inclined to interpret light production, as did Kutschera. Newton Harvey was examined Acholoe astericola in ultraviolet light and observed brightly yellowish fluorescence over the scales after stimulation of bioluminescence, while the non-bioluminescent Lepidonotus clava (Montagu, 1808),show only a bluish fluorescence in the skeletal parts under ultraviolate light, both in the upper and lower sides of the elytra (Harvey, 1926). Next studies were done by Bonhomme in 1940 and 1942 on Harmothoe impar with a large fluorescent crescent on each scale and Malmgreniella lunulata with a very small crescent. He designated as a photogenic area fluorescent group of cells on the ventral surface of the scale, called them "photocytes". These were cells with rather large elliptical nuclei and are packed with large secretion granules, deformed by their closeness to each other. He held some biochemical tests of this granules and reaction to fat and mucus stains was negative, but gave a positive reaction for protein tests and concluded that it should be a protein-substrate combination. J.-M. Bassot in 1966 continues to work on Acholoe astericola bioluminescence. He supported idea of Bonhomme that photogenic tissue located in the ventral epithelium of scale and described the ultrastructure of photosomes, special organelles that made of tubules of endoplasmic reticulum, curved and disposed regularly in a paracristalline array. Later Bassot and Bilbaut in 1977 describing the distribution of bioluminescence and fluorescence over the scale ofAcholoe astericola show that fluorescence can be used to follow the centripetal migration of active zone through the luminous cells of photogenic area that is covering entire scale [2] and not just group of cells in the zone of connecting of elytra to elytrophor. In 2009 Plyuscheva and Martin [3] demonstrated that major bioluminescent signal arriving from the dorsal side of scale and thus ventral epithelium cannot be a source of light production. They described fluorescence of tubercles of "bioluminescent" and "non-bioluminescent" species and proved the presence of bioluminescent protein in "non-bioluminescent" Lepidonotus squamatus. In our work we decided to make a revision of ultrastructural organisation of dorsal and ventral epithelium of scale worms Harmothoe imbricata Linneus 1767 and Harmothoe areolata and compare it with the epithelium structure of two more boiluminescent annelids, Chaetopterus variopedatus and Fredericia heliota. According to our results, the photosomes, special organelles that made of tubules of endoplasmic reticulum can not be observed in all the epithelial cells of ventral epithelium and attributed to tiny zone arou
nd elytrophor connection (Fig. ) and can't be found at all in dorsal epithelium of scales (Fig. ). Thus it is even in controverting with the paper of Bassot and Bilbaut from 1977 where they are describing glow spread over the entire scale. It means that we have to search for some other candidates for the role of "photosomes". According to our comparative analyses and taking into account paper of Thuesen et al [4] on morphology of bioluminescent organs of Chaetognata we can say that in charge of bioluminescent reaction should be secretory granules of epithelial cells that can be observed in all the studied species (Fig. ). It can be proved by presence of autofluorescense in epithelium of Chaetopterus variopedatus (unpublished data). Up to the moment we need to prove the hypothesis with further immunohistochemistry analyses on TEM level with special antibodies against the luciferase of photoprotein involved in bioluminescent reaction. References 1. Nicol JAC, Mar J. Biol. Ass. UK 1958;37:33-41. 2. Bassot JM, Nicolas MT. 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. 5. Thuesen EV, Goetz FE, Haddock SHD. Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on bioluminescence in chaetognaths. Biological Bulletin 2010;219:100-111. [Figure: see text] [Figure: see text] O0056 Bioluminescence colors in beetle luciferases are determined by interactions in the benzothiazolyl pocket of the luciferin binding site and excited oxyluciferin phenolate Vadim R. Viviani(a,b) , Deimison R. Neves(a) , Danilo T. Amaral(b) , Rogilene A. Prado(a) , T. Matsuhashi(c) , Takashi Hirano(c) (a) Laboratory of Biochemistry and Biotechnology of Bioluminescence, Dept. Physics, Chemistry and Mathematics, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Car, Sorocaba, Brazil (b) Graduate Program of Evolutional Genetics and Molecular Biology, Sao Carlos, Brazil (c) Tokyo Electrocommunication University, Tokyo, Japan Beetle luciferases emit different bioluminescence colors from green to red, however, the structural determinants and mechanisms of bioluminescence colors remain unsolved. It is not clear what part of the luciferin binding site is involved in interactions with oxyluciferin responsible for modulating BL spectra, neither the identity of the oxyluciferin emitters. In the past decade, we have cloned a large set of beetle luciferases from the 3 main families of bioluminescent beetles (Elateridae, Phengodidae, Lampyridae) emitting all range of colors from green to red and different pH-sensitivities. Through site-directed mutagenesis, kinetic characterization and modelling studies we showed that important interactions are provided by side-chains (R218, E309, R337) and main-chain carbonyls (C/T/S314) of residues located in the benzothiazolyl pocket of the luciferin binding site. Fluorescence probing of the active site of these luciferases showed that polarity plays a major role in modulating BL spectra. Recently, the use of amino-luciferin analogs with this large set of luciferases and mutants provided strong evidences that the keto form of excited oxyluciferin is the emitter of different BL colors, and that interactions of oxyluciferin phenolate in the luciferin binding site provide the basis for distinct emission spectra. The results support that bioluminescence colors are modulated by the microenvironment polarity and specific acid/base interactions of excited oxyluciferin phenolate in the benzothiazolyl pocket of the luciferin binding site. These interactions are affected by conformational changes mediated by pH and mutations in different luciferases. (Financial support; FAPESP and CNPq) O0057 Three-Dimensional structure of Giant-Mealworm Luciferase-Like enzyme; Illuminating the Origin of Oxygenase activity in Amp-Coa-Ligases and the Evolution of Beetle Luciferases Vadim R. Viviani(a,b) , Rogilene A. Prado(a) , Mario Murakami(c) (a) Laboratory of Biochemistry and Biotechnology of Bioluminescence, Dept. Physics, Chemistry and Mathematics, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Car, Sorocaba, Brazil (b) Graduate Program of Evolutional Genetics and Molecular Biology, Sao Carlos, Brazil (c) National Lab. of Synchrotron Light (LNLS), Campinas, Brazil The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases evolved from AMP-CoA ligases. How the new oxygenase activity originated from AMP-ligases leading to evolution of beetle luciferases is one of the most challenging mysteries of bioluminescence. The luciferase-like enzyme from the Malpighian tubules of the non-luminescent Zophobas morio mealworm offers a unique model to investigate this issue, since it is an AMP-ligase with the ability to oxidize D-luciferin producing weak luminescence. The oxygenase activity emerged as a stereoselective oxidative drift with D-luciferin, a substrate that can be adenylated but can not be easily thioesterified to CoA, as in the case of L-isomer. These data advocate that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases on carboxylic xenobiotics. Kinetic studies indicate that the rate constant (klig ) of adenylation with the new oxidizable D-luciferin substrate was the main enzymatic property that underwent optimization during evolution of luciferases, whereas the oxidation constant (kox ) and quantum yield of bioluminescence changed less. The luciferase-like enzyme and typical luciferases boost up the rate of luciferyl-adenylate chemiluminescent oxidation by a factor of 10(6) and 10(7) , respectively, as compared to the substrate spontaneous oxidation in buffer. Similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophylic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could act as basic groups for proton abstraction. Recently, we solved the 3D structure of the unliganded luciferase-like enzyme by X-ray crystallography. The 3D structure and site-directed mutagenesis studies of the luciferase-like enzyme and beetle luciferases confirm a critical role for T/S345 in the luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue-shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of mutations I327T/S and N-terminal fusion, we developed a new orange emitting luciferase with kinetic and luminescence properties comparable to some beetle luciferases. These results open the possibility to engineer luciferase activity in a set of AMP-CoA-ligases. (Financial support; FAPESP 12/04857-0 and CNPq) O0058 Engineering Chemiluminescent Properties by Means of Non-Adiabatic Computational Chemistry Daniel Roca-Sanjuán(a) , Pooria Farahani(b) , Antonio Francés-Monerris(a) , Roland Lindh(b) (a) Instituto de Ciencia Molecular, Universitat de València, València, Spain (b) Department of Chemistry-Angstrom, Theoretical Chemistry Programme, Uppsala University, Uppsala, Sweden Chemiluminescence is the phenomenon in which a thermally activated reaction produces a product in an electronically excited state via an internal conversion (IC) or an inter-system crossing (ISC) process. It is widely present in living beings and also relevant in medicine and bio-imaging technology. While experiments provides an external perspective of chemiluminescence, computational chemistry allows a description at the molecular level which facilitates the establishment of the chemical functionalities of chemiluminescence.(1) Conical Intersections (CIs) and singlet-triplet crossings (STCs) are the theoretical concepts associated with IC and ISC, respectively. In order to accurately characterise these crossing regions (CIs and STCs) between potential energy surfaces a
nd determine the most relevant reaction paths, multiconfigurational methods and the reaction path approach, based on minimum energy path (MEP) or intrinsic reaction path (IRC) computations and the location of the most accessible CI and STC points, are mandatory. A reductionist approach is employed in the present contribution in which small models of well-known bioluminescent and chemiluminescent molecules, such as luminol, the firefly luciferin, and coelenterazine,(2,3,4,5) are accurately studied with the high-level complete-active-space second-order perturbation theory//complete-active-space self-consistent field (CASPT2//CASSCF)(6) method and by using the reaction path approach. Relationships between molecular structure and chemi-excitation properties are analysed. The findings allow a better understanding of the chemiluminescent mechanism and shall help to engineer chemiluminescent properties, such as lower reaction energy barriers and different ratios of the fluorescence/phosphorescence quantum yields. References 1. Navizet I, Liu YJ, Ferré N, Roca-Sanjuán D, Lindh R. ChemPhysChem 2011;12:3064-3076. 2. Farahani P, Roca-Sanjuán D, Zapata F, Lindh R. J. Chem. Theor. Comput. 2013;9:5404-5411. 3. Roca-Sanjuán D, Lundberg M, Mazziotti DA, Lindh R. J. Comput. Chem. 2012;33:2124-2126. 4. Yue L, Roca-Sanjuán D, Lindh R, Ferré N, Liu YJ. J. Chem. Theor. Comput. 2012;8:4359-4363. 5. Roca-Sanjuán D, Delcey MG, Navizet I, Ferré N, Liu YJ, Lindh R. J. Chem. Theor. Comput. 2011;7:4060-4069. 6. Roca-Sanjuán D, Aquilante F, Lindh R. WIREs Comput. Mol. Sci. 2012;2:585-603. O0059 Design, synthesis and characterization luminescent new materials by self-assembly of copper nanoclusters/Cu(I) complexes in nanoporous materials Céline Rosticher(a) , Luisa De Cola(a,b) , Loïc Donato(b) (a) Hybrid Nanomaterials Research Unit - Institute of Nanotechnology, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany (b) Laboratoire de Chimie et des Biomatériaux Supramoléculaires, Institut de Science et d'Ingénierie Supramoléculaires (I.S.I.S.), Université de Strasbourg, Strasbourg, France In this work, we focus on the development of functional nanostructures with outstanding properties, controlled shape and size through self-assembly. Rare-earth based phosphors are commonly used for application as phosphors in lighting applications, since these are the only commercially viable materials sufficiently photostable upon UV excitation. However, they are expensive, scarce and environment-unfriendly. Our main goal is the encapsulation of copper clusters and copper (I) complexes in inert nanoporous hosts to develop new materials with promising optical properties, low cost, non-toxicity to replace them. These nanostructured hosts are mesoporous silica nanoparticles (MSN) and zeolites-L, synthesized using a sol gel method inspired by Stöber process([1,2]) . They are used to guide the synthesis and to ensure electronic and structural insulation. We wish to create luminescent copper nanoclusters stabilized in nanoporous silica nanoparticles (NPS) as a novel type of phosphors. Indeed, luminescence of Cu clusters has been reported for 8 Cu-atoms clusters([3]) . Those clusters have to be synthetized in a confined area to lead to such a small size. We expect a similar luminescence behavior from copper clusters stabilized inside the pores of mesoporous materials, such as mesoporous silica nanoparticles or zeolites, where sizes between 2 and 10 atoms can be realized without capping agents. The principle of the incorporation of copper nanoclusters in MSN is a 3 steps procedure that leads to materials exhibiting a blue emission under UV. Our second aim is the encapsulation of Cu(I) complexes in mesoporous hosts. Luminescent complexes based on Cu(I) have recently attracted interest due their promising photophysical properties([4]) as they present several advantages; low cost, environmentally friendly, an emission that can be tuned from blue to red, possibility to be neutral/charged. As its luminescence comes mainly from metal-to-ligand charge-transfer states, the metal center changes formally its oxidation state from Cu(I) to Cu(II) during the excitation process([5,6]) . This triggers undesired non-radiative pathways by distortions of the tetrahedral conformation, caused by flattening of the ligand around the metal center (fig. ). Our strategy used to prevent these drawbacks is to develop copper complexes in constrained space, i.e. in the pores of zeolites and MSN in order to stabilize their coordination geometry, minimize aggregation, limit exposure to oxygen. We managed to obtain a nice green emission for Cu(I) complexes encapsulated in MSN. We managed to obtain samples containing Cu(I) complexes encapsulated in mesoporous hosts exhibiting intense yellow-green emission. [Figure: see text] All the compounds were characterized by TEM, XRD, XPS, IR and spectrofluorometry. References 1. Stober W, Fink A, Bohn E. J. of Colloid and Interface Science 1968;26:62. 2. Rahman IA, Padavettan V. J. of Nanomaterials 2012, 1 (2012). 3. Wei W, Lu Y, Chen W, Chen S. J. Am. Chem. Soc. 2011;133:2060-2063. 4. Costa RD, Ort E, Bolink HJ, Monti F, Accorsi G, Armaroli N. Angew. Chem. Int. Ed. 2012;51:8178-8211. 5. Yang L, Feng JK, Ren AM, Zhang M, Ma YG, Liu XD. Eur. J. Inorg. Chem. 2005;1867-1879. 6. Kabehie S, Xue M, Stieg AZ, Liong M, Wang KL, Zink JI. J. Am. Chem. Soc. 2010;132:15987-15996. O0060 Nanoparticle-mediated GFP delivery into Huh7 cell line; a novel platform for gene delivery into human cancer cells Majid Sadeghizade, Marzieh Karimi Tarbiat Modares, Tehran, Iran Highlights PEG-OA nanoparticle offers a new approach for GFP delivery into human cells. Searching for novel, efficient and safe gene delivery systems is of paramount importance for biomedical research. Within the recent years, the emergence of nontechnology-inspired delivery platforms has provided a new promising perspective to the territory of therapeutic gene delivery. In the current study, we introduced a new nanoparticle, PEG-OA as an amphipathic and biodegradable nanomaterial, for the delivery of green fluorescent protein (GFP) into human cancer cells. DNA was loaded onto PEG-OA nanoparticles. Characterization of the structure was conducted through atomic force microscopy (AFM) and dynamic light scattering (DLS). MTT assay was performed to investigate the toxicity of nanocarrier on Huh7 cells. Furthermore, the efficiency of GFP transfection into target cells was analyzed by fluorescence microscopy. MTT assay indicated non-toxicity of our carrier system. On the other hand, the results indicated that PEG-OA carrier system has the capacity to deliver GFP-encoded DNA into cancer cells. Taken together, PEG-OA nanoparticle represents the potential to be used as a suitable system for DNA delivery into human cells. [Figure: see text] References 1. Luo D, Saltzman WM. Synthetic DNA delivery systems. Nature biotechnology 2000;18(1);33-37. 2. Patil SD, Rhodes DG, Burgess DJ. DNA-based therapeutics and DNA delivery systems; a comprehensive review. The AAPS journal 2005;7(1);E61-E77. O0061 Advances in the investigation and synthesis of novel chemiluminescent Benzofuran-2(3H)-ones Stefan Schramm, Dieter Weiß, Rainer Beckert Friedrich-Schiller University Jena, Jena, Thüringen, Germany Based on latest research ([1]) we were able to synthesize several new structures out of the class of the Benzofuran-2(3H)-ones (Figure ). These are all urethane derivatives. The reaction parameters of the synthesis could be optimized, so that the cyclization after the Tscherniak-Einhorn (TE) reaction became obsolete. This led to a significant increase in the yield of the reaction. The ability of the Benzofuran-2(3H)-ones to show chemiluminescence was noticed very early after their discovery ([2]) . The here presented new urethane derivatives standing out for an especially intense chemiluminescence. The reason for this may be suspected in the possible formation of an Azacumarine within the chemiluminescence reaction, which is initiated by the addition of a strong base like DBU. Some of the new urethane derivatives exhibit
even without the addition of bases long lasting chemiluminescence in DMF and acetonitrile. This process, callen Autoluminescence, could already be enhanced by the addition of even a weak base like diluted aqueous ammonia. For this reason a chemiluminescence evenin aqueous media is possible. The specific triggering of chemiluminescence in aqueous medium can be accomplished, just like in the luminol reaction, by the addition of peroxidase (HRP). These new discoveries open up a wide field of possible applications in the region of the biological sciences. [Figure: see text] References 1. a) Schramm S, Weiß D, Beckert R. Luminescence 2012;27:159; b) Schramm S, Weiß D, Brandl H, Beckert R, Görls H, Roca-Sanjuána D, Navizet I. ARKIVOC 2013;3:174-188. 2. a) Lofthouse GJ, Suschitzky H, Wakefield BJ, Whittaker RA, Tuck B. Journal of the Chemical Society, Perkin Transactions 1 1979;1634-1639; b) Matuszczak B. Monatshefte für Chemie / Chemical Monthly 1996;127:1291-1303. O0062 Transcriptome of the New Zealand glowworm, Arachnocampa luminosa Miriam Sharpe(a) , Peter Dearden(a) , Gregory Gimenez(b) , Kurt Krause(a) (a) Department of Biochemistry, University of Otago, Dunedin, New Zealand (b) Otago Genomics & Bioinformatics Facility, Health Sciences, University of Otago, Dunedin, New Zealand Background and Purpose The New Zealand glowworm, Arachnocampa luminosa, has fascinated people for centuries with its star-like display of lights in caves and sheltered gorges throughout the country. The glowworm is a type of fungus gnat (order; Diptera; family; Keropatidae); its carnivorous larval form attracts prey using a bioluminescent light organ at the end of its tail. Revealing the molecular mechanism behind this lightshow will extend our understanding of the biochemistry of bioluminescence, and may provide new tools for research. We aim to study the gene expression of the glowworm light organ by sequencing its transcriptome. Comparing the difference in type and abundance of transcripts from the light organs and the bodies of A. luminosa may reveal proteins involved in bioluminescence. Methods mRNA was extracted from both the light organ and the rest of the body of three glowworms to make up a total of six samples. The mRNA was reverse-transcribed into cDNA, and samples were sequenced using the Illumina HiSeq-2000 sequencer, which generated 200bp paired-end reads. On average, 40 million reads were produced per sample. Since there is no A. luminosa reference genome available for mapping, all quality-assessed reads were combined together and used to assemble a reference dataset de novo using the assembly software Trinity (Broad Institute, Hebrew University of Jerusalem). The abundance of each type of transcript was calculated for each of the six different samples, using the de novo assembly as a reference dataset. Abundance values were normalised to accommodate the difference in sequencing depth between the samples. Differential expression analysis was carried out using edgeR software (Bioconductor). Results The de novo assembly contains a total of 196,766 transcripts, covering 187, 289,921 bases (N50 of 1828). The average length of the transcripts is 951bp. We found six protein-encoding transcripts that are expressed significantly higher in the light organ than in the rest of the glowworm tissue (see table O0062;1; cut-off set at 10% false discovery rate). [Table: see text] Intriguingly, the proteins encoded by three of these transcripts are very similar to the well-characterised luciferase of the firefly beetle Photinus pyralis (luciferin 4-monooxygenase; 31-37% amino acid sequence identity), even though bioluminescence has evolved separately in the two insects, the ancestors of which would have diverged at least 250 million years ago. Functional classification of these proteins as luciferases awaits verification using biochemical studies. Conclusions This is the first study to provide high-throughput cDNA sequence data for A. luminosa. Sequencing this transcriptome has helped to identify putative genes involved in the bioluminescence of A. luminosa and lays the foundation for future biochemical and genomics studies. O0063 Bioluminescence as an educational tool Irina Sukovataya(a) , Valentina Kratasyuk(a,b) , Josef Gitelson(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Bioluminescence is unique tool to teach students state-of-the-art biochemistry and microbiology, molecular biology and biotechnology methods employing various bioluminescent systems [1-3]. 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. Osama Shimomura to create and elaborate demonstration methods, experimental and laboratory works based on bioluminescence, to show the basic manifestations of life at molecular, biochemical, physiological and ecological levels, to get students closer to the modern science. The key academic activities of IFB&BT include higher professional education at three levels (Bachelor's, Master's and Doctorate School). The new academic programs for Bachelor and Master degree levels, the educational complex for the subjects, lecture courses on molecular mechanisms of bioluminescence and construction of bioluminescent analytical systems and practical training courses were developed [4, 5]. For more effective study special web-site with complete content of bioluminescence courses was developed [6]. The educational complex for the subject «Physics and Chemistry of Bioluminescence», includes a Work Program, Guidelines for Seminars, Guidelines for Students' Independent Studies and Methodical Guidelines for Mastering the Subject, presentation in ppt-fomat and video-lecture [6]. The Tutorial considers the luminous bacteria environment and basic properties, the structure of their bioluminescent system, the kinetics of bioluminescent reaction mechanisms, the diversity and the use of bioluminescent systems, the latest data on little-known bioluminescent systems are provided. In particular, the textbook «Physics and chemistry of bioluminescence» [4] covers the essence of bioluminescence in the life circle of sea bacteria, coelenterates, crustaceans, fungi, and the unique bioluminescence of earth worms in Siberia and others. Practical training courses [5] on biochemistry, microbiology, molecular biology and biotechnology including demonstration of luminous organisms, proteins and coding genes and the instrument-engineering kits to conduct practical training classes were designed. Academic and scientific priorities are closely connected. A great number of graduate and postgraduate students from the SibFU and other Russian universities are involve in the bioluminescence research team. Student`s researches take place both on campus and jointly with the Institutes of Russian Academy of Sciences (Institutes of Biophysics, Institute of Physics, Institute of Computational Modelling etc.) making use of knowledge and skills of the faculty doing research at nationally and internationally recognized level. Practical courses are usually developed for different levels of education. So there are several variants of one practical course which are suitable for children from 7 to 15, children from 15 to 17 too. Special practical courses are intended for the school teachers and for the education of adults. The up-to-date high-school practical training course on biology based on recent achievements of sciences using a set of biological and bioluminescent methods for visualization of sophisticated physics, chemical and biological phenomena in illustrative format was developed. The bioluminescent school laboratory consisting of manual with 35 practical lessons, simple portable bioluminometer and immobilized multicomponent reagent "Enzymolum" is produced by small company "Prikladnie biosistemi" (Russia). The work was financially supported by the sta
te contract between Ministry of Education and Science and Siberian Federal University, № 1762, 2011-2013. References 1. Gitelson JI, Kratasyuk VA. Bioluminescent visualization of vital processes and its application in biophysical education. Prog Biophys Mol Biol 1996;65(SuppI 1);24. 2. Kratasyuk VA, Kudinova IY. Practical enzymology course based on bioluminescence. Luminescence 1999;14;189-92. 3. Sukovataya IE, Kratasyuk VA, Esimbekova EN et al. Lecture course on photobiophysics based on bioluminescence. Luminescence 2008;23;93. 4. Physics and chemistry of bioluminescence; textbook / Bondar VS, Visotsky ES, Esimbecova EN [et al.]; ed. by Shimomura O, the Nobel Laureate, Gitelson JI, Academician of RAS. Krasnoyarsk; Siberian Federal Univ., 2012. - 218 p. ISBN 978-5-7638-2729-3. 5. Special biophysical training courses; biology, physics and chemistry of bioluminescence; textbook / Frank LA, Eremeeva EV, Petushkov VN [et al.]; ed. by O. Shimomura, the Nobel Laureate, I. I. Gitelson, Academician of RAS. Krasnoyarsk; Siberian Federal Univ., 2012. - 218 p. ISBN 978-5-7638-2728-6. 6. Web-site of the laboratory " Bioluminescent Biotechnologies" - http;//biolum.sfu-kras.ru/ O0064 "Visualization of cancer therapy in preclinical tumor models and in human cancer patients treated with light emitting oncolytic vaccinia virus strains" Aladar A. Szalay, PhD University of Würzburg, Germany, Department of Radiation Medicine and Applied Sciences, Rebecca and John Moores Comprehensive Cancer Center, University of California, San Diego, USA, Genelux Corporation. In 1986 at the 4th International Symposium on Bioluminescence and Chemiluminescence (ISBC) in Freiburg, Germany, we reported the expression of bacterial luciferase subunit proteins from heterologous promoters. These reports were followed by generation of bacterial luciferase fusion genes encoding a fully functional Lux F fusion polypeptide suitable for exogenously added decanal vapor dependent visualization of gene expression in both prokaryotic and eukaryotic cells and organisms (bacteria, yeasts, plants, insects, mice) (Refs 1 and 2). In addition to imaging of gene expression in single cells, the location of intracellular bacteria was visualized based on expression of bacterial luciferase from the nitrogenase promoter in Rhizobium infected soybean root nodules (plant tumors) activated by the anaerobic bacterioid environment (Refs 3 and 4) with the aid of the Argus 100 low light imaging system from Hamamatsu. In 1989, in a collaborative effort with Siemens AG, Germany, our laboratory constructed the Night Owl low light imager which is commercialized by Berthold Technologies GmbH, Germany. In 1996 at the 9(th) ISBC in Woods Hole, we reported the construction of a functional Renilla luciferase - Aequorea GFP fusion protein (ruc-gfp) resulting in photon emission, as well as GFP fluorescence, and in luminescence resonance energy transfer in both prokaryotic and eukaryotic cells. Here we report the insertion of the ruc-gfp expression cassette into the vaccinia virus genome for imaging light emission in virus infected tumors and the visualization of location of solid tumors and metastases in vaccinia colonized tumor xenografts in mice (Ref 5). Furthermore, we are now able to monitor oncolytic vaccinia based immunotherapy of tumor regression in the experimental animals after a single injection based on light extinction (Ref 6). The detection, therapy and monitoring of therapy with vaccinia virus strains encoding the Renilla luciferase-GFP fusion protein or TurboFP635 protein has been demonstrated in solid tumors, lymph node metastases, as well as in circulating tumor cells. The demonstration of light emission in tissues from vaccinia virus injected human cancer patients will be discussed together with safety following dose escalation and efficacy. Therapeutic efficacy of virus treatment is determined in human patient samples by a second vaccinia virus carried transgene product, β-glucuronidase, which is released from lysed cancer cells into the blood stream, ascites and CSF. The β-glucuronidase is converting C1-MUG1cU substrate resulted in a very sensitive, fluorigenic, pharmacokinetic assay. Lastly, the combination of virus encoded light emitting proteins with virus encoded deep tissue imaging facilitating proteins (PET, MRI and optoacoustic imaging) will also be presented. References 1. Escher A, O'Kane DJ, Lee J, Szalay AA. Bacterial luciferase aß fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc. Nat'l. Acad. Sci, USA 1989;86:6528-6532. 2. Koncz C, Olsson O, Langridge WHR, Schell J, Szalay AA. 1987. Expression and assembly of functional bacterial luciferase in plants. Proc. Natl. Acad. Sci. USA, 84; 131-135. 3. Legocki RP, Yun AC, and Szalay AA. 1984. Expression of ß-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra. Proc. Natl. Acad. Sci. USA, 81; 5806-5810. 4. Legocki RP, Legocki M, Baldwin TO, and Szalay AA. 1986. Bioluminescence in soybean root nodules; Demonstration of a general approach to assay gene expression in vivo using bacterial luciferase. Proc. Natl. Acad. Sci. USA, 83; 9080-9084. 5. Yu YA, Shabahang S, Timiryasova T, Zhang Q, Beltz R, Gentschev I, Goebel W, and Szalay AA. 2004. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat. Biotech. 22:313-320. 6. Zhang Q, Yu YA, Wang E, Chen N, Danner RL, Munson PJ, Marincola FM, Szalay AA. 2007. Eradication of Solid Human Breast Tumors in Nude Mice with an Intravenously Injected Light-Emitting Oncolytic Vaccinia Virus. Cancer Research. 67(20);10038-46. O0065 Genome sequence of the luminous mushroom Mycena chlorophos for searching fungal bioluminescence genes Yugaku Tanaka(a) , Daisuke Kasuga(a) , Yumiko Oba(b) , Sumitaka Hase(a) , Kengo Sato(a) , Yuichi Oba(b) , Yasubumi Sakakibara(a) (a) Keio University, Yokohama, Japan (b) Nagoya University, Nagoya, Japan Although many eukaryotic genome projects have been carried out with the rapid development of sequencing technology, few mushroom genomes have been sequenced so far. Luminous species are widely distributed across biological kingdoms, but no sequence homology among bioluminescence genes of different organisms makes it harder to elucidate novel bioluminescent genes and mechanisms. Fungal bioluminescence is proposed to be one of the luciferin-luciferase reactions, which includes more than two enzymes. Airth and Foerster [1,2] proposed a two-step bioluminescent mechanism involving an initial reduction of the luciferin precursor present in the hot extract by a NADPH-dependent reductase, leading to the formation of the fungal luciferin, and the following reaction of reduced luciferin with luciferase in the presence of molecular oxygen yielding light and oxyluciferin. In fact, the reconstruction of luciferin-luciferase reaction was succeeded by our experiment using the hot-extract and cold-extract (containing two active enzymes) from a luminous mushroom Mycena chlorophos. However, their coding genes and substrates are still unidentified. We sequenced the whole genome DNA and messenger RNA of M. chlorophos by next-generation sequencing, and applied it to searching and cloning bioluminescence genes to reveal fungal bioluminescent mechanisms. Genomic DNA was extracted from fruiting bodies of M. chlorophos and sequenced by Illumina Genome Analyzer IIx. Messenger RNA from four different developmental stages was also sequenced. To determine the draft genome, various parameters for preprocess of short reads and assembly programs were examined. N50, defined as the length for which the collection of all scaffolds of that length or longer reaches half of the total length, and map rate of mRNA short reads were calculated to assess the draft genome. Prediction of all genes coded in the assembled genome was held considering both mapping result of mRNA and parameters of the gene prediction program. Also, mRNA short reads were assembled into transcripts. In order to select candidates for fungal bioluminescence genes, the profile search based on
Hidden Markov Model (HMM) was employed. The statistical model was made from protein families chosen from Pfam database. For two-step bioluminescent mechanism, the profile PF000724 (NADH;flavin oxidoreductase / NADH oxidase family) and the profiles PF00296 (luciferase-like monooxygenase) and PF00743 (flavin-binding monooxygenase-like) were chosen. Similarly, other families were chosen for proteins suspected to promote fungal luminescence. The total of 398,113,885 gDNA reads, whose length is 51 bp or 54 bp, and the 297,135,716 mRNA reads, whose length is 60 bp, were generated. The draft genome has 42.0 Mbp of total length and its N50 was 59.1Kbp. Statistics about the draft genome are shown in Table . The 90.6% of mRNA reads were mapped to the draft genome. The 19,161 protein coding genes were predicted, and had similar functional compositions to those of the closest species, Laccaria bicolor, whose genome was determined. The 29,145 transcripts were assembled from mRNA reads. The results of bioluminescence profile search for the predicted genes and mRNA transcripts are shown in Table . [Table: see text] [Table: see text] 1. Airth RL, Foerster GE. The isolation of catalytic components required for cell-free fungal bioluminescence. Arch Biochem Biophys 1962;97:567-573. 2. Desjardin DE, et. al. Fungi bioluminescence revisited. Photochem Photobiol Sci, 2008;7(2);170-82. 3. Martin F, et. al. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 2008;452(7183);88-92. 4. Finn RD, et. al. The Pfam protein families database. Nucleic Acids Res 2010;38:D211-D222. O0066 Chemiluminescence derived from cigarette smoke; mechanistic features and characterization Galina Fedorova, Valery Menshov, Aleksei Trofimov, Yuri Tsaplev, Rostislav Vasil'ev Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia It has long been known that cigarette smoke is prone to an excited-state generation followed by light emission (chemiluminescence) (1). However, to date the nature of such a phenomenon has remained unclear but is thought to link to smoke free radicals, many of which are reactive oxygen and nitrogen species. The latter species cause oxidative damage of biomolecules and cell structures and thereby lead to pathogenic developments (2-4). Elucidation of the nature of the excited-state generation in the smoke is required if simple chemiluminescence assays are to be developed as analytical tools for studying the free radical content of cigarette smoke and its oxidative potential. The present study used commercial cigarettes of different tar levels as well as experimental cigarettes made from pure Burley and Virginia tobaccos. The mainstream smoke was generated using either laboratory syringes and or a Borgwaldt peristatic-pump A14 smoke engine, with parameters of 35 mL puff volume, 2 second puff duration and one puff every 60 s (the ISO standard). The chemiluminescence measurements were performed using a photon-counting apparatus incorporating a Hamamatsu photosensor module H7467 and RS-232C interface, as well as with a Lum 577 luminometer. The experimental results provided insight into the mainstream cigarette smoke-derived chemiluminescence, in which excited states are generated in unimolecular transformation of smoke-borne free radical species. However, the concentration of these radicals was found to obey bimolecular (second-order) kinetics and depended on the amount of total particulate matter (tar) of the smoke. Thus, the chemiluminescence intensity time profile, i(t), for all cigarette brands studied obeyed the law expressed by Eq. 1, in which i0 is the initial light intensity and is [Formula: see text] the empirical parameter unique to each cigarette type and, according to Eq. 2, equal to the product of the [Formula: see text] effective rate constant (k) of the self-reaction of free radicals in a smoke sample and the total initial concentration ([r(.) ]o ) of free radical species responsible for the excited-state generation followed by light emission. The value decreased from 0.08 s(-1) to 0.005 s(-1) upon the increase of the tar yield from 1.0 to 17.8 mg per cigarette. Surprisingly, no energy transfer took place from the primary excited light-emitting species to luminophoric molecules in the smoke. Chemiluminescence of the mainstream smoke paralleled the chemiluminescence emission derived from cigarette filters. This finding may be harnessed as an analytical tool for estimating the efficiency of cigarette filters towards retention of oxidants formed in the cigarette smoke. For this purpose, comparison of the light-emission intensities from the tobacco side and the mouth side of the cigarette filter should be used (Fig. ). Chemiluminescence emission derived fromthe filter sides depended not merely on the amount of the retained tar, but most importantly on its ability to react with ambient oxygen to form oxidation products of molecular and free-radical nature. The observed difference in the intensities of light emitted from the tobacco and the mouth sides of a cigarette filter depended also on cigarette design and type of filter. These results may enable comparative analysis of the filter retention efficiency towards smoke-borne oxidants exhibited by diverse cigarette brands and allow rapid screening of potential adsorbent materials to be used in cigarette filters. The reported work was generously funded by the British American Tobacco Group Research and Development. [Figure: see text] References 1. Seliger HH, Biggley WH, Hamman JP. Long-lived chemiluminescence in cigarette smoke. Science 1974;185:253-6. 2. Pryor WA. Cigarette smoke and the role of free radical species in chemical carcinogenicity. Environ Health Perspect 1997;05(Suppl 4);875-82. 3. Menshov VA, Trofimov AV. Hydrogen peroxide derived from cigarette smoke; "Pardon impossible, to be sent to Siberia?" Mini-Rev Org Chem 2011;8:394-400. 4. Palmina NP, Maltseva EL, Chasovskaya TE, Kasparov VV, Bogdanova NG, Menshov VA, Trofimov AV. Effects of different phases of cigarette smoke on lipid peroxidation and membrane structure in liposomes. Aust J Chem 2014; published online; http;//dx.doi.org/10.1071/CH13663. O0067 Spectral kinetic measurements in a chemiluminescence experiment; a novel approach Yuri Tsaplev, Rostislav Vasil'ev, Aleksei Trofimov Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia Chemiluminescence spectral studies are of prime importance for an identification of the light emitter. A novel approach disclosed herein is developed for case of more than one chemiluminescence emitting species, whose contributions to the overall light-emitting process are time-dependent. Acquisition of chemiluminescence spectra at low light intensities constitute a formidable task. Indeed, the use of spectrofluorimeters is limited by giant losses of light in their monochromator unities, while the experimental approach based on filtering the light through colored glasses is extremely time-consuming, and its accuracy is limited by the instability of measured signals derived from imperfection of mixing the reagents. To obtain the spectral kinetic information with minimal losses in sensitivity, we offer the two-channel chemiluminometer scheme (Fig. ). In such a scheme, the two photodetectors simultaneouslyrecord the light emitted in different spectral channels. The signal (minus the background) in each spectral channel is proportional to the integral ∫λ1λ2Stλ⋅Pλ⋅Fλdλ, in which S(t, λ) is the chemiluminescence spectrum at time t, P(λ) is the spectral sensitivity of photodetector, while F(λ) stays for the transmission spectrum of the color light filter installed between the chemiluminescence source and the photodetector. To avoid significant losses of light, the spectral bands of the filters should be sufficiently broad, but with a minimum overlap of the transmission bands. The ratio of the signals in the spectral channels, Yt=∫λ1λ2Stλ⋅Pλ⋅F1λdλ/∫λ1λ2Stλ⋅Pλ⋅F2λdλ, is sensitive to changes in the chemiluminescence spectrum. If only one emitter is involved, then Y(t) is constant, and its value is completely determined by
the spectrum of the chemiluminescence emission, S(λ). We call this quantity the spectral ratio, which is the characteristic of an emitter. When Y(t) is not constant, this means, that a system is more complicated and needs further in-depth investigation. It is noteworthy that the spectral ratio can be determined not only experimentally but also by calculations using the known S(λ), P(λ), F1 (λ) and F2 (λ) data. Fig. provides the pertinent illustration of the dual-channel chemiluminescencemethodology presented herein. The displayed results resolve the controversy in the literature data concerning such a chemiluminescence system. Before, the use of the light filters (1) has led to the conclusion that such a chemiluminescence process comprises green (ca. 80%) and blue (ca. 20%) emissions. Conversely, only blue emission was observed with the use of spectrofluorimeter (2). From Fig. O0067;2, the time profiles of the blue and green chemiluminescence channels as well as the Y(t) quantity are clearly seen. Funding by the Russian Academy of Sciences is gratefully appreciated. [Figure: see text] [Figure: see text] References 1. Tsaplev YB. Chemiluminescence from the oxidation of linear hydrazides of carboxylic acids with hypochlorite. Russ J Phys Chem 1999;73:1495-8. 2. Francis PS. The emitting species formed by the oxidation of hydrazides with hypohalites or N-halosuccinimides. Luminescence 2004;19:205-8. O0068 Microchip Capillary Chromatography with Chemiluminescence Detection Based on Tube Radial Distribution Phenomenon Takafumi Matsuda(a) , Masahiko Hashimoto(a) , Kazuhiko Tsukagoshi(a,b) (a) Doshisha University, Kyotanabe, Kyoto, Japan (b) Tube Radial Distribution Research Center, Kyotanabe, Kyoto, Japan Our group has reported the tube radial distribution phenomenon (TRDP) of carrier solvents under microfluidic flow conditions since 2009 [1-4]. When the ternary mixed solvents of water-hydrophilic/hydrophobic organic solvent mixture are delivered into a microspace, such as a microchannel or a capillary tube, the solvent molecules are radially distributed in the microspace generating inner and outer phases. A capillary chromatography system based on the TRDP in which the outer phase acts as a pseudo-stationary phase under laminar flow conditions has been developed. We call the separation method "tube radial distribution chromatography" (TRDC) [1-4]. All of TRDC systems have been performed by using various types of capillary tubes. Miniaturization on a microchip incorporating the microchannels has not been applied to the TRDC system. Here, we tried to carry out the chromatographic system, TRDC, on a microchip incorporating the open-tubular microchannels, based on the TRDP. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC-labeled biomolecule was injected to the double T-junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 m deep, 100 m wide, and 22 cm long) with the ternary water-acetonitrile-ethyl acetate mixture carrier solution (3;8;4 volume ratio, the organic solvent-rich or 15;3;2 volume ratio, the water-rich). The analyte, free-ILITC and labeled bovine serum albumin (BSA) mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo-stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction. 1. Murakami M, Jinno N, Hashimoto M, Tsukagoshi K. Anal. Sci. 2011;27:793-798. 2. Jinno N, Murakami M, Mizohata K, Hashimoto M, Tsukagoshi K. Analyst 2011;136:927-932. 3. Fujinaga S, Jinno N, Hashimoto M, Tsukagoshi K. J. Sep. Sci. 2011;34:2833-2839. 4. Tsukagoshi K. Anal. Sci. 2014;30:65-73, and references cited therein. O0069 Functional complementation of luciferase half-reactions; application to a robust protein-protein interaction assay FlimPIA Hiroshi Ueda(a) , Takahiro Yamashita(b) , Makoto Kurihara(b) , Yuki Ohmuro(a,c) (a) Tokyo Institute of Technology, Yokohama, Kanagawa, Japan (b) The University of Tokyo, Bunkyo-ku, Tokyo, Japan (b) JSPS, Tokyo, Japan The method to detect protein-protein interaction (PPI) is a key technology in biotechnology. Although FRET and BRET have been frequently used to detect PPI in vitro and in vivo, they have limitations in detectable distance and orientation between the interacting partners, and attainable signal/background (S/B) ratios. Although protein-fragment complementation assay (PCA) generally displays higher S/B ratio, it is hardly applicable to PPI detection in vitro, due to limited probe stability. As an alternative, recently we developed a novel PPI assay principle named FlimPIA (Firefly luminescent intermediate-based Protein Interaction Assay), based on the functional complementation of two mutant firefly luciferases (Fluc)[1]. In short, the assay is based on the functional division of two Fluc half-reactions; an adenylation reaction to produce reaction intermediate LH2 -AMP, and subsequent oxidative reactions that consume LH2 -AMP to produce OxL, which then emits light, by combining known mutations [2-5]. When the two mutant Flucs (Donor and Acceptor of adenylate) come close by the interaction, the overall reaction rate increases due to more efficient adenylate transfer. Accordingly, the assay enables rapid and sensitive detection of PPIs (Fig. ). For example, when rapamycin-dependent FKBP12-FRB interaction was detected in vitro, stronger signal than in PCA was obtained with a shorter time period less than 1 s. Moreover, it showed higher probe stability than in PCA and tolerance to longer interaction distance over 7 nm, which disabled FRET between FPs [6]. Also, the same assay was successfully performed in the cells transfected with the probe genes. However, there was a problem in the assay that the background signal due to the second mutant (Acceptor) resulted in lower S/B ratio. To address this issue, we have tried and succeeded in reducing the background activity of the Acceptor by introducing mutations at the hinge region that inhibits adenylation conformation. Also, we optimized the reaction condition including lower substrate (ATP) concentration that effectively suppressed background signal due to auto-luminescence of the Acceptor. As a result, high S/B ratio of ~50 was attained within 0.5 s. We also succeeded in improving S/B ratio by trappingthe Acceptor conformation to that only allows oxidative reactions by chemical and disulphide bonds, that supports "C-terminal domain alteration" mechanism of this enzyme. Results of other PPI detection including phosphorylation dependent p53-MDM2 interaction will also be shown. [Figure: see text] [Figure: see text] References 1. Ohmuro-Matsuyama Y et al. Anal. Chem. 2013;85:7935-40. 2. Branchini BR, et al. Biochemistry 1998;37:15311-9. 3. Branchini BR, et al. ibid 2000;39:5433-40. 4. Branchini BR, et al. ibid 2005;44:1385-93. 5. Ayabe K et al. FEBS Lett. 2005;579:4389-94. 6. Ohmuro-Matsuyama Y et al. Anal. Chem. 2014;86:2013-8. O0070 Novel phenothiazine enhancers in chemiluminescent enzyme immunoassay Marina M. Vdovenko(a) , Alexandra S. Demiyanova(a) , Anastasia V. Gribas(a) , Evgeny E. Efremov(b) , Ivan Yu. Sakharov(a) (a) Lomonosov Moscow State University, Moscow, Russia (b) Institute of Experimental Cardiology, Moscow, Russia The most sensitive enzyme immunoassay (EIA) is the assay with chemiluminescent (CL) detection of enzyme activity. Traditionally in CL-EIA a light is formed upon the oxidation of luminol with hydrogen peroxide catalyzed by peroxidase using as a label of immunoreagents. Since plant peroxidases are poor catalysts in this reaction, certain compounds known as enhancers are added to the substrate mixture to increase CL intensity. For long time the most popular enhancer for peroxidase was 4-iodophenol. In this work we analyzed some phenothiazine derivatives and showed that phenothiazines carrying groups with negative charge are potent primary enhancers in peroxidase-catalyzed CL, whereas phenothiazines with groups with positive c
harge have no the enhancing ability. The most efficient primary enhancers, whose the enhancing activity is higher in many times than that of p-iodophenol, are 3-(10'-phenothiazi-nyl)propane-1-sulfonate (SPTZ) [1] and 3-(10'-phenothiazinyl)propionic acid (PPA) [1,2]. As it is shown previously [3], some pyridine derivatives are secondary enhancers increasing the enhancing ability of phenothiazines. Screening of some pyridines showed that N-morpholi-nopyridine (MORPH) is the most active secondary enhancer. The mechanism of its enhancing action was proposed [4]. The conditions of the performance of the enhanced CL reaction using SPTZ or PPA in combination with MORPH as primary and secondary enhancers were optimized. Under these conditions the light intensity was practically unchanged for a long time. The detection systems with SPTZ/PPA and MORPH were applied successfully in construction of ultrasensitive EIA kit for determination of human thyroglobulin [5] and methylglyoxal-modified low density lipoprotein [2]. The obtained results open good perspectives for use of ECR with phenothiazines/MORPH in the development of ultra-sensitive immunoassay kits. The authors thank Dr. Leopoldo Della Ciana (Cyanagen, Italy) for help in the performance of the work and the Russian Foundation for Basic Research for financial support (11-04-92005_NNS_a). 1. Vdovenko MM, Vorobiev AK, Sakharov IY. Phenothiazine derivatives as enhancers of peroxidase dependent chemiluminescence. Russian J. Bioorg. Chem. 2013;39:176-180. 2. Sakharov IY, Demiyanova AS, Gribas AV, Uskova NA, Efremov EE, Vdovenko MM. 3-(10'-Phenothiazinyl)propionic acid is a potent primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low density lipoprotein. Talanta 2013;115:414-417. 3. Marzocchi E, Grilli S, della Ciana L, Prodi L, Mirasoli M, Roda A. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts. Analyt. Biochem 2008;377:189-194. 4. Sakharov IY, Vdovenko MM. Mechanism of action of 4-dialkylaminopyridines as secondary enhancers in enhanced chemiluminescence reaction. Analyt. Biochem. 2013;434:12-14. 1. Viviani VR, Hastings JW, Wilson T. Two bioluminescent diptera; the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems. Photochem Photobiol 2002;75(1);22-7. O0071 Insight into bioluminescence mechanism of Ca(2+) -regulated photoproteins from spatial structures and site-directed mutagenesis Eugene Vysotski(a) , John Lee(b) (a) Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia (b) Department of Biochemistry and Molecular Biology, University of Georgia, Athens, USA Bioluminescence is a widely distributed phenomenon among marine dwellers, many of which generate light by a common chemiluminescent mechanism of oxidation of coelenterazine [1, 2]. On the basis of biochemistry however, these bioluminescence systems are divided into two classes. One is the luciferase-luciferin reaction and the other, the Ca(2+) -regulated photoproteins, which constitute a unique class of protein biochemistry [2]. All Ca(2+) -regulated photoproteins consist of a single polypeptide chain (ca. 22 kDa) to which a high-energy intermediate, 2-hydroperoxycoelenterazine, is stabilized by tight, non-covalent binding [3]. Photoproteins therefore, can be regarded as luciferases with a stabilized reaction intermediate that can be triggered for bioluminescence on the addition of Ca(2+) . It has been suggested that this stabilization if achieved by hydrogen bonds to a Trp-His-Tyr triad within the binding cavity (Fig. ). Small shifts of these residues resulting from Ca(2+) co-ordination, allow an oxidative decarboxylation of the 2-hydroperoxycoelenterazine to proceed to yield the bioluminescence emitter, protein-bound coelenteramide in its S1 excited electronic state. Over the past decade, the crystal structures of the hydromedusan photoproteins as well as their ligand-dependent conformation states, as well as some mutants with altered bioluminescence properties, have been determined. From these spatial structures along with comprehensive mutagenesis studies, the function of residues within the substrate-binding cavity has been revealed, in stabilizing the 2-hydroperoxy adduct of coelenterazine, in accounting for the different bioluminescence spectra from photoprotein types, and their role in formation of an active photoprotein from the apophotoprotein and coelenterazine. Furthermore, the observations have allowed a proposal for a proton-relay mechanism for triggering the bioluminescence reaction by Ca(2+) [4, 5]. The work was supported by RFBR grant 12-04-00131 and the 'Molecular and Cellular Biology' Program of the RAS. [Figure: see text] References 1. Haddock SH, Moline MA, Case JF. Bioluminescence in the sea. Annu Rev Mar Sci 2010;2:443-493. 2. Shimomura O. Bioluminescence; Chemical Principles and Methods. World Scientific Publishing Co.; Singapore, 2006. 3. Vysotski ES, Markova SV, Frank LA. Calcium-regulated photoproteins of marine coelenterates. Mol Biol 2006;40:355-367. 4. Eremeeva EV, Natashin PV, Song L, Zhou Y, van Berkel WJ, Liu ZJ, Vysotski ES. Oxygen activation of apo-obelin-coelenterazine complex. ChemBioChem 2013;14:739-745. 5. Eremeeva EV, Markova SV, van Berkel WJ, Vysotski ES. Role of key residues of obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct. J Photochem Photobiol B 2013;127:133-139. O0072 The unique bioluminescent chemistry of the New Zealand glowworm (Titiwai, Arachnocampa luminosa) Oliver Watkins, Miriam Sharpe, Kurt Krause, Nigel Perry Perry Otago University, Otago, New Zealand The New Zealand (NZ) glowworm Arachnocampa luminosa Keroplatidae, Diptera (Skuse) is found all over NZ in damp areas of forest, by streams and in caves. The larvae, the pupa and the adult forms all bioluminesce with a blue light. We have been researching the chemistry behind this glow. A new bioluminescent system, with unique light emitting properties, would be an important scientific and biotechnological advance and could improve current, and generate novel, bioluminescent applications. The system has previously been studied by Lee, Shimomura and Hastings, among others, but the mysteries of its chemical mechanism remain unelucidated(1,2,3) . Our experiments have confirmed that the glowworm luminescent system is composed of a novel ATP dependent luciferin-luciferase reaction separate from any known system. An enzyme mix containing glowworm luciferase was prepared using a desalting spin column to remove luciferin from a crude glowworm (GW) lysate. This crude enzyme mix, when combined with a GW luciferin containing fraction and ATP under assay conditions, produced light (Fig. ). Neither the luciferin containing fraction nor the luciferase containing fraction on their own produced light. This luminescence assay, as modified by us from the Hastings protocol could be used to repeatedly and reliably detect GW luciferin, and was used to track luminescent compounds as they were purified by chromatography. [Figure: see text] Two distinct luciferin containing fractions were separated by bench reverse-phase C18 chromatography. Both fractions produced light under assay conditions. One of these fractions was successfully further purified by preparative C18 HPLC. Analysis of this sample by liquid chromatography mass spectrometry led to an aromatic small molecule compound, termed Compound X by us, being put forward as an Arachnocampa luciferin candidate. Compound X was then synthesised by us according to a published method and was found to glow with the same luminescent wavelength maxima and the same time duration as the extracted crude glowworm luciferin when tested by luminescence assay (Fig. ). This compound X is a new class of luciferin derived from a distinct metabolic pathway and is not related to other known luciferins, including that from the firefly, Photinus pyralis. The structure is being kept confidential pending patent protection. Future work includes elu
cidation of the full chemical mechanism, and the identity and mechanism of the other participating luminescent compound found in this remarkable organism. 1. Viviani VR, Hastings JW, Wilson T. Two bioluminescent diptera; the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems. Photochem Photobiol 2002;75(1);22-7. 2. Shimomura O, Johnson FH, Haneda Y. Observation on the biochemistry of luminescence in the New Zealand glowworm Arachnocampa luminosa. In Bioluminescence in progress, Johnson FH, Haneda Y (eds). Princeton University Press; Princeton, 1966;487-94. 3. Lee J. Bioluminescence of the Australian glow-worm Arachnocampa richardsae Harrison. Photochem Photobiol 1976;24(3);279-285. O0073 Chemiluminescence with Supermarket Products Dieter Weiß, Herbert Brandl, Rainer Beckert FSU Jena, Jena, Germany Well prepared and presented experiments are highlights in classes and lectures. There are many publications in which such experiments are described [1]. Most of these experiments are provided as demonstrating experiments, carried out by a professional chemist or teacher and are too difficult or too complex for highschool- or undergraduate students. Such experiments often require expensive equipment, organic solvents or toxic chemicals. Additionally there are many problems regarding the time intensive preparation and the disposal of resulting waste. This also applies for experiments containing luminescence and chemiluminescence. Although many of these experiments are pretty impressive and beautiful, it is impossible to realize these experiments for all students in a class or a practical course. Beside this fact the laws in schools are very restrictive in Germany and many things are forbidden. This creates the curious situation that more and more students leave high school without having done any chemical experiment in their life. Hence we were searching for commercial products, easily available in supermarkets, drugstores or collectable from natural products. To our own surprise we found a wide range of chemicals and technical products usable for chemiluminescence experiments. Of course it is not possible to replace Luminol or TCPO by supermarket products, but oxidation agents, non toxic solvents and dyestuffs usable for chemiluminescence experiments [2]. Oxidating agents; Laundry detergents often contain sodium percarbonate (2Na2 CO3 /3H2 O2 ) in concentrations suitable for chemiluminescence experiments. In addition they contain optical brightener, blue emitting fluorescence dyes. Some whiteners may also contain pure sodium percarbonate as a white powder. Dissolving this powder in warm water forms concentrated and alkaline hydrogenperoxide solutions. Solvents; It is difficult to substitute solvents but we found that biodiesel is a suitable and non toxic solvent for the peroxioxalate chemiluminescence Peroxides; The chemiluminescence of peroxides is well known [3] but working with peroxides is not easy. We found that old plant oils contain organic peroxides in concentrations large enough for chemiluminescence experiments. Dyes; Chlorophyll is an acceptable emitter dye for the peroxyoxalate chemiliminescnece as well as for the chemiluminescence of peroxides. Pumpkin Seed Oil is one of the best chlorophyll sources we have found. The flowering plant St. John's word (Hypericum perforatum) is the source of hypericin, an orange emitting luminescence dye. Chlorine; The reaction of chlorine with hydrogenperoxide forms singulett oxygen with a red chemiluminescence emission. Some cleaning agents contain hypochlorite in high concentrations. Acidification with acetic acid results in the formation of chlorine gas which may react with solid whitener or a solution of whitener in water. Catalysts; The Luminol reaction needs a catalyst, normally haemin or blood traces. Horseradish (Armoracia rusticana) and other brassicaceae containing peroxidases in different concentrations and are perfect catalysts for this chemiluminescence reaction. Polyphenols; Green tea is a source of polyphenols and in combination of these with a whitener solution and paraformaldehyde starts the Trautz-Schorgin-Reaction [4]. 1. Brandl H. Trickkiste Chemie 2. Auflage.; Aulis/Stark, 2006, Wagner, G. Chemie in faszinierenden Experimenten, Aulis, 1997. 2. Brandl H, Weiß D, Albrecht S. MNU 64/3, 2011;160 - 165. 3. Helberger JH, Hever DBB 66;1933;11-15 4. Panzarasa G, Sparnacci K. J. Chem. Educ. 89;2012;1297-1300 O0074 Sure we have lots of bioreactor solids… but how many are active? Patrick Whalen, David Tracey, Jeremy Duguay LuminUltra Technologies Ltd., Fredericton, NB, Canada The Problem Biological wastewater treatment processes rely on having living biomass of good quality present to consume the biodegradable contaminants present in wastewater. Unfortunately, the conventional methods of monitoring biomass concentrations have much interference, such as inert substances or even dead biomass. Because of this, wastewater treatment plants are prone to debilitating upsets and do not routinely achieve a high operating efficiency. Objectives & Methodology To overcome these problems, new technologies have been developed to measure the living biomass directly and as such provide a new basis for process control. One such monitoring tool is Adenosine Triphosphate (ATP). ATP is present in all living cells and cannot exist without life. In addition, ATP is very easy to measure and provides near real-time feedback. As such, it is an ideal supplement to current monitoring practices. This paper will focus on the advantages of Cellular ATP (cATP) as a superior measure of living biomass concentration and how it has been used to correct process stability issues and identify opportunities for improvement. Results The results will focus on three industries using different types of wastewater treatment unit operations. Research indicates that MLSS measurements typically consist of between 15 and 40 percent living biomass. Because cATP measures only living biomass, it is possible to evaluate this ratio by first converting cATP numbers to Mixed Liquor Active Volatile Suspended Solids (MLAVSS) using an established conversion factor of 0.5. The ratio of living biomass concentration to total solids concentration (i.e. MLAVSS / MLSS) is termed the Active Biomass Ratio, or ABR. [Figure: see text] Figure shows the average ABR values of sites for which both MLSS and cATP data was available. The shaded area represents the 15 to 40 percent range of living to total solids cited by research, and it is apparent that the majority of values fall into this range. This finding essentially confirms the accuracy of earlier predictions. Analysis of the data from a 'Reference Mill' showed that ATP parameters related better to process performance than did conventional measurements. For this process, a BOD removal efficiency of 92.3% was the routine target. [Table: see text] Table shows how sorting MLSS, MLVSS, and MLAVSS alongside performance data helps to demonstrate that to achieve high BOD removal, a sufficient concentration of living biomass is required. There is a significant difference between MLAVSS levels for different targets, whereas MLSS and MLVSS do not show as much variation. Furthermore, the trend of performance versus F/M using MLAVSS is linear - the more 'M' per unit of 'F', the greater the performance of the process. These findings suggest that by maintaining an MLAVSS concentration of 3,000 mg/L (i.e. cATP of 6,000 ng/mL) and an F/M of approximately 0.93 or less in the bioreactor, the probability of meeting effluent discharge regulations will be greatly improved. To verify these findings, the new guidelines for F/M ratio can be applied against the percentage of BOD removal over the number of days for which data is available. The percentage of process failures (i.e. BOD removal < 88.6%) was analyzed. On the days for which data was available and the F/M was less than 0.93, only once did the BOD removal fall below the acceptable range. As F/M increased, the rate of failure also increased. For tracking cause-and-effect relationships
with regards to process performance, plant stability, and optimization opportunities, this is valuable information. Benefits and Significance The most important benefit to having an accurate measure of living biomass in biological systems is the ability to develop meaningful cause and effect relationships with key operating parameters, such as aeration, macronutrient, or biostimulant supplementation requirements, food-to-microorganism ratio, solids retention time, or temperature. Establishing meaningful relationships can result in opportunities for process improvement in terms of upset avoidance and cost savings with minimal investment. O0075 A novel ATP-dependent bioluminescent system from the Siberian earthworm Fridericia heliota; structure elucidation of luciferin and its analogs Valentin Petushkov(b) , Maxim Dubinnyi(a) , Aleksandra Tsarkova(a) , Natalja Rodionova(b) , Mikhail Baranov(a) , Osamu Shimomura(b) , Ilia Yampolsky(a) (a) Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia (b) Laboratory of Photobiology, Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk, Russia We report structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota. This luciferin represents a key component of a novel ATP-dependent bioluminescence system. The UV, fluorescence, NMR and HRMS spectral studies were performed on 5 mkg of the isolated substance, and gave four isomeric structures, conforming with spectral data. These isomers were chemically synthesized and one of them was found to produce light in the reaction with a protein extract from Fridericia. The novel luciferin was found to have an unusual deeply modified peptidic nature, implying an unprecedented mechanism of action. [Figure: see text] Four isomeric structures 1-4 were consistent with NMR and mass spectra of luciferin. These isomers differ only by the order of peptide bonds connecting the four residues, identified as the building blocks of Fridericia luciferin; CompX, lysine, GABA and oxalate. We synthesized all the isomeric peptides and explored their ability to produce light upon addition to the crude Fridericia luciferase in the presence of ATP and MgSO4 . Only synthetic compound 1 showed luminescence in these conditions, with the luminescence spectrum (fig. ) and intensity-concentration dependence identical to those of the natural luciferin. [Figure: see text] Our further efforts will be focused on structural characterization of luciferin biosynthetic precursors and oxyluciferin, evaluation of the role of ATP and on sequencing and cloning of Fridericia luciferase. 1. Petushkov VN, Dubinnyi MA, Tsarkova AS, Rodionova NS, Baranov MS, Kublitski VS, Shimomura O, Yampolsky IV. A novel-type luciferin from Siberian luminous earthworm Fridericia heliota; structure elucidation by spectral studies and total synthesis, submitted. 2. Petushkov VN, Dubinnyi MA, Rodionova NS, Nadezhdin KD, Marques SM, Esteves da Silva JCG, Shimomura O, Yampolsky IV. AsLn2, a luciferin-related modified tripeptide from the bioluminescent earthworm Fridericia heliota. Tetrahedron Lett. 2014;55:463-465. 3. Petushkov VN, Tsarkova AS, Dubinnyi MA, Rodionova NS, Marques SM, Esteves da Silva JCG, Shimomura O, Yampolsky IV. CompX, a luciferin-related tyrosine derivative from the bioluminescent earthworm Fridericia heliota. Structure elucidation and total synthesis. Tetrahedron Lett. 2014;55:460-462.
PMID: 24913697 [PubMed - as supplied by publisher]
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