Bioluminescence and Chemiluminescence: Light Emission: Biology and Scientific Applications, Proceedings of the 15th International Symposium

Proceedings of the 15th International Symposium on BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and S...

Author: Xua Shen | Xiao-lin Yang | Xin-rong Zhang | Zong Jie Cui | Larry J. Kricka

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Proceedings of the 15th International Symposium on

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications

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Shanghai, P. R. China 13 – 17May 2008

Proceedings of the 15th International Symposium on

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications edited by

Xun Shen Chinese Academy of Sciences, P. R. China

Xiao-Lin Yang People's Hospital of Peking University, P. R. China

Xin-Rong Zhang Tsinghua University, P. R. China

Zong Jie Cui Beijing Normal University, P. R. China

Larry J Kricka University of Pennsylvania, USA

Philip E Stanley Cambridge Research & Technology Transfer Ltd, UK

World Scientific NEW JERSEY * ~ O ~ O O* NSINGAPORE

BElJlNG * SHANGHAI * HONG KONG * TAIPEI * CHENNAI

Published by

World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications Copyright © 2009 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN-13 978-981-283-957-2 ISBN-I0 981-283-957-7

Printed in Singapore by World Scientific Printers

PREFACE These are the Proceedings of the 15 th Symposium on Bioluminescence and Chemiluminescence held at the Shanghai Galaxy Hotel on 13-17 May, 2008. This series of symposia started in Brussels in 1978, and a list of the other Proceedings volumes appears at the end of this Preface. As in previous symposia, participants came from far and wide and in all 19 countries were represen ted. The Organizing Secretariat was fortunate to have the continued association with the International Society for Bioluminescence & Chemiluminescence. The organizers are thankful for the kind support of the society. We also thank John Wiley & Sons for publishing the regular abstracts in the journal Luminescence Vol. 23(2) 2008. Editorial Note This volume was compiled without peer review from camera-ready manuscripts of lectures and posters presented at the Symposium. The Editors have, in the interest of rapid publication, made only minor stylistic changes. They take no responsibility for scientific or priority matters. The Editors: Xun Shen, Xiao-Lin Yang, Xin-Rong Zhang, Zong Jie Cui, Larry J Kricka, Philip E Stanley.

THE MARLENE DELUCA PRIZE The Marlene DeLuca prizes were again generously given by Dr Fritz Berthold, together with Berthold Technologies. Dr. Berthold has provided these prizes at each symposium since the 1988 Symposium in Florence. The prize can be awarded to symposium participants under the age of 35 on the day before the starting date of the symposium. The prize is given in memory of Dr. Marlene DeLuca who made major contributions to the science of bioluminescence (see Stanley PE. Dedication to Marlene DeLuca: Journal oj Bioluminescence and Chemiluminesceence 1989;4:7-11 (includes list of her papers). Similarly to previous years' selections, the President of the International Society, Professor Xun Shen (Institute of Biophysics, Chinese Academy of Sciences, China), assembled a selection committee from the society to choose the four winners based on their presentations. The 2008 prize winners were: Zhijuan Cao, School of Pharmacy, Fudan University Shanghai. G-rich sequence-functionalized polystyrene microsphere-based instananeous derivatization for the chemiluminescence-amplified detection of DNA. Julien Claes, Laboratory of Marine Biology, Catholic University of Louvain. Bioluminescence of sharks, a case study: Etmopterus spinax. v

vi

Preface

Elena Eremeeva, Photobiology Laboratory, Institute of Biophysics Krasnoyarsk and Laboratory of Biochemistry, Wageningen Univers ity. The kinetics of coelenterazine binding with apo-obelin and apo-aequorin. Michael Koksharov, Department of Chemistry, Lomonosov State University Moscow. pH-tolerant mutants of Luciola mingrelica luciferase created by random mutagenesis.

INTERNATIONAL SOCIETY FOR BIOLUMINESCENCE AND CHEMILUMINESCENCE 2006-2008 ISBC COUNCIL Council Members: B. Branchini (President), A. A. Szalay (Past President), M. Aizawa (President Elect), Y. Ohmiya (Secretary), P. Pasini (Past Secretary), E. Hawkins (Treasurer & Membership Secretary), L. J. Kricka (Publications Officer). Councilors: H. Akhavan-Tafti, L. Brovko, R. Hart, P. Hill, O. Nozaki, A. Roda, E. Widder, K. Wood 2008-2010 ISBC COUNCIL Council Members: M. Aizawa (President), B. Branchini (Past President), Larry J Kricka (President Elect), Y. Ohmiya (Secretary), P. Pasini (Past Secretary), E. Hawkins (Treasurer & Membership Secretary), L. J. Kricka (Publications Officer). Councilors: H. Akhavan-Tafti, L. Brovko, R. Hart, P. Hill, O. Nozaki, A. Roda, E. Widder, K. Wood

LOCAL ORGANIZING AND PROGRAM COMMITTEE CHAIRMAN: Xun Shen VICE CHAIRMEN: Zong Jie Cui, Xin-Rong Zhang MEMBERS: Guo-Nan Chen, Hua Cui, Zong-Jie Cui, Wei-Jun Jin, Xiang-Gui kong, Jin-Miong Lin, Ya-Ning Liu, Xun Shen, Da Xing, Xiao-Lin Yang, Guo-Qiang Yang, Xin-Rong Zhang, Zhu-Jun Zhang, Hui-Sheng Zhuang SECRETARIAT: Xiao-Lin Yang (Secretary), Ya-Ning Liu (Co-Secretary), JinLing Min (Co-Secretary) MANUSCRIPT EDITORS: Larry J Kricka and P E Stanley

ACKNOWLEDGEMENTS We wish to express our sincere appreciation to the following for their generous support of this symposium.

Preface

vii

HOSTED BY: The Commission for Photobiology, Biophysical Society of China. CO-HOSTED BY: The Commission for Analytical Chemistry, The Chinese Chemical Society, The Commission for Luminescence, The Chinese Physical Society. LOCAL SPONSORS: China Association for Science and Technology, National Natural Science Foundation of China. The Institute of Biophysics, The Chinese Academy of Sciences. SPONSORS: John Wiley & Sons, Ltd, China Medical Technologies, Prom ega Corporation. EXHIBITORS: Chemclin Biotech Co, Ltd. (Beijing); Hamamatsu Photonics K.K. (Beijing); Berthold Technologies GmbH& Co. KG; Berthold Detection Systems GmbH; Prom ega Corporation; Perkin Elmer Instruments (Shanghai) Co., Ltd.; Nature Gene Life Sciences Company Ltd. (Hong Kong); Longmed Bio-Tech. Ltd. (Beijing); Thermo Fisher Scientific (Shanghai) Co., Ltd.; Olympus (Beijing) Sales and Service Co., Ltd.; China Medical Technologies; Nikyang Enterprise Ltd (Hong Kong).

NEXT SYMPOSIUM The next Symposium will be held in Lyon, France in 2010. Details of the 16th BL&CL Symposium will be posted on, http://www.isbc.unibo.it. PROCEEDINGS OF PREVIOUS SYMPOSIA 14th 2006 San Diego, CA, USA Bioluminescence & Chemiluminescence: Chemistry, Biology and Applications. Editors: Szalay AA, Hill PJ, Kricka LJ, Stanley PE. Singapore: World Scientific 2007. pp. 283. ISBN 981-270-816-2. 13 th 2004 Yokohama, Japan Bioluminescence & Chemiluminescence: Progress and Perspectives. Editors: Tsuji A, Matsumoto M, Maeda M, Kricka LJ, Stanley PE. Singapore: World Scientific 2004. pp. 520. ISBN 981-238-156-2. 12th 2002 Cambridge, UK Bioluminescence & Chemiluminescence: Progress & Current Applications. Editors: Stanley PE, Kricka LJ. Singapore: World Scientific 2002. pp. 520. ISBN 981-238-156-2. 11 th 2000 Monterey, CA, USA Proceedings of the 11th International Symposium on Bioluminescence & Chemiluminescence. Editors: Case JF, Herring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE. Singapore: World Scientific 2001. pp. 517. ISBN 98102-4679-X.

viii Preface 10 th 1998 Bologna, Italy Bioluminescence and Chemiluminescence: Perspectives for the 21 51 Century. Editors: Roda A, Pazzagli M, Kricka LJ, Stanley PE. Chichester: Wiley 1999. pp. 628. ISBN: 0-471-98733-6. 9 th 1996 Woods Hole, MA, USA Bioluminescence and Chemiluminescence: Molecular Reporting with Photons. Editors: Hastings JW, Kricka LJ, Stanley PE. Chichester: Wiley 1997. pp. 568. ISBN: 0-471-97502-8. 8 th 1994 Cambridge, UK Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. Editors: Campbell AK, Kricka LJ, Stanley PE. Chichester: Wiley 1994. pp. 672. ISBN: 0-471-95548-5. 7th 1993 Banff, Canada Bioluminescence and Chemiluminescence: Status Report. Editors: Szalay AA, Kricka LJ, Stanley PE. Chichester: Wiley. 1993, pp. 548. ISBN: 0-471-94164-6. 6th 1990 Cambridge, UK Bioluminescence and Chemiluminescence: Current Status. Editors: Stanley PE, Kricka LJ. Chichester: Wiley 1991. pp. 570. ISBN: 0-471-92993-X. 5th 1988 Florence, Italy Bioluminescence and Chemiluminescence: Studies and Applications in Biology and Medicine. Editors: Pazzagli M, Cadenas E, Kricka LJ, Roda A, Stanley PE. Chichester: Wiley 1989. pp. 646. (published as volume 4, issue 1 of the Journal a/Bioluminescence and Chemiluminescence, 1989). ISBN: 0-471-92264-1. 4th 1986 Freiburg, Germany Bioluminescence and Chemiluminescence: New Perspectives. Editors: Sch61merich J, Andreesen R, Kapp A, Ernst M, Woods WG. Chichester: Wiley 1987. pp. 600. ISBN: 0-471-91470-3. 3 rd 1984 Birmingham, UK Analytical Applications of Bioluminescence and Chemiluminescence. Editors: Kricka LJ, Stanley PE, Thorpe GHG, Whitehead TP. London: Academic Press 1984. pp. 602. ISBN: 0-12-426290-2. 2 nd 1980 San Diego, CA, USA Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications. Editors: DeLuca MA, McElroy WD. New York: Academic Press 1981. pp.782. ISBN: 0-12-208820-4. 1st 1978 Brussels, Belgium International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Proceedings 1978. Editors: Schram E, Stanley PE. Westlake Village, CA: State Printing & Publishing, Inc., 1979, pp. 696.

INTRODUCTION On behalf of the Organizing Committee of 15th International Symposium on Bioluminescence & Chemiluminescence, held May 13-17, 2008, I would like to thank the International Society of Bioluminescence and Chemiluminescence (ISBC) for their trust and support to host this exciting meeting. The symposium brought scientists from different parts of the world to Shanghai, China's most comprehensive industrial and commercial city. Since the first symposium was held in 1978 in Brussels, Belgium, the symposium has subsequently been held every two years in Europe, America and Japan. This is the first time that this symposium has been held in China. Thus, it gave Chinese scientists, interested in bioluminescence and chemiluminescence, an opportunity, to interact closely with the international bioluminescence and chemiluminescence community. It also gave the scientists from Europe, America and other parts of Asia an opportunity to learn that Chinese scientists are catching up the world in all aspects of science, including research and application of bioluminescence and chemiluminescence. In the last decade, great advances have been made in fundamental research and in the applications of bioluminescence and chemiluminescence. Bioluminescence imaging has emerged as a powerful new optical imaging technique. It offers realtime monitoring of spatial and temporal progression of biological processes in living animals. The bioluminescence resonance energy transfer (BRET) methodology has also emerged as a powerful technique for the study of protein-protein interactions. Luciferase reporter gene technology represents one of the major recent achievements of molecular biology. Luciferase genes can be artificially introduced into a cell to monitor gene expression and used to explore molecular mechanisms in the regulation of gene expression. Furthermore, chemiluminescence detection and analysis have been more and more applied to life science research. For example, chemiluminescent labels and substrates have been widely used to replace radioisotope-labeling and have become the most efficient and sensitive method for detecting proteins in various immunoassays. In this symposium, five outstanding experts delivered keynote lectures describing recent advances in molecular imaging using bioluminescence, chemical mechanisms involved in squid bioluminescence, novel applications of electrochemiluminescence, luminescence-based point-of-care testing devices in biomedical diagnostics, and molecular imprinted chemiluminescence imaging sensors. In the final plenary session, Professor J. Woodland Hastings, the world renowned pioneer in understanding bioluminescence, reviewed the history of the discoveries in bioluminescence and its applications. We were fortunate to have oral and poster presentations given by scientists from 19 countries, as well as active participation from industrial exhibitors. The sessions included luciferase-based bioluminescence, photoprotein-based bioluminescence, fundamental aspects and applications of chemiluminescence, luminescence imaging, fluorescence quantum dots and other inorganic fluorescent materials, phosphorescence and ultraweak luminescence, instrumentation and new methods. ix

x

Introduction

On May 12, 2008, just one day before the symposium, a major earthquake measuring 8.0 on the Richter scale hit Wenchuan County in southwest China's Sichuan province. It is the biggest disaster in Chinese history. As many as 70,000 people died, 20,000 people were missing and millions of people became homeless. To express our sympathy and help the people in the earthquake area, the symposium participants benevolently donated more than 1200 US dollars during the symposium. On behalf of the Organizing Committee, I would like to thank all of the donors for their kind support to the people in earthquake area. The organizers and I are grateful to all the generous sponsors for their financial support of the symposium. Special thanks are owed to the China Association for Science and Technology and the National Natural Science Foundation of China for their sponsorship, and Promega Corporation and China Medical Technologies for their financial support. I would like to thank my co-organizers, Drs. Xiaoping Yang, Zong Jie Cui, Xinrong Zhang, Yaning Liu and all my competent and friendly staff, Shunyi Wei, Yue Wang and Wenli Xu, who aided the participants of the 15th International Symposium. In particular, I would like to thank Dr. Larry J. Kricka for his great effort in editing the manuscripts. Without them, this symposium would not be so successful.

Cordially,

Xun Shen President The 15th International Symposium on Bioluminescence and Chemiluminescence

CONTENTS

Preface

v th

Introduction to the 15 Symposium

ix

PART 1. BASIC BIOLUMINESCENCE Plenary lecture - Progress, perspectives and problems in basic aspects of bioluminescence HastingsJW Bioluminescence of sharks, a case study: Etmopterus spinax Claes JM and Mallefet J Chemiexcitation mechanism for Cypridina (Vargula) and Aequorea bioluminescence Hirano T, Ohba H, Takahashi Y, Maki S, Kojima S, Ikeda H andNiwaH

3

15

19

Site-directed mutagenesis of Lampyris turkestanicus luciferase: The effect of conserved residue(s) in bioluminescence emission spectra among firefly luciferases Hosseinkhani S, Tafreshi N Kh, Sadeghizadeh M, Emamzadeh R, Ranjbar Band Naderi-Manesh H

23

Chemiluminescent and bioluminescent analysis of plant cell responses to reactive oxygen species produced by a new water conditioning apparatus equipped with titania-coated photo-catalytic fibers Kagenishi T, Yokawa K, Lin C, Tanaka K, Tanaka R and KawanoT

27

pH-tolerant mutants of Luciola mingrelica luciferase created by random mutagenesis Koksharov MI and Ugarova NN

31

xi

xii Contents Bacterial bioluminescence with flavinmononuc1eotide activated by N-methylimidazole Krasnova 01, Tyulkova NA and Doroshenko 10 New method of measuring bacterial bioluminescence Krasnova 01, Tyulkova NA and Doroshenko 10

35

39

Enhancement of thermostability of Luciola mingrelica firefly luciferase by mutagenesis of non-conservative residues CYS62 and CYS146 Lomakina GY, Modestova YA and Ugarova NN

43

Web-resource: "Bioluminescence and luminous organisms" of the IBSO culture collection Medvedeva SE, Kotov DA and Rodicheva EK

47

Chemistry of symplectin bioluminescence with fluorodehydrocoelenterazine Nakashima Y, Kongjinda V, Tani N, Kuse M and Isobe M

51

Mechanisms of heavy atom effect in bioluminescent reactions Nemtseva EV, Kirillova TN, Brukhovskih TV and Kudryasheva NS

55

Theoretical analysis on the absorption spectra of intermediates of firefly luciferin in deoxygenated dimethyl sulfoxide Sakai Hand Wada N

59

Biophoton emission of biological systems in terms of odd and even coherent states Kun SI, Liu C and Jia H- Y

63

Study on ATP-dependent luminescence reaction of the arm light organs of the luminous squid Watasenia scintillans Teranishi K and Shimomura 0

67

Mechanism of bacterialluciferase: Energetic and quantum yield Considerations TuS-C

71

Contents

Mechanism responsible for the spectral differences in firefly bioluminescence UgarovaNN Luminous mushrooms Vydryakova GA, Psurtseva NV, Belova NV, Gusev AA, Pashenova NV, Medvedeva SE, Rodicheva EK and Gitelson JI Use of Cypridina luciferin analog for assessing the monoamine oxidase-like superoxide-generating activities of two peptide sequences corresponding to the helical copper-binding motif in human prion protein and its model analog Yokawa K, Kagenishi T and Kawano T

xiii

75

79

83

PART 2. APPLIED BIOLUMINESCENCE Bioluminescent assay of antibiotic susceptibility of clinical samples Frundzhyan VG and Ugarova NN

89

BART: Smart biochemistry, bright bioluminescence, low-cost hardware Gandelman GA, KiddIe G, McElgunn CJ, Rizzoli M, Murray JAH and Tisi LC

93

BART applications in medical and food diagnostics Gandelman GA, KiddIe G, Rizzoli M, Murray JAH and Tisi LC

97

Change of expression efficiency of natural and cloned lux-operon in conditions of famine GusevAA

. 101

Construction of recombinant luminescence bacteria vector to evaluate genetoxic environmental pollutants Huang X-X; He M, Shi H-C and Cai Q

105

Development ofa novel bioluminescent assay for nitric oxide by using soluble guanylate cyclase Sano Y, Seki M, Suzuki S, Abe S, Ito K and Arakawa H

109

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PART 3. BASIC CHEMILUMINESCENCE Mass spectrometric approach to elucidation of chemiexcitation of dioxetanes Ijuin HK, Ohashi M, Tanimura M, Watanabe Nand MatsumotoM Theoretical considerations on the roles of hydrogen bonding in thermal decomposition of peroxides lsobe H, Yamanaka S, Okumura M and Yamaguchi K A new bright chemiluminescent reaction: Interaction of acetone with solid-phase potassium monoperoxysulfate in the complex of europium nitrate Kazakov DV, Safarov FE, Schmidt Rand Kazakov VP Study of novel aryloxalate chemiluminescence reaction without addition of hydrogen peroxide Kishikawa N, Ohyama K, Nakashima K and Kuroda N

115

119

123

127

Nucleophilic acylation catalysts effect on luminol chemiluminescence Marzocchi E, Grilli S, Della Ciana L, Mirasoli M, Simoni P, Prodi Land Roda A

131

Effect of surfactants on peroxyoxalate chemiluminescence reaction Nakashima K, Abe K, Nakamura S, Wada M, Harada S andKurodaN

135

Solvent-promoted chemiluminescent decomposition of bicyclic dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl Tanimura M, Watanabe N, ljuin HK and Matsumoto M Synthesis and characterization of near-infrared chemiluminescent probes Teranishi K

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Generation of high-energy chemiluminophores in ambient light Tsaplev Yu B, Vasil' ev RF and Trofimov A V Alkaline metal ion enhanced chemiluminescence of bicyclic dioxetanes bearing a 3-hydroxynaphthalen-2-yl group Watanabe N, Kakuno F, Hoshiya N, Ijuin HK and Matsumoto M

xv 147

151

PART 4. APPLIED CHEMILUMINESCENCE Plenary lecture - Analytical challenges for luminescence-based point-of-care testing devices in biomedical diagnostics Roda A, Guardigli M, Mirasoli M, Michelini E, Dolci LS, and Musiani M

157

Plenary lecture - Molecular imprinted polymer-based chemiluminescence sensors Zhang Z

161

Flow injection chemiluminescence determination of hydroxylamine hydrochloride Baezzat MR and Izadpanah M

173

Study on gold-sensitised chemiluminescence for the determination of norfloxacin Bao J-F, Jiang Z-H and Yu X-J

177

Conjugates of (acridinium)x-BSA-anti-HCV core to enhance the detection of HCV core antigen Chang CD, Chang KY, Jiang L, Sablilla VA and Shah DO

181

Chemiluminescence determination of rutin based on a micelle-sensitizing N-bromosuccinimide-H20 2 reaction Du JX, Hao Land Lu JR

185

Luminol-dependent chemiluminescence increases with formation of phenothiazine cation radicals by horseradish peroxidase Hadjimitova VA, Traykov T and Bakalova R

189

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Contents

Variety of chemiluminescent methods for antioxidant activity: Investigation of Crataegus oxicantha extract Hadjimitova VA, Traykov Tand Bakalova R

193

Simultaneous mUltiplex bio- and chemiluminescent enzyme immunoassay for PCR products derived from genetically modified Papaya Ito K, Tanaka Y, Maeda M, Gomi K, Inouye S, Akiyama H and Arakawa H

197

Effect of sugars on aluminum-induced oxidative burst and cell death in suspensions of tomato cells Kadono T, Kawano T, Yuasa T and Iwaya-Inoue M

201

Chemiluminescence determination of sparfloxacin using Ru(bipY)32+-Ce(IV) system Karim MM, Choi JH, Alam SM and Lee SH

205

Flow injection analysis with chemiluminescence detection: Determination of gatifloxacin using the KMn04-formaldehyde system Khan MA, Alam SM and Lee SH

209

Determination of ciprofloxacin in pharmaceutical formulation by chemiluminescence method Khan MA, Lee SH, Alam SM, Wabaidur SM and Chung HY

213

Chemiluminescence flow-through biosensor for hydrogen peroxide based on enhanced HRP activity by gold nanoparticles Lan D and Li B

217

Flow injection chemiluminescence determination of thiamine by the enhancement of luminol- K3Fe(CN)6 system Li YH, Yang Y and Lu JR

221

Chemiluminescent and electron spin resonance spectroscopic measurements of reactive oxygen species generated in water treated with Titania-coated photocatalytic fibers Lin C, Tanaka K, Tanaka L and Kawano T

225

Contents

A sensitive micellar-enhanced chemiluminescence method for the determination of ofloxacin by flow injection analysis Ma H, Zhang Y, Miao L and Sun X Excessive extracellular chemiluminescence and necrosis of neutrophils in bovine neonates and potentially supportive role of vitamin C Mehrzad J, Mohri M and Burvenich C Chemiluminescence of 9-benzylidene-l O-methylacridans with electron-donating groups by chemically generated singlet oxygen - Application to metal ion sensing using azacrowned compound Motoyoshiya J, Tanaka T, Kuroe M and Nishii Y Effects of l,4-butanediol dimethacrylate on HL-60 cells metabolism Nocca G, De Sole P, De Palma F, Martorana GE, Rossi C, Corsale P, Antenucci M, Giardina Band Lupi A Determination of pyrogallol by imidazole chemiluminescence enhanced with hydrogen peroxide Nozaki 0, Munesue M, Momoi H, Shizuma M, Kawamoto H and Ikeda T

xvii

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233

237

241

245

Chemiluminescence study on the regulation of NADPH oxidase activity by thioredoxin reductase in vascular endothelial cells Shen X and Liu Z-B

249

Quantitative detection of singlet oxygen with a chemiluminescence probe during photodynamic reactions Wei Y, Xing D, Luo S, Xu Wand Chen Q

253

Flow-injection chemiluminescence determination of human serum albumin based on fluoresceinyl Cypridina luciferin analog-'02 reaction Xu W, Wei Y, Xing DA, Luo S and Chen Q

257

Charge-transfer-induced luminescence (CTIL) mechanisms of chemi- and bioluminescence reactions Yamaguchi K, Isobe H, Yamanaka S and Okumura M

261

xviii Contents A novel synergistic enhancer for HRP-Luminol-H 20 2 based chemiluminescence and its application in immunoassay Yang X and Sun X

265

Separation and detection of amino acids with a novel capillary electrophoresis chemiluminescence system Yin DG, Xie CJ, Liu BH and Wu MH

269

A novel chemiluminescent immunoassay of total thyroxine using the acridinium ester 2' ,6' -dimethyl-4' -(N-succinimidyloxycarbonyl) phenyl-1O-methyl-acridinium-9-carboxylate methosulfate as label Yin DG, He YF, Liu YB, Shen DC, Han SQ, Luo ZF, Xie CJ, Zhang L, Liu BH and Wu MH

273

Determination of ascorbic acid by a flow injection chemiluminescence method with a novel rhodanine Yu J, Zhang C, Tan Y, Ge S, Dai P and Zhu Y

277

Study of superweak luminescence in plants and application to salt tolerance in alfalfa Zhou H, Yang Q and Liu Y

281

Development and optimization of a quantitative western blot and dot blot procedure for the determination of residual host cell proteins present in inactivated polio vaccine using a GZll based signal reagent Zomer G, Hamzink M, De Haan A, Kersten G and Reubsaet K Development and optimization of a fast and sensitive ELISA for polio D-antigen using a GZll based signal reagent Zomer G and Hamzink M

287

291

PART 5. APPLIED ELECTROLUMINESCENCE Detection of Xanthomonas oryzae pv. Oryzicola by electrochemiluminescence polymerase chain reaction method Wei J and Zhang L

297

Contents

xix

A novel electrochemiluminescent sensor based on cationic polymer/chitosan for ultrasensitive detection of hydrogen peroxide Wu X, Wang Y, Dai H and Chen G

301

Capillary electrophoresis - electrochemiluminescence detection of ciprofloxacin in biological fluids Zhou X and Jia L

305

PART 6. BIOMEDICAL APPLICATION OF FLUORESCENT PROTEINS A novel multicolor fluorescent protein from the soft coral Scleronephthya gracillima Kuekenthal Kato Y, Jimbo M, Sato C, Takahashi T, lmahara Yand Kamiya H

311

Fluorescence from STlevel of complexes of tryptophan with europium (III) in water-ethanol solution Osina 10, Ostahov Sand Kazakov V

315

Identification of developmental enhancers using targeted regional electroporation (TREP) of evolutionarily conserved regions Pira CU, Caltharp SA, Kanaya K, Manu SK, Greer LF and Oberg KC

319

PART 7. DEVELOPMENT AND BIOMEDICAL APPLICATIONS OF QUANTUM DOTS AND OTHER INORGANIC FLUORESCENT MATERIALS Quantum dots as fluorescent resonance energy transfer donors in antibody-antigen systems Hu S, Yang H, Cai R, Zhang Q and Yang X

325

Synthesis and photoluminescence of green-emitting X2-(Y,GdhSiOs:Tb3+ phosphor under VUV excitation Zhang ZH, Wang YH and Li XX

329

Luminescent properties of Na2CaMg2Si401s:Tb3+ nano-sized phosphor Zhou L-Y, Yi L-H, Huang J-L, Wei J-S and Gong F-Z

333

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Contents

PART 8. BIOLUMINESCENCE, CHEMILUMINESCENCE AND FLUORESCENCE IMAGING The measurement of cytosolic ATP during apoptosis: Bioluminescence imaging at the single cell level Akiyoshi R and Suzuki H Bioluminescence imaging of bacteria-host interplay: Interaction of E. coli with epithelial cells Brovko LY, Wang H, Elliot J, Dadarwal R, Minikh 0 and Griffiths MW Ultrasensitive chemiluminescent immunochemicallocalisation of protein components in painting cross-sections Dolci LS, Sciutto G, Rizzoli M, Guardigli M, Mazzeo R, Prati S and RodaA Development of a new device for ultrasensitive electrochemiluminescence microscope imaging Dolci LS, Rizzoli M, Marzocchi E, Zanarini S, Della Ciana L and RodaA

339

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347

351

Visualization of sequential response in intra cellular signal transduction cascade by fluorescence and luminescence imaging in the same living cell Hatta-Ohashi Y, Takahashi T and Suzuki H

355

Bioluminescence imaging of intracellular calcium dynamics by the photoprotein obelin The! MM, Sugiyama T and Suzuki H

359

Applications of delayed fluorescence and laser confocal scanning microscope techniques in monitoring artificial acid rain stress on plants

363

Zhang H, Wen F and Zhou X Delayed fluorescence and optical molecule imaging techniques for detecting the stress response of plants to high temperature Zhang Land Wen F

367

Contents

xxi

PART 9. ASPECTS OF FLUORESCENCE AND PHOSPHORESCENCE

The interaction of Tb 3+-protocatechuic acid complex with nucleic acids and its application in determination of nucleic acids based on fluorescence quenching Chen Y, Yang Yand Yang J

373

Fluorescence enhancement of KI for the morin-fsDNA system and its analytical application 377 Ding H, Wu X, Yang J and Wang F Microemulsion sensitized determination of BSA with 3-(4'-methylphenyl)-5-(2'-sulfophenylazo) rhodanine by resonance Rayleigh scattering method Ge S, Dai p, Yu J, Li B and Tan Y Fluorimetric determination of rutin using rutin-Fe(IlI) system Karim MM, Jean CW, Lee SH and Wabaidur SM Micelle enhanced fluorimetric determination of benserazide in pharmaceutical formulations Lee SH, Kim WH, Meea K and Khan MA Improvement in carbaryl assay by fluorescence in a micellar medium Lee SH, Jean CW, Kim WH, Chung HY, Wabaidur SM, Park HW, Suh YS and Khan MA

381

385

389

393

Study of the interaction between human serum albumin and 7-ethyl-1Ohydroxycamptothecin Li G and Liu Y

397

Resonance Rayleigh scattering method for determination of alginic sodium diester with methylene blue Liu Yand Li G

401

Effects of metal ions on peroxynitrite nitrifying protein Luo Y, Cui S, Zhang L and Zhong R

405

xxii Contents Mechanism and properties of bio-photon emission and absorption of protein molecules in living systems Pang X-F The mechanism of photon emission of bio-tissues and its properties Pang X-F and Cao X-Y Synthesis of a novel fluorescence probe of P-CD and cuprous iodide pyridine and its application Qiao J, Dong R, Li D, Dong C and Shuang S Phosphorescence properties of 2-bromoquinoline-3-boronic acid in sodium deoxycholate and its potential application in recognition of carbohydrates Shen QJ, Zou WS, Jin WJ and Wang Y

409

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421

425

Study on the interaction between methyl blue and HSA in the presence of P-CDIHP-P-CD by molecular spectroscopy Song S, Hou X, Shuang S and Dong C

429

Study on the interaction of kaempferol with human serum albumin by spectroscopy and molecular modeling Tian J, Liu J, Hu Z and Chen X

433

Selection of salt-tolerant rice variety using light-induced delayed fluorescence Wang J, Xu W, Xing D and Zhang L

437

Effects of LMWOA on biodegradation of phenanthrene studied by fluorimetry Wei XY, Sang LZ, Zhu YX and Zhang Y

441

Alleviation effects of salicylic acid and lanthanum on ultra weak bioluminescence in maize leaves under cadmium stress Wei ZL, Jiao CZ, Su YN and Tian ZH

445

Rhodamine B-quinoline-8-amide as a fluorescent "ON" probe for Fe3+ in acetonitrile Xiang Y, Li ZF and Tong AJ

449

Contents

xxiii

Studies on determination of deoxyribonucleic acid by second order scattering with a novel rhodanine Yu J, Li B, Zhu Y, Cheng X and Zhang L

453

Fluorescence characteristics of novel chlorophenyl-arsenoxylphenylazo rhodanines and application in the determination of thallium (I) Yu J, Cheng X, Ge S, Tan Y and Li B

457

Molecular recognition of amino acids by hematoporphyrin and metallohematoporphyrin receptors Zhang Y, Lei Y-C and Liu D-S

461

Determination of BSA by its enhancement effect on second order scattering of 3-(4'-methyl phenyl)-5-( 4'-methyl-2'-sulfophenylazo) rhodanine Zhu Y, Yu J, Dai P, Zhang C and Li B

Index

465

469

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PARTl BASIC BIOLUMINESCENCE

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PROGRESS, PERSPECTIVES AND PROBLEMS IN BASIC ASPECTS OF BIOLUMINESCENCE JWHASTINGS Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

INTRODUCTION th It is a great pleasure to participate in this 15 Symposium on Bioluminescence and Chemiluminescence, thirty years after the first, brilliantly conceived and organized in Brussels by Eric Schram and Philip Stanley, later to be joined by Larry Kricka, and to express my gratitude to the organizing committee for inviting me. It is also an overwhelming experience to see the greatly transformed Shanghai. There has 'also been a profound transformation in the field of bioluminescence over these thirty years, progressing from the vision in Brussels that luciferase systems could be used for analytical purposes in biochemistry and medicine) to the now widespread use of genes of luciferases and GFP as reporters to track expression of other genes in time and location. 2 In parallel, there have been many important advances is basic aspects. 3 Color mutants of both luciferases and green fluorescent protein have been put to great advantage in studies where they are used as reporters and, along with other mutants, contribute to our understanding of reaction mechanisms. Crystal structures have been obtained for luciferases from four systems- bacterial, firefly, coelenterate and dinoflagellate, and much has been elucidated concerning the structures of emitters and reaction intermediates. Here I will discuss specific aspects of each of the four systems for which luciferase structures are available, starting with the coelenterate system and the use of the term photoprotein. Coelenterates: Aequorin & photoproteins are luciferase intermediates. For many years the biochemistry of the brilliantly luminescent jellyfish Aequorea was a real enigma. Cold-water extracts gave bright and long-lived emission, but the luciferin-Iuciferase test was frustratingly negative. Shimomura made the seminal discovery that the reaction requires calcium, and found that cold-water extracts made in the presence of EDTA yielded a protein that gave light upon the addition of excess caIcium. 4 He named the protein aequorin, and later dubbed it a photoprotein, the precise nature of which was not well appreciated at first. It was later shown to be a luciferase intermediate, effectively the "substrate" in the assay because turnover is slow, and is destroyed in hot water extracts of the luciferin-Iuciferase test. 5 Sessions at this symposium are divided into luciferase-based bioluminescence and photoprotein-based bioluminescence. But both use luciferases; the photoprotein aequorin is simply a stable luciferase-peroxy-Iuciferin intermediate in which a 67 subsequent reactant has been withheld, as confirmed by its crystal structure. • Such intermediates in this or other systems, when accumulated, can provide the substrate 3

4

Hastings JW

for a rapid flash in living cells if the lacking reactant is rapidly added, thus calcium for aequorin. The flash decay will thus be first order and attributable to the rate constant for the decay of the intermediate formed after calcium addition (Fig. I), and the total light emitted in the flash will be proportional to the amount of intermediate. Also, it should be noted that for the flash to decay to baseline, the prior enzymatic reaction step(s) must be very slow so that little if any more intermediate will be reformed during the course of the flash, during which time the triggering substance can be withdrawn so that new intermediate can be accumulated.

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Fig. 1. Kinetics of the reaction of aequorin with calcium mixed in a stopped-flow apparatus at 23 0 C. Firefly: the regulation of the flash. Although the luciferin-Iuciferase reaction appeared to "work" in firefly extracts, it turned out that the components were not those specified in the long-established protocol. McElroy discovered 8 that ATP is the component exhausted in cold water extracts of fireflies, while both luciferin and luciferase remain (Fig. 2), while the hot-water extract contains ATP. In McElroy's lab, we established that the reaction of ATP and lucifer in with purified luciferase involves two steps;9 the first forms an active intermediate, later determined to be the adenylate, and the second is the reaction with oxygen, leading to an excited state and light emission. The prompt decline of luminescence over the first minutes was shown to be due to luciferase inhibition, not substrate exhaustion. All evidence indicates that the flash of the firefly is initiated by the introduction of oxygen into the photocytes, triggered by a nerve impulse, which actually does not end on the photocytes, but on adjacent cells. IO- 12 More recently, nitric oxide (NO)

Progress, Perspectives and Problems in Basic Aspects of Bioluminescence

5

has been proposed to be a humoral agent involved in transmission of the signal from the nerve ending to the photocyte to initiate a flash. 13 •14 The evidence for this is not strong, and I believe the proposed mechanism to be incorrect.

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lime (min.) Fig. 2. Depiction of the steps and conditions for a luciferin-Iuciferase reaction in which an exhausted cold-water extract is mixed with a hot-water extract to give light emission. How it differs in firefly extracts is also noted.

Briefly, the NO mechanism postulates that mitochondrial oxygen consumption maintains photocytes anaerobic in spite of a continuous input of oxygen from tracheoles. A flash is initiated through a cascade of transduction steps from the nerve ending that result in NO production in the photocytes, where it inhibits this respiration, allowing oxygen to reach luciferase and initiate the reaction. As NO production ceases, along with some other possible factors, the mitochondrial utilization of oxygen resumes and the luciferase reaction declines. The kinetics of the rise phase of the flash, which in many species is less than 100 msec, seems difficult to attribute to a cascade of signal transduction events. But the extinction of the flash is most certainly not caused by the withdrawal of a reactant. Instead, it has kinetics attributable to the reaction of a luciferase intermediate whose

6

Hastings JW

precursor is accumulated in the absence of oxygen, comparable to the case of the jellyfish flash. Some years ago I demonstrated that such a "biochemical" flash can be produced in the test tube. 9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate "active" intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with lucifer in. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate.

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Fig. 3. Flashes in response to the rapid addition of oxygen to firefly luciferase reactions initiated in the complete absence of oxygen. 9 A: Time course of normal reaction in air. B,C,D: started under strict anaerobic conditions; oxygen added later at times indicated.

Fig. 4. Kinetics of a flash obtained by addition of oxygen, as described in Figure 3. 9

Bacteria: A peroxide intermediate, quorum sensing and milky seas. Although the luciferin-Iuciferase test in bacterial extracts was negative, Strehler 16 discovered that light emission in extracts could be obtained by adding reduced pyridine nucleotide, underlining the fact that bioluminescence is not a phenomenon separate

Progress, Perspectives and Problems in Basic Aspects of Bioluminescence

7

from all other cell biochemistry, but linked to it in different ways in different systems. Light emission in bacteria is continuous, deriving electrons for the reduction of flavin, the luciferin in this system, from the respiratory pathway, as indicated in Fig 5. Reports that it occurs as pulses have not been confirmed. 17 This luciferase reaction also forms a semi-stable peroxide intermediate, which we demonstrated some years ago 1S and later isolated. 19 It is reasonably stable in the absence of aldehyde and might, in principle, be accumulated in the cell and triggered to emit a flash by aldehyde addition. Indeed, bioluminescence in tunicates, which utilizes a bacterial luciferase system 20 derived from endosymbionts,21 emits light as flashes, the biochemical basis for which has not been investigated. An important phenomenon, now called quorum sensing, was discovered from studies of bacterial bioluminescence, in which it was found that growth and luminescence are controlled separately.22 After inoculating a culture into fresh medium, growth is exponential with no lag, but the amount of luciferase remains constant for the first three hours, after which its synthesis and light emission increase very, very rapidly (Fig. 6). This was shown to be due to the production and release into the medium of a substance that we named auto inducer; upon reaching a critical concentration, it induces the synthesis of luciferase and other proteins involved in the bioluminescence. Eberhard and colleagues determined the structure to be a homoserine lactone and synthesized it. 23 SUBSTRATE ~ NAOH ~ CYTOCHROMES ~ O2

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Fig. 5. Pathways and intermediates in the bacterialluciferase reaction.

8 Hastings JW 100 t:..O.D.- 660 NM IN VIVO LUM. IN VITRO LUM .

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Fig. 6. Time courses showing that the development of luminescence and luciferase (both in vitro activity and by antiluciferase, CRM) lag cell growth. 22 For many years this phenomenon was believed to be simply a special curious feature of luminous bacteria, but when DNA sequences became available, genes homologous to those responsible for auto inducer production were found to occur widely in the bacterial world. Up to then it had been generally believed that bacterial cells are mostly loners, essentially autonomous in their activities. But this discovery demonstrated that bacteria produce substances that control expression of different genes in many other bacteria, both in the same and different species, thus constituting chemical communication. 24,25 A major function of luminescence in bacteria is to provide light when cultured in specialized light organs of a higher organism. There, the production of luciferase and light are delayed until cell numbers are high enough for the light to be visible to other organisms. In some pathogenic bacteria toxin production may be delayed until the invading population is high: a surprise attack can produce massive amounts of toxin and overwhelm before resistance can be mounted. Luminous bacteria can be isolated from sea water almost anywhere in the world, but the number is typically very few, so the autoinducer in the water should and does not reach the concentration needed to induce luciferase in isolated cells. 26 Yet ever since records of ship voyages have been kept, there have been repeated reports of continuous luminous light emission in the ocean, all around the ship as far as the eye

r r(}v r",.s_

Perspectives and Problems in Basic Aspects of Bioluminescence

9

can see.27 This has been called "Milky Sea", for it does indeed look like the ship is on a sea of milk! Although no explanation of the phenomenon had been reported in the literature, a group of scientists wondered if earth-imaging satellite cameras might be able to detect the light emission. Checking the archives, they found a ship log reporting the phenomenon in 1995 when a camera had been overhead. They retrieved the satellite and detected a weak signal on three consecutive nights; with background subtracted it revealed a luminous area of about 14,000 km 2 , its exact structure changing from night to night (Fig. 7).28,29 The reported positions of the ship when it entered and exited the area corresponded exactly to the coordinates obtained from the satellite data. The location off the Horn of Africa is where reports in of Milky Seas have been most frequent. 27 Because the emission is continuous it had been speculated, and many scientists that luminous bacteria might be responsible. But, if so, how might the auto inducer concentrations needed be achieved? The answer to this is not nor is it certain that the light is actually due to lum inous bacteria of the kind cultured. But a clue comes from reports of merchant sailors, who from time to time what they saw in a bucket of water from the milky sea. A was that the water " ... contains thousands of very thin lines of

7. Bioluminescence of milky seas recorded by satellite imaging for 3 consecutive nights. Raw data, A,B,C; with background subtracted, locations of images. 28

10 Hastings JW

approximately 13 mm long ,,27 If bacteria are concentrated on a substrate, perhaps a filamentous of some autoindueer could accumulate. Future studies should give the answer.

be

triggers the flash; two functions in one These unicellular marine plankton, which my laboratory has studied for many years, are for the sparkling oceanic luminescence, earlier called pn,osrmclre,;cence Most of our work has with the photosynthetic species, Gonyaulax polyedra), which emits brief (0.1 s) flashes from small named scintillons. 3,3o They contain two major luciferase and a luciferin binding protein (LBP); the activities of both ""If'''''''''''''' The luciferin is a tetrapyrrole, probably derived from The sequences of the N-terminal -100 residues of the two nr,>tpl!1c identical but the remaining regions have no similarities 31 In the molecule 37kDa) is comprised of three repeat homologous each with a located independent catalytic site, where the sequences are about 95% identical. Each individual domain has luciferase and each has four conserved which have been shown to be involved in the by

8. Structure for Noctiluca luciferase (top) showing that it occurs as tandem of a gene possessing a sequence homologous to a domain luciferase (bottom) together with a sequence homologous to a full luciferin protein. The Noctiluca protein lacks the first N-terminal -100 amino acids found in both Lp proteins. 34

Progress, Perspectives and Problems in Basic Aspects of Bioluminescence

11

A crystal structure of one of the domains reveals a catalytic pocket and residues 33 responsible for regulation by pH. The LBP has four homologous domains, but their sequence similarities are not great. 34 The luciferase genes and proteins are very similar in seven different luminous photosynthetic species. They are about the same length and all have three domains, and occur as tandem repeats but with very different intergenic sequences. 35 ,36. The individual domains of different species are more similar to each other than to either of the other two domains of the same species. But in the heterotroph Noctiluca sc intillans the catalytic and luciferin binding sequences are both found in a single gene, and are expressed as a single protein (Fig. 8). The N-terminal -100 sequences found in L. polyedrum, which might be functional for protein-protein association, are completely absent. There is only a single luciferase domain, and it is truncated on the N-terminal side, with three of the four histidines found the three-domain luciferases absent. Aside from the N-terminal -100 sequences, the luciferin binding sequence is similar in size and homologous to the LBP in L. polyedrum, including the four domain structure. Bioluminescence originated independently many different times in evolution From a biological point of view bioluminescence is truly unusual by virtue of its evolutionary origins. As well illustrated by the four systems described, the genes, proteins and substrates involved are altogether different, as are the regulatory and functional aspects of the systems. This is most readily explained by assuming that the different systems arose independently,37 some being related to genes coding for proteins with completely different functions (coelenterates, fireflies), others with no known affinities (bacteria, dinoflagllates). How could this have been? Why is luminescence different in this respect from many, perhaps most, other genes, which have relationships to genes with similar functions in phylogenetically distant organisms? I propose that this is because the different bioluminescence systems actually have different functions, thus not subject to being carried out by the same proteins. For the systems reviewed, coelenterate flashes may startle predators and deter predation; fireflies communicate in courtship by flash patterns; bacteria provide light for various uses for hosts that culture them in different specialized organs, and dinoflagellates flash in response to mechanical stimulation by their predators, thus revealing their presence to their own predators (the burglar alarm theory). Some years ago I estimated that there may be up to 30 different bioluminescent systems.37 Researchers interested in luciferases, as well as mechanisms and functions of light emitting organisms, will thus still find a diversity of new systems for exploration with the prospect of many new and different applications. I hope that researchers will pursue such studies with vigor in the years to come.

12

Hastings JW

REFERENCES 1.

2. 3. 4.

5. 6. 7.

8. 9.

10.

II. 12. 13.

14. 15. 16. 17. 18.

Schram E, Stanley P. eds. International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Westlake, CA: State Printing & Publishing, Inc. 1979: 696 pp. Hastings JW, Johnson C. Bioluminescence and chemiluminescence. Meth Enz. 2003;360:75-104. Wilson T, Hastings JW. Bioluminescence. Annu Rev Cell Devel BioI 1998;14:197-230. Shimomura 0, Johnson F, Saiga Y. Extraction, Purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962;59:223-39. Shimomura 0, Johnson F. Regeneration of the photoprotein aequorin. Nature 1975;256:236-8. Head J, Inouye S, Teranishi K, Shimomura 0. The crystal structure of the photoprotein aequorin at 2.3 angstrom resolution. Nature 2000;405:372-6. Liu Z-J, Vysotski E, Rose J, Lee J and Wang B. De novo structure determination of the photoprotein obelin at 1.7 angstrom resolution using single wavelength sulfur anomalous scattering data. Protein Sci 2000;9:2085-93. McElroy WD. The energy source for bioluminescence in an isolated system. Proc Natl Acad Sci 1947;342-5. Hastings JW, McElroy WD, Coulombre J. The effect of oxygen upon the immobilization reaction in firefly luminescence. J Cell Comp Physiol 1953;42:137-50. Case J, Strause L. Neurally controlled luminescent systems. In: Herring P. Ed Bioluminescence in Action. London: Academic Press, 1978:331-45. Timmins G, Robb F, Wilmot C, Jackson S, Swartz H. Firefly flashing is controlled by gating oxygen to light-emitting cells. J Exp BioI 2001 :2795-2801. Ghiradella H, Schmidt J. Fireflies at 100: A new look at flash control. Integrat Comp BioI 2004;44:202-12. Trimmer B, Aprille D, Dudzinski D, Lagace C, Lewis C, Michel T, Qazi S, Zayas R. Nitric oxide and the control of firefly flashing. Science 2001 ;292:2486-8. Aprille J, Lagace C, Modica-Napolitano J, Trimmer B. Role of nitric oxide and mitochondria in control of firefly flash. Integrat Comp BioI 2004;44:213-19. McElroy WD, Hastings JW. Initiation and control of firefly luminescence. In: Prosser C. Ed. Physiological Triggers. New York, NY:Ronald Press, 1956:80-4. Strehler B. Luminescence in cell-free extracts of luminous bacteria and its activation by DPN. J Am Chern Soc 1953;75:1264. Haas E. Bioluminescence from single bacterial cells exhibits no oscillation. Biophys J 1980; 31: 301-12. Hastings JW, Gibson Q. Intermediates in the bioluminescent oxidation of reduced flavin mononucleotide. J BioI Chern 1963;238:2537-54.

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13

19. Hastings JW, Balny C, Le Peuch, C, Douzou P. Spectral properties of an oxygenated luciferase-tlavin intermediate isolated by low-temperature chromatography. Proc Natl Acad Sci 1973 ;70:3468-72. 20. Nealson K, Hastings JW. Luminescent bacterial endosymbionts in bioluminescent tunicates. In: Schwemmler W, Schenk J, eds. Endocytobiology, Berlin: Walter de Gruyter & Co, 1980: 461-6. 21. Mackie G, Bone Q. Luminescence and associated effector activity in Pyrosoma (Tunicata pyrosomida). Proc Roy Soc London Ser B 1978;202:483-95. 22. Nealson K, Platt T, Hastings JW. The cellular control of the synthesis and activity of the bacterial luminescent system. J Bact 1970;104:313-22. 23. Eberhard A, Burlingame A, Eberhard C, Kenyon G, Nealson K, Oppenheimer N. Structural identification of autoinducer of Photobacterium jischeri luciferase. Biochemistry 1981 ;20:2444-9. 24. Fuqua C, Winans S, Greenberg EP. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu Rev MicrobioI1996;50:591-624. 25. Bassler B, Losick R. Bacterially speaking. Cell 2006;125:237-46. 26. Booth C, Nealson K Luminous bacteria from the ocean emit no light. Biophys J 1975;15:56a. 27. Herring P, Watson M. Milky seas: a bioluminescent puzzle. Marine Observer 1993;63:22-30. 28. Miller S, Haddock S, Elvidge C, Lee T. Detection of a bioluminescent milky sea from space. Proc Natl Acad Sci 2005;102:14181-4. 29. Nealson K, Hastings JW. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Appl Environ Microbiol 2006;72:2295-7. 30. Hastings JW. Bioluminescence, microbial. Encyl Microbiol 2000; 1:520-9. 31. Li L, Hong R. Hastings JW. Three functional luciferase domains in a single polypeptide chain. Proc Natl Acad Sci 1997;94:8954-8. 32. Li L, Liu L, Hong R, Robertson D, Hastings JW. N-terminal intramolecularly conserved histidines of three domains in Gonylaulax luciferase are responsible for loss of activity in the alkaline region. Biochemistry 2001 ;40: 1844-9. 33. Schultz W, Liu L, Cegielski M, Hastings JW. Crystal structure of a pHregulated luciferase catalyzing the bioluminescent oxidation of open tetrapyrrole. Proc Natl Acad Sci 2005;102:1378-83. 34. Liu L, Hastings JW. Two different domains of the luciferase gene in the heterotrophic dinotlagellate Noctiluca miliaris occur as two separate genes in photosynthetic species. Proc Nat! Acad Sci 2007; 104:696-70 1. 35. Liu L, Wilson T, Hastings JW. Molecular evolution of dinotlagellate luciferases, enzymes with three catalytic domains in a single polypeptide. Proc Natl Acad Sci 2004;101:16555-60.

14

36.

37.

Hastings JW

Liu L, Hastings JW. Novel and rapidly diverging intergenic sequences between tandem repeats of the luciferase genes in seven dinoflagellate species. J Phycol 2006; 42:96-103. Hastings JW. Biological diversity, chemical mechanisms and evolutionary origins of bioluminescent systems. J Mol Evol 1983; 19:309-21.

BIOLUMINESCENCE OF SHARKS, A CASE STUDY: ETMOPTERUS SPINAX 1M CLAES,I,2 1 MALLEFET 1,2 1Laboratory of Marine Biology, Catholic University of Louvain, 3 Place Croix du Sud, Kellner Building, B-1348 Louvain-la-Neuve, Belgium 2 Biodiversity Research Centre Email: [email protected]

INTRODUCTION Bioluminescence arose independently in a wide range of species, from bacteria to fishes, which are the only luminous vertebrates. Consequently, luminescent species demonstrate a great diversity in the structure, in the control, as well as in the function of their photogenic system.! Among luminous organisms, cartilaginous fishes are probably the least investigated group and incredibly few information is available concerning their bioluminescence.' Even if it has been once suggested for some sharks of the genius Somniosus and Megaschasma,J·4 symbiotic luminescence, common in teleosts, seems unlikely in chondrichtyes, This group contains however numerous self-luminous species, with at least one species of ray (Benthobatis moresbyi), and probably more than 50 different sharks (-13% of current shark species).,,6 Luminescent sharks belong to 2 squalid families, the Etmopteridae (lantern sharks) and the Dalatiidae (dwarf mesopelagic sharks), which evolved separately 90 million years ago, it is therefore possible that the bioluminescence arose 2 times independently in sharks: Until now, only information regarding the photogenic structures of these sharks is available in the literature. Dalatiidae have photophores constituted of a single photocyte (=photogenic cell) placed in a pigmented cup and covered by a lens formed by a group of small cells, while photogenic organs of Etmopteridae are more elaborated, composed of a pigmented sheath containing several photocytes, one of several lens cells, and an iris-like structure which has been suggested to allow a control of light emission:· 7 In both groups photocytes have granules supposed to contain the luminescent materia!.",8" Luminous sharks have also a specialized squamation allowing photophore accommodation in the skin.' The physiological control, the biochemistry, and the function of bioluminescence in these fishes remain totally unknown due to a lack of experimental data. Based on simple observation of the luminous pattern, authors have suggested that Dalatiidae would use their luminescence for counterillumination while Etmopteridae could in addition use it as a schooling aid. The aim of this work is to use morpho-physiological techniques to investigate the control and the function of bioluminescence in the velvet belly lantern shark Etmopterus spinax, a common etmopterid species. 15

16

Claes JM & Mallefet J

MATERIALS AND METHODS In February and December 2007, specimens of E. spinax (22.5-52.5 cm TL, total length) were collected in the Raunefjord, Norway. Light microscopy, fluorescence microscopy, and digital imaging analysis software were used to investigate bioluminescence of embryos and free-swimming specimens. We followed the elaboration of the luminous pattern and the development of photophores to determine when they become able to produce light. The density, the size of photophores, as well as the ventral surface occupied by photophores and luminous tissues were calculated for all the sharks. Peroxide-induced luminescence was also recorded from luminous tissues of 30 different sharks, grouped by 10 cm categories, via a luminometer Berthold FB12. Light response was standardized using the maximal intensity of light in megaquanta per second per square centimetre for each luminous zone (Lmax in Mq.s-l.cm-\ A theoretical visual model was equally performed using these data as well as photophore density to estimate maximum visual range of luminous zones and the depth at which these zones match the downwelling light in adult sharks (> 30 cm). A first screening of classical neurotransmitters and hormonal drugs was performed on adult sharks to investigate the control of luminescence in E. spinax. RESULTS AND DISCUSSION We have established the sequential visualization of 9 different luminous zones during E. spinax embryogenesis (Fig.l). We followed the organogenesis of photophores which is a well controlled process whose the last observable event is the apparition of fluorescent vesicles inside the photocytes. These vesicles are also observed in photophores of adult E. spinax and E. lucifer (Fig. 2A). At this moment photophores can emit light after peroxide application. Spontaneous luminescence in embryos confirms that they are able to luminesce before birth (Fig. 2B). During embryogenesis the ventral surface covered by photophore and luminous zone increase, and attains 38% and 82%, respectively. During this period, the diameter of photophores increases while their density decreases. Although the number of tested embryos is limited, it seems that light capabilities induced by peroxide application attained its maximum just before birth (Fig. 3). All these results strongly suggest camouflage by countershading in juveniles, more subject to predation than adults. The maximum theoretical visual ranges were obtained at 700 m when the shark is on its back, a behaviour frequently observed in aquarium. Even though these ranges were relatively weak «1.5 m) they could be an aid for species recognition, for mating, and for schooling in E. spinax. All the zones would match the downwelling light around 600 m, a depth at which adults of this species are found in the Mediterranean Sea which would therefore be also able to counterilluminate. 1o

Bioluminescence of Sharks

17

Fig. 1. Luminous pattern of E. spinax. Numbers correspond to appearance order of zones: I, rostral; 2, ventral; 3, caudal; 4, infra-caudal; 5, mandibular; 6, pectoral; 7, pelvic; 8, lateral; 9, infra-pelvic.

2. (A) Photocytes' fluorescent vesicles (arrow) present in the centre of a photophore of E. lucifer microcospy). Scale bar = 50 J.l.m. (B) Self glowing embryo (11 em TL) of E. spinax. Arrow indicates the insertion of the yolk sac. Scale bar = I em. s:>..

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Fig. 4. Drugs triggering light in E. spinax. SNP = Sodium nitroprusside (NO-donor). Mt 7 (except for Melatonin. Pt = prolactin. N prolactin for whieh N 3). Concentrations: KCI = 0.2 M, others 10-3 M. Control= H20, 0.35 M.

18

Claes JM & Mallefet J

Results of the pharmacological screening studies are shown in Fig. 4. Response to KCI as well as to GABA and 5HT strongly suggests a nervous control of luminescence in E. spinax. Moreover, high responses to melatonin and prolactin are in favour of an additional hormonal control of luminescence, which has never been highlighted in a fish before. NO-donor (SNP), could have a modulator role in control of luminescence of E. spinax as in Argyropelecus hemigymnus, a luminous teleost." ACKNOWLEDGMENTS Research is supported by a F.N.R.S. grant to JM Claes. J Mallefet is Research associate for the F.N.R.S. (Belgium). We would also like to thank EJ Warrant and DE Nilsson for their help in evaluating the luminescence visual range of E. spinax. Contribution to Biodiversity Research Centre. REFERENCES 1. Wilson T, Hastings JW. Bioluminescence. Annu Rev Cell Bioi 1998;14:197230. 2. Reif WE. Functions of scales and photophores in mesopelagic luminescent sharks. Act ZooI1985;66:111-8. 3. Berland B. Copepod Ommatokoita elongata (Grant) in the eyes of the Greenland shark - a possible cause of mutual dependence. Nature 1961;191:829-30. 4. Herring PJ. Tenuous evidence for the luminous mouthed shark. Nature 1985; 318:238. 5. Alcock A. A naturalist in Indian seas. London: Murray 1902:236. 6. Hubbs CL, Iwai T, Matsubara K. External and internal characters, horizontal and vertical distribution, luminescence, and food of the dwarf pelagic shark Euprotomicrus bispinatus. Bull Scripps Inst Oceanogr 1967;10:1-64. 7. Oshima H. Some observations on the luminous organs of fishes. J Coli Sci, Imp Univ, Tok 1911;27:1-25. 8. Seigel JA. Revision of the dalatiid shark genus Squaliolus: Anatomy, systematics, ecology. Copeia 1978;4:602-14. 9. Munk 0, Jorgensen JM. Putatively luminous tissue in the abdominal pouch of a male dalatiine shark, Euprotomicroides zantedeschia Hulley & Penrith, 1966. Act Zool 1988;69:247-51. 10. Coelho R, Figueiredo I, Bordalo P, Erzini K. Depth distribution of the velvet belly lantern shark, Etmopterus spinax, in southern Portugal. Abstract of the 2005 Annual ICES Conference, Aberdeen, UK. 11. Kronstrom J, Holmgren S, Baguet F, Salpietro L, Mallefet J. Nitric oxide in control of luminescence in hatchetfish Argyropelecus hemigymnus. J Exp BioI 2005;208:2951-61.

CHEMIEXCITATION MECHANISM FOR CYPRIDINA (VARGULA) AND AEQUOREA BIOLUMINESCENCE

T HIRANO, 1 H OHBA, 1 Y TAKAHASHI, 1 S MAKI, 1 S KOJIMA, 1 H lKEDA,2 H NIWA 1 JDept of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan; 2Dept of Applied Chemistry, Grad School of Engineering, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Email: [email protected]

INTRODUCTION Bioluminescence of the ostracod Cypridina (Vargula) and the jellyfish Aequorea produce light with the substrates, Cypridina luciferin and coelenterazine, which have the imidazo[1,2-a]pyrazin-3(7H)-one (imidazopyrazinone) ring. A remarkable characteristic of the bioluminescence is a high quantum yield of light production (
Oy-O-oH

~~"(0 Cypridina luciferin

coelenterazine

coelenteramide

(phenolate anion)

(phenolate anion)

MA TERIALS AND METHODS Light emission from chemiluminescence reactions was monitored with a luminometer (PMT: Hamamatsu R5929) at 25 ± 1°C. Chemiluminescence quantum yields (
20

Hirano T et al.

Lichrospher ODS column. Quantum chemical calculations [B3LYP/6-31G(d)] were performed using the Gaussian 03 program. RESUL TS AND DISCUSSION Chemiexcitation quantum yield. As a chemiluminescence reaction condition for 1, we used aerated diglyme solutions containing acetate buffer (pH 5.6, 0.66% v/v). This is the solvent system discovered by Goto et ai, in which Cypridina luciferin 3 chemiluminesces with a high
0t{

~"9

.chemiexcitation

0""'r ~~~ ~J( _ jV Ar N a:Ar=~M" Ar ~ +

+

N

NH

H

1

b: Ar

=3-indolyl

c: Ar =

-©-oH

3 (N-H form of

-co2

~

N

•

Ar

0Yl

jV ~ 1

2

NH

* __

2 + hv

"

dioxetanone)

Fig. 1. Chemiluminescence reaction mechanism of 1 in diglyme/acetate buffer Chemiexcitation mechanism for Aequorea bioluminescence. The indolyl at C6 of Cypridina luciferin plays an essential role as an electron-donating group for the efficient chemiexcitation mechanism, the ICT TS --+ SI route in the CTIL , mechanism. ,2 Because 4-oxidophenyl (O--C 6 H4 ) at C6 of coelenterazine phenolate anion is also a good electron-donating group, the above mechanism will be applicable to the Aequorea system. We have already clarified that the SI state ofcoelenteramide

Chemiexcitation Mechanism for Cypridina and Aequorea Bioluminescence

21

phenolate anion is the bioluminescence light-emitter with an leT character. 4 To evaluate the character of the transition state (TS) of the dioxetanone decomposition, we performed DFT calculations of dioxetanones having 4-hydroxyphenyl [3e(OH)] and 4-oxidophenyl [3e(0-M+)] and of the corresponding TSs as Aequorea-model molecules and states (Table 1). We chose Li+ and Na+ as counter cations in 3e(0-M+) for changing the electron-donating ability of 0-. Energies (DE) are the values relative to each 3, which indicate the activation energies of the dioxetanone decompositions. The J.1, qDo, qNHPy, and qAr values are dipole moment and the total Mulliken charge densities of the atoms constituting the dioxetanone, NH-pyrazine, and aryl moieties, respectively. Because the Lewis acidity of Li+ is stronger than that of Na+, the electron-donating ability ofO-Li+ is weaker than that ofO-Na+. Then, the order of the electron-donating ability ofthe substituents (R) is O-Na+ > O-Li+ > OH. The order of D.E, 3e(0-Na+) < 3e(0-Li+) < 3e(OH), indicates that the electron-donating R accelerates the thermal decomposition of 3. The J.1 and q data indicate that the leT character of 3-TS becomes strong with increase of the electron-donating ability of R. The leT character of 3e(0-Ln-TS is similar to that of the reported 3a-TS having an electron-donating 4-(dimethylamino)phenyl, while 3e(OH)-TS has a weak leT character.! The evidence that s for Ie in diglyme/acetate buffer is smaller than that for Ia indicates that the electron-donating ability of 4-hydroxyphenyl of Ie is not enough to increase s. To reproduce the high efficiency of Aequorea bioluminescence, we can postulate that the chemiexcitation occurs from the dioxetanone intermediate having an electron- donating 4-oxidophenyl, not 4-hydroxyphenyl. Table 1. Relative energies (D.E), dipole moments (J.1) and Mulliken charge densities (q) for dioxetanones 3 and transition states 3-TS of the dioxetanone decompositions calculated by 83LYP/6-31 G(d)

Substrate or state 3c(OH)

D.E I kcal mol-! 0.00

3c(OH)-TS

29.32

3c(O Lij

0.00

3c(O-Lt)-TS 3c(O Na+) 3c(O-Na+)-TS

25.07 0.00 22.42

J.1ID (D.J.1t 2.35 5.85 (+3.50) 9.21 18.79 (+9.58) 12.68 23.90 (+ 11.22)

qDo (D.qt 0.049 -0.128 (-0.177) 0.037 -0.244 (-0.281) 0.031 -0.285 (-0.316)

qN~)ta (M: -0.181 -0.062 (+0.119) -0.219 -0.058 (+0.161) -0.238 -0.066 (+0.172)

qAr (D.qt 0.074 0.152 (+0.078) 0.130 0.296 (+0.165) 0.158 0.358 (+0.200)

Differences between the values for 3-TS and the corresponding 3. 4 The results of the previous fluorescence study on coelenteramide phenolate anion

a

22

Hirano T et al.

and the quantum chemical calculations described here indicates that the 4-oxidophenyl of coelenterazine plays important roles as an electron-donating group for the efficient chemiexcitation (Figure 2). The 4-oxidophenyl induces the strong ICT character ofTS and SI for preventing the intersystem crossing to the triplet state during chemiexcitation. Generation of the ground state of coelenteramide from TS will not be preferred, because of the difference in their ICT character. Therefore, the 4-oxidophenyl of coelenterazine leads the chemiexcitation process in Aequorea bioluminescence to the efficient mechanism, the ICT TS-- 8 1 route in the CTIL mechanism.

~':'O]\

O?'f-R

1.'1

O-,R N

B·H

d ~,

$

9

0

,..

[

E9 B-H

NH

;r"

N~H,C.H5

,0

Nx.NH

11'''(6 ..Q,.!T ,

c'

CH,C,H,

~~NH

~

--------

N-H form dioxetanone (phenolate anion)

0yR 1*

0'

0

transition state (T5) (strong ICT)

$

B.H

90

'T ~ I

N~H,C.H,

;:,.::

_ coelenteramide 51 + CO 2 phenolate anion (strong ICT)

c9 o'

SO + cO 2 (weak ICT)

E9 B·H

d ""

eo ,..

I

0yR ,Nx.NH N'

CH,C,H,

Fig. 2. The ICT TS---+S 1 route in the CTIL mechanism for Aequorea bioluminescence [R = 4-hydroxyphenyl]

REFERENCES 1.

2.

3. 4.

Hirano T, Takahashi Y, Kondo H, Maki S, Kojima S, Ikeda H, Niwa H. The reaction mechanism for the high quantum yield of the Cypridina (Vargula) bioluminescence supported by chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (Cypridina luciferin analogues). Photochem Photobiol Sci 2008; 7: 197-207. Isobe H, Okamura M, Kuramitsu S, Yamaguchi K. Mechanistic insights in charge-transfer-induced luminescence of 1,2-dioxetanones with a substituent of low oxidation potential. J Am Chern Soc 2005; 127: 8667-79. Goto T. Chemistry of bioluminescence. Pure Appl Chern 1968; 17: 421-41. Mori K, Maki S, Niwa H, Ikeda H, Hirano T. Real light emitter in the bioluminescence of the calcium-activated photoproteins aequorin and obelin: light emission from the singlet-excited state of coelenteramide phenolate anion in a contact ion pair. Tetrahedron 2006; 62: 6272-88.

SITE-DIRECTED MUTAGENESIS OF LAMPYRlS TURKESTAN/CUS LUCIFERASE: THE EFFECT OF CONSERVED RESIDUE(S) IN BIOLUMINESCENCE EMISSION SPECTRA AMONG FIREFLY LUCIFERASES SAMAN HOSSEINKHANI, NARGES KH T AFRESHI, MAJID SADEGHIZADEH, RAHMAN EMAMZADEH, BIJAN RANJBAR, HOSSEIN NADERI-MANESH Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran, 14115-175. Email: [email protected]

INTRODUCTION Bioluminescence (BL) is the emission of visible light in living organisms. Firefly luciferases catalyze a two-step oxidation of luciferin in the presence of A TP, Mg2+ and molecular oxygen to produce light, oxyluciferin, CO 2 and AMP. I Since even a few photons can be detected using available light-measuring technology, luciferase based technology is a powerful tool, e.g., red-emitter luciferases are suitable for imaging and for multiple labeling in whole cells as well as for dual reporter applications. 2 Emission of red bioluminescence is unusual among beetle luciferases. Differences in bioluminescence color are caused by: (1) natural species variations in luciferase structure;3 (2) amino acid substitutions introduced by mutagenesis techniques;4 (3) in vitro substitutions of analogues of luciferin and ATP. 5 Most investigations on light emission changes to red wavelengths have been focused on the North American 6 firefly Photinus pyralis. Based on these results, four mechanisms have been proposed to explain color variations in beetle luciferases. 6 Despite the determination of the structure of P. pyralis and Luciola cruciata, with and without ligand, respectively, detailed mechanism for the bioluminescence color change is still unclear. 6 ,7 The sequence alignment of primary structure of Phrixothrix in comparision with green light emitters, showed the presence of Arg 353 in Ph RE luciferase. In this regard, an Arg was inserted in L. turkestanicus luciferase. 8 In addition, a set of red-emitter mutants of L. turkestanikus luciferase on the basis of sequence homology and similar mutation in other species were made by site directed mutagenesis. MATERIAL AND METHODS Site directed mutagenesis. The mutants including S284T, H245N, H431 Y and insertion mutagenesis were prepared by SOE-PCR. Mutagenesis primers, F-Cloning containing Bam HI restriction site (5' -CGT TGG ATC CAT GGA AGA TGC AAA AAA TAT TAT G-3') and R-Cloning containing HindIII restriction site (5'-CAG CAA GCT TIT ACA ATT TAG ATT TTT ITC 23

24

Hosseinkhani S et al.

CCA TC-3') along with F- and R-mutant primers were designed. The overlapping mutagenesis primers containing the mutation codon were made for each mutant. The plasmid carrying the native luciferase was used as template. Two PCRs were carried out using F-mutantR-cloning and F-cloning:R-mutant by Pfu polymerase. The primary amplicons were purified (Qiagen, USA) and mixed in a I: I molar ratio and second PCR performed. The mutagenesis products, digested with BamHIIHindIlI, were inserted into the BamHIIHindIlI restriction sites of digested/dephosphorylated pET28a high expression vector and ligated mixtures were transformed into the competent cells of Escherichia coli BL21 byelectroporation. Protein expression and purification. E. coli colonies harboring the expression plasmid of native or mutant luciferases were inoculated and grown at 37°C. The purification of6X His-tagged fusion protein was performed by Ni-NTA spin column as described by the manufacture (Qiagen, USA). Determination of kinetic parameters. ATP and luciferin kinetic parameters were measured at 25°C with injection of 50 JlL of diluted enzyme to the substrate solution in various concentrations of ATP and luciferin. 8 Bioluminescence spectra. BL spectra were recorded using a Cary-Eclipse luminescence spectrophotometer (Varian) from 400-700 nm wavelengths. Sequence alignment and homology modeling. Sequence alignment and homology modeling were done using Ebi (www.ebi.ac.uk) and WISS-PROT (http://swissmodel.expasy.orgl) servers. RESUL TS AND DISCUSSION Multi-alignment showed the presence of Arg353 in Ph RE luciferase, which corresponds to the deleted residue in firefly luciferases (Fig. 1A). Moreover H245, S284 and H433 are in the conserved regions (data not shown). Bioluminescence spectra. As is depicted in Fig 1B amongst mutants, only the S284T mutant exhibits a single peak in the red region which is also reported for a similar mutant of P. pyralis luciferase,2 suggesting that a single substitution at this position (284) is sufficient to cause a complete shift to the red region. As indicated in Fig 1B, H245N, H431 Y and Luc (Arg) exhibit a bimodal spectrum with a maximum in the red region (at 615 nm) and a smaller shoulder at 560 nm in the green region, whilst the native luciferase exhibits a spectrum with only a peak at 555 nm. Kinetic properties of native and mutant luciferases. As is shown in Table 1, mutations have adversely affected the performance of the enzyme activity in S284 T and H431 Y. However, the specific activity of H245N and Luc (Arg) mutant luciferases is higher than other known mutants (76.6% and 81% of wild type, respectively). This may indicate (similar to P. pyralis) that the imidazole ring of His245 is not necessary to maintain highly efficient decay of the oxyluciferin excited state. 6 Arg 356 (in Luc (Arg) mutant) has been inserted in a region containing a flexible loop 352TPEG-DDKP359. Structural and molecular modeling studies indicate (not

Site-Directed Mutagenesis of Lampyris Turkestanicus Luciferase

25

shown) that the flexible loop is engaged in a network of many intermolecular and ionic bonds with the other residues in the backbone. Table 1. Kinetic and spectral properties of wild type and mutant enzymes. Asterisks identity minor peaks. Error associated with Km ± 10% Mutants Wild type S284T H245N H431Y rLuc(Arg)

Quantum Specific . Amax(nm) Yield *1014 activity*IOI3 RelatIve pH = 5.5 (RLU/s/mg) ~tlVlty pH = 7.8

LUCI'f,' ATP enn Km Km(IlM) (IlM) 16.12 30 23 26 24

135 248.4 168 187.2 142

I 0.5 0.9 0.14 OJ

1.5±0.IS 0.36 ± 0.04 1.15±0.14 0.25 ± 0,07 0.73 ± 0.14

100 24 76.6 16.6 81

560 555 618 619 572*,617 617 564*,612 619,564* 558*,616 618,560·

Optimum temperature ("C) 24 30 24 24 34

ITPEG-GWLHT ITPEG-GWLHT ITPEG-GWLHT ITPEG-GIiIKHT ITPEG"'GWLHT ITPEG-GWLHT ITPEG-GWLHS ITPEG-GWLHS ITPEC~ClIILlIS

ITPEG.,GWLHS

lTAEG;;'CWIHS LSPNDl.!.GWLHT

.

. *"";::

1. (A) Partial multiple sequence alignment (for more data refer to the bioluminescence emission spectra produced by the wild-type and mutant luciferases-catalyzed oxidation of lucifer in at pH 7.8.

26

Hosseinkhani S et al.

However, insertion of Arg in a loop changes the peptide backbone conformation and makes the emitter site more accessible to the polar solvent. Substitution of His 431 with Tyr changed the color to red. It seems that the mutation of His 431 to Tyr which is 12 A from the active site has a strong effect on the catalytic activity of the enzyme. The X-ray data for luciferase showed that the His431 residue is located in a region containing a flexible loop Tyr425-Phe433 6 The imidazole ring of His431 forms a hydrogen bond with the carboxyl group of Asp429. This hydrogen bond fixes the position of the imidazole ring and increases the rigidity of the flexible loop and upon its mutation to Tyr and disruption of H-bond makes the color red. Our results emphasize the importance of certain specific residues and regional structure in determination of bioluminescence color among firefly luciferases,' i.e., in spite of differences in primary structure of firefly luciferases and variation in their color, some of conserved residues among different species are critical for color determination. REFERENCES 1. White EH, Rapaport E, Seliger HR, Hopkins T A. The chemi- and bioluminescence of firefly luciferin: An efficient chemical production of electronically excited states. Bioorg Chem 1971; 1: 92-122. 2. Branchini BR, Southworth TR, Khattak NF, Michelini E, Roda A. Red and green emitting firefly luciferase mutants for bioluminescent reporter application. Anal Biochem 2005; 345:140-8. 3. Viviani VR, Bechara EJH, Ohmiya Y. Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structure. Biochemistry 1999;38:8271-9. 4. Ohmiya Y, Hirano T, Ohashi M. The structural origin of the color differences in the bioluminescence of firefly luciferase. FEBS Letts 1996;384:83-6. 5. DeLuca M, Leonard NJ, Gates BJ, McElroy WD. The role of I,N6_ ethenoadenosine triphosphate and 1,~ -ethenoadenosine monophosphate in firefly luminescence. Proc Nat! Acad Sci USA 1973;70: 1664-6. 6. Branchini BR, Southworth TL, Murtiashaw MH, Boije H, Fleet S E. A Mutagenesis study of the putative luciferin binding site residues of firefly luciferase. Biochem 2003; 42: 10429-36. 7. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature 2006; 440:372-6. 8. Tafreshi N Kh, Sadeghizadeh M, Emamzadeh R, Ranjbar B, Naderi-Manesh H, Hosseinkhani S. Site-directed mutagenesis of firefly luciferase: Implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases. Biochem J 2008:in press.

CHEMILUMINESCENT AND BIOLUMINESCENT ANALYSIS OF PLANT CELL RESPONSES TO REACTIVE OXYGEN SPECIES PRODUCED BY A NEW WATER CONDITIONING APPARATUS EQUIPPED WITH TITANIA-COATED PHOTO-CATALYTIC FIBERS TKAGENISHI,' K YOKAWA,' C LIN",2 K TANAKA,2 R TANAKA,2 T KAWANO' I Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan; 2K2R Inc" Kitakyushu 807-0871, Japan Email: [email protected].}p

INTRODUCTION A water conditioning photo-catalytic apparatus (exPCA W1.2, K2R Inc., Kitakyushu, Japan) equipped with the sheets of Ti0 2-coated photo-catalytic fibers was applied for preparation of water rich in reactive oxygen species (ROS). Interestingly, the conditioned water has an unusual long-lasting ROS-generating nature. One likely use of the conditioned water is controlling the biological responses of living plant cells. It is known that various physiological and biochemical events during the plant life cycle, such as germination of seeds, induction of defense mechanism against pathogenic microorganisms and adaptation to severe environments, are controlled by ROS. To assess if the level of ROS produced in the conditioned water remained at the level actively inducing the responses of living plant cells, we tested the responses of tobacco cell suspension culture (BY-2, expressing aequorin gene) to addition of the water treated with exPCA Wl.2. Presence of superoxide anion in the conditioned water-treated cell suspension culture was detected with Cypridina lucifer in analog (CLA) chemiluminescence and the movement of calcium ion (mediated with ROS-responsive calcium channels) across the plasma membrane was assessed with aequorin luminescence in the presence and absence of specific inhibitors. MATERIALS AND METHODS Water conditioning photo-catalystic apparatuses (Fig. 1) were fabricated by K2R Inc. These apparatuses have photo-catalystic titanium-coated fibers and UV-A (360 nm) bulbs to enable the photo-dependent excitation of Ti0 2. The exPCA Wl.2 is also equipped with two ultrasonic (USW) generating devices for mixing. When required, O2 alone or O 2 and NO were supplied to the system through artificial lung (equipped to minimize the impacts of bubbles) connected with air pump. Monitoring of the dissolved oxygen (DO) level was required for enabling the optimal generation of superoxide in the water. Water from the water tank (5 L) was maintained at ca. 20°C and circulated at 20 Umin. A superoxide-specific chemiluminescence probe, Cypridina luciferin analog (CLA; 2-Methyl-6-phenil-3, 7 -dihydromidazo[ 1,2-a]pyrazin-3-one) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). All other reagents were from Sigma (St. Louis, MO, USA). Cell suspension-cultured tobacco cells (cell line, BY-2) expressing 27

28

l'ca)~enlsnz

T et al.

aequorin gene were used as the model plant materials to be treated with conditionned ROS-rich water. The cell suspension culture was propagated and cells were harvested 2 weeks after sub-culturing. They were diluted with an equal volume of the fresh culture medium and incubated with I J.lM coelenterazine in the dark for 8 h as previously described. l Water for at least 30 min was sampled and added to tobacco cells (0.1 mL water to 0.5 mL culture). Aequorin luminescence was measured with CHEM-GLOW Photometer (American Instrument Co, MD, USA) and the CLA chemiluminescence was detected with Luminescensor PSN AB-2200-R (Atto Corp., Tokyo, Japan). was Cell death was assessed by staining cells with Evans Blue. Quantitative by (6 times) counting of 50 randomly chosen cells.

l. Water conditioning photo-catalytic apparatus. Two exPCA Wl.2s connected in

tandem (left) and the diagram of water conditioning system (right).

RESULTS AND DISCUSSION In the cell suspension culture treated with the processed water, CLA chemiluminescence (Fig. 2) and aequorin luminescence (Fig. 3) of superoxide and increase in cytosolic calcium ion concentration respectively, were measured. We observed the spikes of CLA chemiluminescence in the cell suspension culture after addition of photo-catalytically processed waters, that photo-catalytic process generated or conditioned the waters enabling the stimulation of plant cells with oxidative stress.

Chemiluminescent and Bioluminescent Analysis of Plant Cell Responses

~L

29

Q) (.)

10min

~ u

8000 7000 6000 c: 5000 .4000 S' 3000 - i: 2000 1000 Q) 0 J: u

o without fiber

~

uv +

uv

USW

+ 02

~._J. ",. ~_

_j'~USW •

• Control

•

+

USW

I

i

+ 0

Ii

J' / • ,I

~

§ 'E -

with fiber

Control

« ..J U

UV

UV

+

+

+

USW

USW

USW

+ 02

+ 02 +

I~ NO

UV

NO

Fig. 2. Detection of CLA chemiluminescence reflecting the superoxide generation in the cell suspension culture after addition of photo-catalytically processed waters. The effect of photo-catalytic fiber, UV, USW, O 2 and NO on superoxide generation were examined. Typical CLA chemiluminescence profiles after addition offour differently processed waters (treated in the presence of photo-catalytic fibers) or non-treated water (control) to tobacco cell suspension are shown (left). Comparing the yield of superoxide in the presence and absence of the photo-catalytic fibers (right). Arrows (left) indicate the timing of water addition.

~L uV+USW+02 (+ Tlron)

60

;;

1~

~

~ •

.. uv+ usw + 02 3 ~ (+ La +) ~

..... ,.....,'1 ,.

~" ",".~

'''1_

(+ Ai3+)

II..

•

;;;

50

~ 40

....

:;l

30

'ii o

20

-0

10

Control

uv + USW

UV +

usw + 02

Fig. 3. Effect of photo-catalytically processed waters on induction of calcium influx into tobacco cells and cell death. Aequorin luminescence reflecting the changes in [Ca2+]c (left) and increase in Evans Blue stained cells reflecting the cell death (right). Arrows indicate the timing of water addition. Superoxide was obviously abundant in water treated by the UV-driven photo-catalytic fiber USW-assisted water processing process in the presence of gaseous O2 . Further

30

Kagenishi T et al.

addition of NO gas did not drastically affect the yield of superoxide. B

45 -;- 40 g 35

.

.

~ 30

.5

~

25 20

'j§ 15 ~ 10 Y

:Jo Control

uv + usw uv + USW + 02

5

0

~~~~~

Control

__~~~-L_ _

UV + USW

UV + USW + 02

Fig. 4. Summary of aequorin luminescence analysis (left) and CLA chemiluminescence analysis (right). Increase in the aequorin luminescence was observed after treatment of the tobacco BY -2 cells with photo-catalytically processed waters (Fig. 3, left). The processed water-dependent increase in aequorin luminescence was inhibited by Tiron, a scavenger of superoxide; La3 +, calcium channel blocker; and AI 3 +,abd TPCI, a known inhibitor of ROS-responsive calcium channels. These data suggest that the photochemically processed water stimulates the opening of ROS-responsive calcium channels through the action of ROS, especially superoxide. 2• 3 Following ROS production and [Ca2+]c increase, we observed that cell death was also enhanced (examined 2 h after addition of water), suggesting that the oxidatively conditioned water can be used in the control of cell viability (Fig. 3, right). Fig. 4 is the statistical confirmation of the actions of oxidatively cond itioned waters on calcium influx and superoxide production through repeated experiments. UV, USW and oxygenation (supply of O 2 gas) may be synergistically stimulating the responses. ACKNOWLEDGEMENTS This study was supported in part by Knowledge Cluster Initiative from Ministry of Education, Culture, Sports, Science and Technology. REFERENCES I. Kawano T, Sahashi N, Takahashi K, Uozumi N, Muto S. Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: The earliest events in salicylic acid signal transduction. Plant Cell Physiol 1998;39:721-30. 2. Kawano T, Kadono T, Furuichi T, Muto S, Lapeyie F. Aluminium-induced distortion in calcium signaling involving oxidative bursts and channel regulations in tobacco BY-2 cells. Biochem Biophys Res Commun 2003;308:35-42. 3. Kawano T, Kadono T, Fumoto K, Lapeyrie F, Kuse M, Isobe M, Furuichi T, Muto S. Aluminium as a specific inhibitor of plant TPCI Ca2 + channels. Biochem Biophys Res Commun 2004; 324: 40-5.

pH-TOLERANT MUTANTS OF LUCIOLA MINGRELICA LUCIFERASE CREATED BY RANDOM MUTAGENESIS MI KOKSHAROY, NN UGAROY A Dept o/Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia E-mail: [email protected]

INTRODUCTION The majority of firefly luciferases demonstrate highly pH-sensitive bioluminescence spectra. Bioluminescence maximum of the luciferase from Luciola mingrelica (Lml) shifts from 566 to 618 nm when lowering pH from 7.8 to 6.0. It is usually assumed that depending on the conformation of the luciferase the reaction product - excited state oxyluciferin - can exist in two distinct molecular forms producing green and red light respectively. I A gene region coding first 225 residues of Lml was subjected to random mutagenesis using an error-prone PCR method. The mutant libraries were screened by in vivo bioluminescence assays to identify color-shifted colonies. Several mutant enzymes with decreased pH-sensitivity were identified and studied. Single and double substitutions were found which make bioluminescence spectra of Lml nearly pH-insensitive.

METHODS General methods. Luciferase activity, kinetic constants and bioluminescence emission spectra were determined as described previously.2 Random mutagenesis and colony screening. The initial 680 bp region ofLml gene was amplified by an error-prone PCR under standard conditions. 3 The concentration of 0.2 mM Mn2+ was used in error-prone PCR that resulted in about 50% active clones. The fragment obtained was digested with NheI and BamHI restriction endonucleases and ligated to pLR3. The plasmid pLR3 is a derivative of pLR2 with three additional cloning sites. Escherichia coli XLI-blue was transformed with resulting ligated DNA and plated on LB agar with ampicillin. Plates with 500-3000 colonies harboring Lml mutants were screened by in vivo bioluminescence using a digital camera. Interesting clones were picked onto LB plates and their bioluminescence was compared with wild-type luciferase (WT). Cell-free extracts were prepared for the mutants with a changed color and adequate brightness and bioluminescence spectra were evaluated at pH about 8 and 6. Protein purification. For a protein expression Lml mutants were subcloned into vector pETL4 encoding Lml with N-terminal 6xHis-tag. This vector was obtained by transferring multiple cloning site from vector pET28a to pET23b ("Novagen", USA) and subsequent ligation of luciferase gene from pLR3. Mutant proteins were purified by chromatography on Ni-IDA Sepharose ("Amersham", Sweden). 31

32

Koksharov MI & Ugarova NN

RESUL TS AND DISCUSSION Screening for different bioluminescence color. Though at pH 7.S Lml produces yellow-green light in vitro, E. coli colonies expressing wild-type Lml have yelloworange color due to lower intracellular pH combined with pH-sensitivity of Lml bioluminescence spectra. During screening, a number of yellow-green, greenyellow, orange-red clones were obtained. Bioluminescence spectra of cell-free extracts confirmed that greenish mutants produce such color due to the lowered pHsensitivity of their bioluminescence spectra. In this study we have focused our attention on this group of mutants. Catalytic and bioluminescent properties. Several mutants with increased pHtolerance which produced more bright or similar colonies compared to WT were selected for sequencing. Mutant enzymes were purified and studied. Corresponding substitutions and properties are given below. Since there were often multiple substitutions relative to WT, presumably key mutations responsible for the change ofbioluminescent properties are shown in bold. Table 1. Catalytic and bioluminescent properties of mutant enzymes

Enzyme WT MTS MT2 MT3 MT4

MT6

SIISC F16L, Il9T Y35N Y35H, KI91R YIIF, F16L, A40S, SI18C

Amax (fwhm), nm

Relative specific activity 100 130

LH2

ATP

pH 7.S

pH 6.1

33 ± 3 34 ± 3

260 ± 30 230 ± 30

566 (76) 566 (75)

610 (96) 610 (94)

60

45 ± 3

155 ± 30

564 (70)

610shoulder

70

34 ± 3

265 ± 30

564 (67)

564 (65)

60

n.d.

n.d.

564 (67)

564 (65)

20

41 ± 4

235 ± 20

564 (67)

566 (70)

Km (/lM)

567 (90)

Mutant MT2 shows significantly lower pH-sensitivity than WT. Since FI4R was shown to lower bathochromic shift of the Photinus pyralis luciferase,4 the corresponding substitution FI6L in MT2 should be responsible for this effect. The substitution Il9T is unlikely to affect the active site judging from the crystal structure of firefly luciferase. 5 Substitution of Y35 appears to be the most effective among other known mutations reducing pH-sensitivity of firefly luciferases: single mutation of Y35 to Asn or His (mutants MT3 and MT4 respectively) resulted in the luciferase with nearly pHinsensitive bioluminescence spectra (Fig. I) without any appreciable contribution of the red emitter. Bioluminescent properties of both mutants were identical at all pH

pH-Tolerant Mutants of L. mingrelica Luciferase Created by Random Mutagenesis

33

studied. Additional substitution of the surface residue K191R in MT4 is unlikely to affect bioluminescent properties. The bioluminescence spectra of Y35 mutants also show increased temperature resistance: at 42°e spectra are similar to that of WT at 25°e. On the other hand the bioluminescence spectrum of WT undergoes a significant increase of red-emitting component at 42°e. 1,0

><

_ '"E 0,5

O,O-+-:;;.-..,~~~~~~~,--.~~~ -f""'>;:'-~~~~~~~,--.~~~-,

500

550

600

A,

nm

500

550

600

A,

650

nm

Fig. 1. The bioluminescence spectra of WT Lml and mutants Y35N,H at pH 7.8 and pH 6.1 (t=25°C) Mutant MT6 was produced on the basis of the previously obtained mutant S 118e which showed bioluminescence spectra similar to WT. Surface residue at the 11th position differs greatly among pH-sensitive firefly luciferases and unlikely to affect bioluminescence spectra. Hence combining F16L with additional mutation A40S also results in almost pH-insensitive luciferase. Analysis of the crystal structures of the wild-type Luciola cruciata luciferase and a red-emitting mutant S286N in complex with a high-energy intermediate analogue (DLSA)5 allows to propose a possible explanation of the striking effect of the single substitutions Y35N,H. In the case of red-shifted mutant the structure is almost identical to that of the WT and differs only in the orientation of N236 and 1288 which resulting in a more opened conformation of the oxyluciferin (LO) microenvironment. The only other significant difference is the orientation of the loop 233-237 (Fig. 2): in green-emitting WT the residue P235 makes a close contact with Y35 while in the "red" mutant they are separated by 3.9 A. It is possible that such loop conformation is necessary for the green emission. The substitution of a bulky Y35 to a smaller Asn or His could stabilize the close packing of P235 and 35 th residue thus sustaining enzyme in the native green-emitting conformation and compensating for deteriorative effects of low pH and elevated temperature. The reason for the effect of substitutions FI6L and A40S is less clear. Perhaps they provide a more dense packing that stabilizes the native conformation of Lml.

34

Koksharov MI & Ugarova NN

structure of WT firefly luciferase) showing the positions of mutations of a loop in "opened" red-emitting conformation

REFERENCES

3.

V. The origin, diversity, and structure function of insect Mol Life Sci 2002;59: 1833-50. Maloshenok Uporov I, Koksharov M. Bioluminescence spectra mutant firefly luciferases as a function of 2005 1262-7. error-prone

BACTERIAL BIOLUMINESCENCE WITH FLA VINMONONUCLEOTIDE ACTIVATED BY N-METHYLIMIDAZOLE OI KRASNOV A, NA TYULKOV A, 10 DOROSHENKO

Institute of Biophysics, Russian Academy of Sciences, Siberian Branch Krasnoyarsk, Russia, E-mail: [email protected]

INTRODUCTION Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide (FMNH 2) and a long chain aldehyde by molecular oxygen to yield FMN, the corresponding acid, H 20 and blue-green light. Luciferase is highly specific in relation to the reduced flavin mononucleotide. I A negative charge on the phosphate is considered necessary for correct orientation of the substrate on luciferase and for Nstabilizing active conformation of the enzyme - substrate complex?,3 methylimidazole derivatives increase the reactivity of a number ofnucleophiles, such as functional amino acid residues of the active center of an enzyme or the site of the flavin binding. 4 In this work the interaction of bacterial luciferase with flavin mononucleotide which was activated on the phosphate group was investigated. MATERIALS AND METHODS Purification of luciferase. Luciferase was isolated from the recombinant strain of Echerichia coli SL60 with lux A and B genes of Photobacterium leiognathi from the collection of the Institute of Biophysics (RAS, SB) and purified by ion-exchange chromatography.5 The purity of the luciferase according to Laemmli electrophoresis was 90-95%. Preparation of flavin mononucleotide. The activation of FMN (Sigma, USA) was realized using N-methylimidazole in the presence of 3-phenylphosphine and dipyridyl disulfide, maintaining a molar ratio according to the method suggested for 0ligonucleotides. 3 The degree of activation was 100%. The photoreduction of the FMN was conducted in a glass "shirt" on a light, at 25 DC in the presence of 0.0 I M EDTA. Chemical reduction of the activated FMN derivative was produced with nicotinamide adenine dinucleotide (NADH) or dithiothreitol (DTT) directly in the reaction mixture. Measurement of luciferase activity. This was conducted by the following two methods: 1. Luciferase activity was determined by a flavin injection assay in which 500 ~L of photo reduced flavin mononucleotide were rapidly mixed with 500 ~L ofa solution containing luciferase, tetradecanal (4.7 mM) and 20 mM of phosphate buffer at pH 7.0; 2. NADH or DTT was added to the cuvette, containing luciferase, tetradecanal and phosphate buffer and FMN. Under these conditions long-lived luminescence stimulated by multiple turnovers of enzyme was observed. Values of the rate constants of inhibition of the first order reaction were determined as a tangent of the angle of declination on the plot of log of intensity versus time. The rate constant of inhibition of the second order reaction calculated in this way, 35

36

Krasnova 01 et al.

approximated the dependence of the rate constant of inhibition of the first order on the concentration of the activated flavin derivative. RESULTS AND DISCUSSION The injection of activated photoreduced FMN into the reaction mixture does not produce luminescence. At the same time luciferase does not lose its catalytic activity. The addition of native photoreduced FMN to the same mixture resulted in light response with appropriate kinetic parameters. The incubation of luciferase with nonreduced activated FMN also results in irreversible inactivation of the enzyme (Fig. 1).

Fig. 1. The second order rate constant for inactivation of the luciferase by nonreduced activated flavin mononucleotide. The activated FMN derivative obviously modifies no more than one functional group on the protein according to the calculated coefficient. The value of the rate constant of inhibition of the first order reaction was about 400 M-1min- l , suggesting low modifying ability of the nonreduced FMN derivate. The addition of the second substrate of luciferase - tetradecanal - to the mixture before addition of the activated FMN derivate did not protect the luciferase from inactivation. The addition of the protector of SH-groups - OTT (8 mM) resulted in the essential removal of inhibition. The inhibition was about 20% within 45 min with the nonreduced form of FMN and was completely removed by incubation with the photoreduced form of activated FMN derivate in the presence of the protectors in the reaction mixture. The activated FMN derivative should react with SH-groups of luciferase which are known to be good nucleophiles. 4 However, the presence of OTT in the reaction mixture, or its direct co-addition with the flavin does not turn the photoreduced activated flavin a substrate of the reaction. The value of light emission intensity with native FMN under the multiple turnovers of the enzyme achieved a maximum when the concentration of OTT in the mixture

Bacterial Bioluminescence with Flavinmononucleotide Activated by N-Methylimidazole

37

was equal to 40 mM. Under these conditions the long-lived luminescence was observed. The use of activated FMN also results in the long-lived luminescence of about 25 % intensity of that with native FMN. Km values determined by LinewearBurk linearization have shown activated FMN to compete with the native form for the active center of luciferase (Km values of 17xl 0- 6 M and 5xlO-7 M, respectively, for activated and native FMN). Km value for tetradecanal in the reactions with native and activated flavin differed negligibly and were 1.4xlO-8 M and 5.6xlO-8 M, respectively. Thus the presence ofN-methylemidazole reduces the affinity offlavin mononucleotide for luciferase by one order of magnitude, but it is not essential for binding of aldehyde. Thus we have shown, that activated FMN derivative can contact not only with SHgroups of luciferase resulting in its inactivation, but can also penetrate into the active Centre of the enzyme producing light-emission. The protection of SH groups is not the determining factor of catalytic activity of luciferase with activated FMN. If NADH is used for the reduction of flavin mononucleotide and the protectors of SH groups are not added into the reaction mixture, the activated FMN can still function as a substrate for luciferase and light-emission is observed. The presence of N-methylinidazole should increase the electronegative properties of the phosphate group, causing some structural modifications, which are suggested by the absorption spectra and the molar absorption coefficient obtained for the activated derivative (Fig. 2).

WanJength(nm)

Fig. 2. Absorption spectra of native FMN (1), nonreduced activated FMN (2), photoreduced form of activated FMN (3). Chemical reduction of activated flavin by DTT or NADH as donors of protons, apparently does not result in the modifications of the structure of the flavin derivate molecule. When using a chemically reduced substrate we have found that the affinity of the aldehyde for luciferase differed insignificantly from the native form. Thus it is

38

Krasnova 01 et al.

possible to assume, that the phosphate group compared to ribityl, does not influence binding of the aldehyde with luciferase. The site of binding the phosphate group is suggested to be an area, of high density of the key residues. 6 It is considered that either hydrogen bonds, or electrostatic interactions are produced between atoms of oxygen of the phosphate group and amino acid residues of the flavin site. 6 We assume, that the introduction of Nmethylimidazole may result in the elimination of the atoms of oxygen and the change of its charge, which can exclude the formation of the one of the bonds, characteristic of native phosphate group. On the other hand, the redistribution of electron density increases electronegative properties of the free oxygen atom of the phosphate, causing therefore, the amplification of electrostatic interactions of the activated substrate with luciferase. The possibility of a covalent bond between the activated phosphate group and functional groups of luciferase also seems probable since amino acid residues in the active center are a good nucleophiles.

REFERENCES l. 2.

3.

4.

5. 6.

Baldwin T, Nicoli M, Becvar J, Hastings JW. Bacterial luciferase: binding of oxidized flavin mononucleotide. J BioI Chern 1975; 250:2763-8. Meighen E, MacKenzie E. Flavine specificity of enzyme-substrate intermediates in the bacterial bioluminescent reaction. Structural requirements of the flavine side chain. Biochem 1973;12:1482-9l. Kurfurst M, Macheroux P, Ghisla S, Hastings JW. Bioluminescence emission of bacterial luciferase with 1-deaza-HMN. Evidence for the noninvolvement of N(l)-protonated flavin species as emitters. Eur J Biochem 1973; 181 :453-7. Buneva VN, Godovikova TS, Zaritova VF. Modification of RNAse by di- and oligodeoxyribonucleotide 5'-phospho-N-methylimidazolide derivatives. Bioorgan Chim 1986;12:906-10. Illarionov BA, Tyulkova NA. Escherichia coli bacterial strain SL-60 - producer ofbacterialluciferase. Invention Patent 1997:N 2073714. Tanner J, Mitchell D, Tu S-C, Miller S. Structure of bacterial luciferase homodimer: Implications for flavin binding. Biochem 1997;36:665-72.

NEW METHOD OF MEASURING BACTERIAL BIOLUMINESCENCE OI KRASNOV A, NA TYULKOV A, 10 DOROSHENKO Institute 0/ Biophysics, Russian Academy o/Science, Siberian Branch Krasnoyarsk, Russia, E-mail: [email protected]

INTRODUCTION Bacterial luciferase catalyzes the oxidation of a long-chain aliphatic aldehyde to the corresponding fatty acid and water with the emission of light in the visible range. The enzyme is interesting not only for its capacity to emit light which is not specific for the flavin monooxygenases, but also for its application in bioluminescent assays. For the bacterial luciferase to show its activity, reduced flavin mononucleotide is required in the reaction mixture. Flavin can be reduced either catalytically with H2 over a platinum electrode or by reaction with sodium dithionite in anaerobic conditions,I.2 and also by bubbling hydrogen in the presence of a few crystals of palladium on activated carbon. 3 Another method is reduction of flavin by Cu(l ).4 In some of these cases the bioluminescent reaction is recorded as short flashes of light. In particular, fast autooxidation of photoreduced FMNH2 in a photoreduced FMNHz-initiated reaction makes the enzyme complete one cycle and this makes possible study of the kinetics of the process. The prolonged glow is produced by reduction of FMN with NAD(P)H-FMN - oxidoreductase,5 thus making it possible to employ the bioluminescent reaction to analyze the content of dehydrogenases in biological fluids and certain xenobiotics and toxic agents. We present the results on FMN reduction using either dithiothreitol (DTT) or nicotinamide dinucleotide (NADH) to produce a steady-state luminescent kinetics in a one-enzyme system. METHODS We used the recombinant luciferase from Escherichia coli SL-60 strain with cloned genes lux A and lux B of the luciferase from Photobacterium leiognathi luminescent bacteria. The bacterial biomass underwent one freezing-thawing cycle at -20 °C followed by cell disruption with ultrasound. The cell debris was discarded by centrifugation at 20,000g for 30 min. The extract was chromatographed with DEcellulose column equilibrated with 0.02 M K-Na-phosphate buffer, pH 7 and eluted with the stepwise gradient (0.3 M ofNaCl). The luciferase-containing fractions were loaded into MonoQ column in FPLC system (Pharmacia, Sweden) equilibrated with O.OIM K-Na-phosphate buffer (pH 7,0) and eluted in the range of 0.2-0.4 M of NaCI. The yield of luciferase was 8-10 mL with NaCl varying from 0.3 to 0.36 M. Protein concentration was evaluated with microbiuret reaction. Luciferase samples were 95% pure on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Luciferase activity was measured with a bioluminometer designed at the Institute of Biophysics (Russian Academy of Sciences, Siberian Branch) and 39

Krasnova OJ et al.

40

calibrated by the luminol technique. Kinetic parameters were measured with LKBWallac 2210 recorder (Finland) in a glass cell with 10 j.tL of luciferase, 50 )lL of FMN, 50 j.tL of aqueous solution of tetradecanal and 840 j.tL of 0.02 M phosphate buffer. The reaction was initiated by the addition of 50 )ll of aqueous solution of NADHorDTT. RESULTS AND DISCUSSION The addition of saturating concentrations of tetradecanal and FMN (0.047 )lmol and 0.038 )lmol, respectively) and 200 mM phosphate buffer pH 7, solutions of dithriothreitol or NADH into a mixture with 5-100 )lg of luciferase resulted in a luminescence, with an intensity-time profile depending on the concentrations of added components (Fig. 1). 70

60

;>

50

~

40

e

NADH

'0

--= Q,j

.S

.c:~ ~

30

DTT

20

10

0.01

0.02

0.03

0.04

0.05

Concentration of reducing agent, mM

Fig. 1. The luciferase activity with the addition of NADH and DTT. With the addition of NADH, the glow was detected after NADH had reached 1 )lmol and it showed maximum at 10-12 )lmol. For DTT the glow was detected starting at a concentration of 2-3 )lmol and it reached maximum at 40 )lmol, with the maximum intensity being an order of magnitude lower than that with NADH. Light emission decay constants also differed considerably. Even though with NADH the luminescence intensity is higher it decayed faster with kr = 1,5x1O -3S-'. With DTT the reaction can run for several hours. During the first 30-40 min the luminescence intensity was constant, and then showed a gradual decay with a decay rate constant kr =4.7 x1O- 5 s-'. The constants were calculated for the reaction of 10 )lg of

41

New Method of Measuring Bacterial Bioluminescence

luciferase and in either case both the intensity and the rate of reaction increased with the enzyme concentration. The specific activity of luciferase was 1.1 x 10 12 quanta S'I per 1 mg of luciferase with DTT and 1.7 x 10 13 quanta S'I per 1 mg of luciferase with NADH, which is 3-4 orders of magnitude lower than in photoreduction of FMN, but, it is correspond to the intensity of luminescnece of a coupled assay with the flavin oxidoreductase. The dependence of luminescence intensity on FMN concentration is governed by Michaelis-Menten kinetics when the reaction is inhibited by excess FMN.

3.8

3.3

-

2.8

..... 0==

Q..

'0"'

2.3

fIJ

,.Q

<

1.8

1.3

0.8 0.3 1..:::...-_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _--1 300

320

340

360

380

400

420

Wavelength, nrn

440

460

480

500

Fig. 2. Effect of DTT addition on the absorption spectra of FMN. 1 min of reduction-I; 5 min of reduction-2; 10 min of reduction-3; 15 min of reduction-4, after shaking-So Under the saturating concentrations of NADH and DTT the values of Km for FMN were 9x 10'7 M and 5x 10'7 M, respectively. These data are close to those in the reaction with photoreduced FMN. It should be emphasized that increasing molarity of the buffer inhibits the light emission reaction, probably, due to decrease of flavin mononucleotide reduction, and in 100 mM phosphate buffer the reaction is completely inhibited.

42

Krasnova O[ et al.

Variation of pH of buffer increased light emission intensity with the shift towards the alkali range from pH 5 to pH 8. At pH 8.0 the intensity of the reaction increased two-fold compared to that observed at pH 7.0. At the same time the light emission decay constant increased by two orders probably due to destabilization of luciferase in alkaline solutions. Light emission was shown by our experiments to require negligibly small amounts of reduced FMN which we failed to detect with a spectrophotometer. Therefore, to detect the reduction ofFMN in the presence of DTT and NADH we have performed spectrophotometric measurements with double the concentrations of FMN and reducing agents. The marked changes observed in FMN differential spectra confirmed the assumption that FMN is directly reduced by these reagents (Fig. 2).

REFERENCES 1. Meighen EA, Bartlet I. Complementation of subunits from different bacterial luciferases: evidence for role of the p-subunit in the bioluminescent mechanism. J BioI Chern 1980; 255:11181-7. 2. Hastings JW, Balny C. The oxygenated bacterial luciferase-flavin intermediate. J BioI Chern 1975; 250:7288-93. 3. Mitchell G, Hastings JW. The effect of flavin isomers and analogues upon the color of bacterial bioluminescence. J BioI Chern 1969; 224:2572-6. 4. Lei BF, Becvar JE. A new reducing agent of flavins and its application to the assay of bacterial luciferase. Photochem Photobiol 1991; 54:473-6. 5. Gunsalus-Miguel A, Meighen EA, Nicoli MZ, Nelson KH, Hastings JW. Purification and properties of bacterial luciferases. J BioI Chern 1972; 247:398-404.

ENHANCEMENT OF THERMOSTABILITY OF LUCIOLA MINGRELICA FIREFLY LUCIFERASE BY MUTAGENESIS OF NON-CONSERVATIVE RESIDUES CYS62 AND CYS146 GY LOMAKINA, Y A MODESTOV A, NN UGAROV A Dept o/Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia, e-mail: [email protected]

INTRODUCTION Beetle luciferases contain from four (Photinus pyralis) to thirteen (Elateridae) free SH-groups. It can be expected that the essential difference in the luciferase stability depends on the SH-group number. The Luciola mingrelica luciferase contains eight Cys residues. Three of them are absolutely conservative (82, 260, 393) and five are non-conservative (62, 86, 146, 164,284). Single replacements of the conservative Cys residues to Ala in L. mingrelica luciferase didn't alter the catalytic properties and stability of the enzyme. I Apparently, significant difference in the luciferase stability is determined by the number of non-conservative Cys residues, especialy those in which SH-groups are located in the vicinity or on the surface of the protein globule and are accessible to oxidation. Titration of SH-groups showed that L. mingrelica luciferase contained three surface CYS.2 The analysis of the luciferase spatial structure showed that Cys62 and Cys 146 residues were located on the surface of the protein globule and had a hydrophilic environment. This is the reason why an oxidation-resistant Ser residue was chosen for point mutation experiments due to its hydrophilic and isosterical to Cys properties. In this study the site-directed mutagenesis of Cys62 and 146 to Ser in L. mingrelica luciferase was performed. The effect of these mutations on thermal inactivation of the enzyme in the absence and in presence of dithiothreitol (DDT) and on the activity of L. mingrelica luciferase was studied. MA TERIALS AND METHODS Materials. Restriction endonuclease Nhel was from Fermentas and BamHI was from Boehringer Mannheim. T4 DNA ligase, Pfu and Pfu Turbo DNA polymerase were from SibEnzyme. Oligonucleotides were from Sinto!. D-Luciferin was synthesized as described earlier. 3 ATP and dithiothreitol (DTT) were from ICN. All other chemicals were of analytical grade. Site-directed mutagenesis was carried out by PCR using PLR plasmid (6270 base pairs) encoding the L. mingrelica luciferase gene (1644 bps). The following primers were used: 5'-GATATTACATCTCGTTTAGCTGAGGCCATG-3' for Cys62Ser mutant and 5'-CCACGATTCTATGGAAACTTTTAT TAAG-3' for Cys146Ser mutant. Escherichia coli competent cells (LE 392) were transformed with mutant plasmids and used for the enzyme purification till homogeneity as described earlier. I High-purity enzymes were stored at -70°C in the working buffer (0.05 mol/L Tris43

44

Lomakina GY et al.

acetate buffer, 2 mmol/L EDTA, 10 mmol/L MgS0 4 , pH 7.8). Luciferase activity was measured as maximal bioluminescence intensity (lmax) at saturated concentrations of substrates (1 mmollL ATP, 0.3 mmollL LH 2) on a FB 12 luminometer (Zylux Corp, USA). Km values for LH2 and ATP were determined by varying substrate concentrations in the range 4-40 umollL LH2 at4 mmollL ATP and 0/04-0.40 mmollL A TP at 1 mmollL LH 2. Measurements were carried out in trip Iicate. Irreversible inactivation of luciferase. Aliquots of luciferase solution (10- 8 , 10-7 and 10-6 mollL), 30 /-lL, in the working buffer were incubated at 37°C in the presence and in absence of 12 mmol/L DTT. At certain time intervals the microtubes were taken out of the thermostat and immediately chilled on ice for 10 min before activity measurements. The residual activity was measured at 22°e. Kinetic constants and statistics were calculated using Origin 6.0 software. Fluorescent spectra were recorded with Perkin Elmer LS-50B spectrofluorimeter at enzyme concentration of 10-6 mol/L in working buffer. RESUL TS AND DISCUSSION Expression, purification and characterization of mutant luciferases. Mutant plasmids encoding point mutations Cys62Ser and Cys 146Ser were prepared by PCR using pLR plasmid carrying the gene of L. mingrelica luciferase and mutations were confirmed by sequence analysis. The wild type (WT) enzyme and its mutant forms were purified to homogeneity (90-95%). The catalytic activity of the Cys62Ser mutant was equal to that of the WT enzyme, while the activity of the Cysl46Ser mutant was 1.5-fold higher. The Km values for both substrates of the WT and mutant enzymes were the similar: K m.LH2 = 20 ± 3 /-lmollL and K m.ATp = 0,18 ± 0,04 mmollL. These mutations also did not change the intrinsic fluorescence spectrum (Amax = 340 nm). Irreversible inactivation of L. mingrelica luciferase at 37°C. Thermal inactivation of the WT luciferase and its mutants was investigated at 37°C at various enzyme concentrations (10- 6 _10- 8 mollL). Two-exponential time-curves of thermo inactivation corresponding to the fast and the slow phases of inactivation were observed. The fast (k/) and slow (k2) inactivation rate constants of the luciferase were shown to be dependent on the enzyme concentration. This phenomenon is typical for oligomeric enzyme when monomers are less stable than 0Iigomers. 4 •5 At the concentration 10-6 mollL, the rate constants k/ and k2 of WT and mutant enzymes were similar. At 10-7 mollL enzyme and lower, the k/ and k2 values increased both for the WT and mutant luciferases, but constants k/ and k2 of mutant forms were four to six times lower that those of the WT (Table 1). Thus, mutations significantly increased the stability of luciferase at both stages of inactivation.

Enhancement of Thermostability of L. mingrelica Firefly Luciferase

45

Table 1. Kinetic rate constants of thermal inactivation for the WT firefly luciferase Luciola mingrelica and its mutant forms at 37°C at the enzyme concentration 10-6 - 10-& mollL. Conditions: 0.05 mol/L Tris-acetate buffer, 2 mmollL EDTA, 10 mmollL MgS0 4 , pH 7.8. WTenzyme

Mutant Cys62Ser

Mutant Cysl46Ser

Enzyme concentration, mollL

kb min

10-·

0,05±0,OJ

0,0 J6±0,005

0,06±0,02

0,020±0,006

0,04±0,OJ

0,OJ2±0,OO3

10-7

0,34±0,02

0,074±0,003

O,JO±O,OJ

0,OJ6±0,005

0,06±0,OJ

0,OJ6±0,004

10-"

O,39±0,04

0,070±0,O09

0,J6±0,03

0,034±0,OO9

0,07±0,02

0,0 J5±0,OO3

-I

-I

k,. min

-I

-I

k/. min

k" min

-I

kb min

-I

k,. min

The protective effect of DTT for SH-containing enzymes is well known. We compared the inactivation of 10-7 mollL WT and mutant enzymes at 37°C in the absence and presence of 12 mmol/L DTT. In the presence of DTT, the kJ value of WT did not change, whereas k2 decreased three-fold. Therefore, the addition of DTT stabilizes the WT only at the second step of inactivation. For mutant Cys62Ser, the addition of DTT caused a two-fold decrease in kJ but almost did not change k2 • For mutant CysI46Ser, DTT had no effect on either kJ or k2 (Fig.I). Our results show that inactivation of firefly luciferase includes two processes: inactivation due to oxidation of SH-groups and denaturation. In the absence of DTT the inactivation rate constant k2 is a sum of two constants: denaturation and inactivation caused by the oxidation of SH-groups. In the presence of DTT, the k2 value. is just a denaturation constant. Replacement of the Cys residues with solvent-exposed SHgroups resulted in the elimination of the oxidative inactivation. Thus, the k2 value of WT in the presence of DTT and that of mutants in the absence of DTT became similar. Thus, Cys62Ser and CysI46Ser mutations led to the significant stabilization of the enzyme at fast and slow phase of inactivation independently of the DTT presence. Elimination of the surface non-conservative Cys residues resulted in the exclusion of the oxidative inactivation effect.

46

Lomakina GY et ai.

In(A/Ao) I!

(l~..

(J

WT

C62S

C146S

I~~

I

J()

20

30

':t~

4()

(]

J

~I 3~1--~__~__~__~_

_ _L I -_ _L -_ _L I -_ _

10

20

30

40

IJ

I()

20

30

40

t, min Fig. 1. Time-courses of enzyme inactivation at 37°C of WT Luciola mingrelica luciferase and its mutant forms with point mutations Cys62Ser and Cysl46Ser in the absence (filled triangles) and presence (open circles) of 12 mmollL dithiothreitol. 7 The enzyme concentration was 10- mollL. For other reaction conditions, please refer to Table 1.

ACKNOWLEDGEMENTS The research was supported by Russian Foundation for Basic Researches (Project 08-04-00624-a).

REFERENCES 1. Dementieva E, Kutuzova G, Zheleznova E, Lundovskikh I, Ugarova N. Physicochemical properties of recombinant Luciola mingrelica luciferase and its mutant forms. Biochemistry (Moscow) 1996;61: 115-9. 2. Brovko L, Ugarova N, Kinetics and mechanism of inactivation and reactivation of immobilized luciferase from fireflies Luciola mingrelica and the role of sulfhydryl in these processes. Biokhimiia (in Russian) 1980;45:794-801. 3. Taleborovskaya I, Katkova V, Ryzhova V, Shchegolev A, Berezin IV Synthesis of D-luciferin, USSR patent 1983: 1192324. 4. Poltorak 0, Chukhray E, Torshin 1. Dissociative thermal inactivation, stability, and activity of oligomeric enzymes. Biochemistry (Moscow) 1998;63 :303-11. 5. Lundovskikh I, Dementieva E, Ugarova N, Kinetics and mechanism of thermo inactivation of firefly luciferase Luciola mingrelica. Moscow Univ Chern Bu112000;41: 362-6.

WEB-RESOURCE: "BIOLUMINESCENCE AND LUMINOUS ORGANISMS" OF THE IBSO CULTURE COLLECTION SE MEDVEDEV A, DA KOTOV, EK RODICHEVA Institute a/Biophysics a/the Siberian Branch a/the Russian Academy a/Science, 660036, Krasnoyarsk, Russia, [email protected]

INTRODUCTION Bioluminescence is one of the most fascinating and unique phenomena in nature. The ability to emit visible light is a peculiar feature of several groups of organisms. The major luminous organisms, both in species composition and in number, are the marine inhabitants. The smallest ones are luminous bacteria and they provide a splendid visual marker that is convenient for basic research and applied studies. The Institute of Biophysics SB RAS hosts and maintains a specialized collection of luminous bacteria (CCIBSO 836) containing over 700 strains isolated from various regions of the World Ocean and recombinant Escherichia coli strains with luxgenes. The wide application of bioluminescence in medicine and ecology brings forth the problem of analyzing the information on the structure and functioning of bioluminescent systems in natural and transgenic microorganisms, as well as features that are closely interrelated with bioluminescence. Luminous bacteria are studied intensively including, their distribution in the World ocean, symbiotic models, metabolic peculiarities and regulation of luminescent system, and application in biotechnology, medicine, and ecological monitoring. CCIBSO is not only a storage of genetic material, but also a resource for fundamental and applied studies, and an important channel for global information interchange. I, 2 The aims of the CCIBSO information network system are: gathering of information on microorganisms with lux-genes, its analysis and free access, and distribution of these data throughout the global network. Bioluminescence is the basic attribute for inclusion of data into the information system. Information in one easy-to-access source will be of great help to researchers in the area of bioluminescence and its applications in biotechnology, bioremediation, biotesting, and molecular biology. WEB RESOURCE The information system contains several modules: the Web-portal "Bioluminescence and luminous organisms", the database "BIOLUMBASE" of natural and genetically modified luminous microorganisms, the electronic catalogue of cultures maintained in CCIBSO, information concerning history of bioluminescence studies, different properties and expressions of bioluminescence, methods and devices to measure bioluminescence, reagents for bioluminescent analysis and also applied programs for administration of the information system (Fig. I ). The developed structure of the database will reflect all types of properties and communications of relevant material,

47

48

Medvedeva SE et al.

to provide a fast way of reception of the information on any interesting attribute, as well as areas of use of natural luminous bacteria and their bioluminescent systems.

Fig. 1. Structure of Web-resource ofCCIBSO The biggest part of the Web-resource is an electronic collection of microorganisms that consists of two parts. One part is the Electronic Catalogue of luminous bacteria stored in CCIBSO 836. Here there is brief information about the history of CCIBSO, list of strains, media and methods of storage. The second part is a database "BlOLUMBASE" (DB). At present the database "BIOLUMBASE" is designed for natural (NM) and genetically modified (TM) luminous microorganisms bearing lux-genes? They form two separate but interconnected sections. DB "BlOLUMBASE" is created to collect information that can be useful in different aspects of microbial ecology, medicine and biotechnology where the luminous bacteria, luminescent systems selected from them and lux-genes cloned in others organisms are used. To create the DB "BIOLUMBASE" we developed appropriate database and client software. 3,4 Each of the DB sections contains logic blocks with detailed information, arranged in a tabular form, on some specific characteristics. The information on the functioning and regulation of luminescent systems and lux-operons is presented in the tables of the key block "Luminescent system". This block includes the subsection "Luminescent system of bacteria" of section NM and the subsection "Operon" of section TM. The key block contains the major characteristics of bacterial luminescent systems and serves as a bridge for access to the other DB sections. Here the variety of data on luminescent systems of bacteria is provided: phenomenology, structure, interaction with the general metabolism of a cell, effect of physical and chemical factors on luminescence etc. Information about mechanisms, regulation and variability of gene expression, and lux-operon structure of the known luminous bacteria, is also included. The database "BIOLUMBASE" contains various information about luminous bacteria, structural organization and regulation mechanisms of luminescence of known species of luminous bacteria, as well as the documents created by other appendices. Users can receive catalogue information about strains and their properties, functions,

Web-Resource: "Bioluminescence and Luminous Organisms"

49

application, bibliography references, to make a search of strains on any set of attributes (Fig. 2). The information contained on pages of this unit is extracted from the database by means of SQL-inquiry, processed by a server and is then displayed as HTML-pages.

Sal

Web

HTMl

Server ; ,

-

users\

Int~:~\~! ;, W~

~~ ~~

I

HTML

!~~

Sal t------_;Adm;n;slralor :

,-------, BIOLUMBASE Server

~Interfaces

Fig. 2. The scheme presents two BIOLUMBASE sections - "Natural Luminous Microorganisms" and "Transgenic Luminous Microorganisms" provided in the database Server. The local IBP users (LAN IBP) have an access to the DB via special software, and the Internet WWW users - via the standard web browsers.

The scientific - educational Web-portal "Bioluminescence and luminous organisms" was developed for remote access to the information stored in the database "BIOLUMBASE" (http://bl.ibp.ru). Access to the test version of Web-portal is now open. Information contained on the Web-portal is mainly devoted to the description of bioluminescence and its use in various areas of science, industry, education. On the Web-portal there are many auxiliary services facilitating access to the information: e.g., a search system allowing searches of the information by key words or complex inquiry; a professional forum for consulting experts and to exchange experience, a catalogue of Web-references facilitating search of the necessary additional information on bioluminescence. The Web-portal has an authorization system of users allowing them to obtain access to additional services and unique information (Fig. 3). Also, authorized users can participate in development of a Web-portal and add their own information (articles, reviews, hyperIinks, responses, etc.). Web-resource can be used as a scientific-educational information resource. The majority of texts are accompanied by a list of the literature in which it is possible to find more detailed information on a specific problem The basic sources to add information are: results of bioluminescence research carried out in the Institute of Biophysics of the Siberian Branch of the Russian Academy of Science; materials as books and other printed and electronic publications; information received from leading experts in the field of bioluminescence, Russian and foreign; forwarding materials of research of bioluminescence of World Ocean; databases of the natural and genetically modified luminous organisms; databases of leading world collections of cultures - holders of luminous organisms; the unspecialized databases concerning luminous organisms (databases of metabolic reactions, gene sequences, etc.); scientifically - the technical information. It provides access to different information about bioluminescence, list of luminous bacteria strains and their

50

Medvedeva SE et at.

and functions, the mechanisms of regulation of bioluminescent the constructs with lux-genes, and applications of bioluminescence in microbiology,

The main html-pages of Web-resource CClBSO "Bioluminescence and luminous organisms" medicine and biotechnology. Noteworthy is that this database will be useful for evaluation of biological hazards of transgenic strains. Users will be able to and strain searches starting from any feature of interest. The information system is accessible through the Internet we are updating the Web-portal and database with new information and the user interface. ACKNOWLEDGEMUINT by the project NQ38 of program of basic researches of the SB Work is

REFERENCES 1. Rodicheva Vydryakova Medvedeva SE. Catalogue of luminous bacteria cultures. Novosibirsk:Nauka, 1997. Medvedeva Boyandin Lankin Kotov D, Rodicheva E, L. BIOLUMBASE-the database of natural and transgenic bioluminescent Luminescence 2005;20:90-6. 3. Maklakov SV. BPwin and ERwin. CASE-tools for information 2000. S, Herbert SJA. PowerBuilder 6.0. Unleashed. Indianapolis: SAMS 4. 1997.

CHEMISTRY OF SYMPLECTIN BIOLUMINESCENCE WITH FLUORODEHYDROCOELENTERAZINE Y NAKASHIMA,' V KONGJINDA,' N TANI' M KUSE,2 M ISOBE' I Lab. Organic Chemistry, Graduated School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan 2Chemical1nstrument Division, Research Center for Materiasl Science, Nagoya UniverSity, Chikusa, Nagoya 464-8601, Japan Email: [email protected]

INTRODUCTION Symplectin is a photoprotein of flying squid (Symplectoteuthis oualaniensis L. Tobiika - Japanese name), which emits blue light (470 nm) in the presence of monocations (Na+, K+) and O 2.1 Symplectin has a chromophore comprising of dehydrocoelenterazine (1: DCL)2.3 that forms a covalent bond with the active site cysteine of apo-symplectin. Fig. 1 describes our postulated mechanism for the bioluminescent reaction of symplectin. The chromophore (2) first reacts with oxygen to produce dioxetanone intermediate (3),4.5 which quickly decomposes to afford the corresponding amide structure (4) with the emission of blue light. Finally, the amide fragments to produce apo-symplectin and coelenteramine (5). ys

apo-symplectin

HS)

Q.\

2: symplectin chromophore

)

0

\':'~

ff I '"

HO

6

)J~-0~NN ~ "" OH r

I

*

I 0'"

coelent~~aZine 1)1N~j tV?'~_ '"

1: Dehydrocoelenterazine

N)

""

I NN

+

I /. 18

(DCl)

HO

I

I""

'---_-basic)

baSjC~

II /

reSid~

-

residue

I

02 pH d.8

ys

hv(470 nm)

s)

N~JH UOH : Coelenteramide

ff ~ I'"

HO

5: coelenteramine

CO 2

O~

hydrolysis

h-

N

?"

~I

oxidized chromophore

3: dioxetanone intermediate

Fig. 1. Postulated mechanism of symplectin bioluminescence. 6 .7 51

52

Nakashima Yet al.

We are interested in identifying the active site cysteine of symplectin. During these studies, we found that an equilibrium exists between DCL and symplectin chromophore by using dithiothereitol (DIT) as a apo-symplectin model (Fig. 2). This equilibrium made it difficult to determine the active site of symplectin, since the chromophore decomposed during analysis. In order to overcome this difficulty, we have synthesized mono-fluorinated analogs (F-DCLs), which formed chromophores irreversibly even after tryptic digestion (Fig. 2).8,9

1_ 'C

HS OH

H.~~SH .)

I~ iN~)

OH (reconstitution)

.

2: symplectm

OH

SJ

o~

apo-symplectin

aU

H

w:J?" ~

chromophore

o~

OTT

dJ,~ '-~C!H

OH MeOH-CH 2 CI 2

\..

HO

I

~ ~~ ~

Y'

~

dissociation

' 1)1 I

HO

~

""

I

* I~---:;

N~N I Ij N' ,J

H) r-

apo-sym'p!~ctin (reconslitulion)

~

I

'____-basic resid~

2: symplectin

chromophore

d I~

HO

""

~

OH

o~~ I.h-

N ...-:N)

~

/.

on

I 51

NI .-/

H

r~

I.

..

diSSOCiatIOn

I

addition

::~OH

OH

H.~~SH o

0H

HO'

MeOH-CH 2 CI 2

d I

~

/./-

HO

0$ N

N

6

N H

Y"

I

""

addition

'"

Fig. 2. Equilibrium between DCL and DTT adducts.

Trypsin digestion of reconstituted symplectin with F-DCL afforded peptides that bound to the active site cysteine (MS identification). However, these results were preliminary, because we could not obtain MS/MS data of these peptides.IO,J J We therefore decided to synthesize two di-f1uoro-DCLs (diF-DCL-l and diF-DCL2) that would make the chromophore structure more stable than F-DCLs and would afford increase of + 18 Da compare to the corresponding F-DCLs peak.12

RESULTS AND DISCUSSION Bioluminescent activity of di-fluoro-dehydrocoelenterazines. We synthesized difluoro-DCLs (diF-DCL-l and diF-DCL-2),13 and then tested their bioluminescent activities. DiF-DCL-l was the most active substance for symplectin bioluminescence. DiF-DCL-l showed a higher luminescent activity than the natural DCL. We therefore concluded that diF-DCL-l is the best probe to analyze the symplectin active site. We are now searching for the position of the active site cysteine by using reconstituted symplectin with diF-DCL-l. In contrast, diF-DCL-2 showed the weakest activity among the DCLs. Our focus then shifted to understand why such differences appeared depending on the position of f1uorines. This investigation would afford fruitful insights into the molecular mechanism of bioluminescence of symplectin. 14

Chemistry of Symplectin Bioluminescence with Fluorodehydrocoelenterazine

53

Reactivity of diF-DCLs to thiol group. From UV Nis spectroscopic measurements, we found that diF-DCLs had a max at 530 nm and existed as a red colored solution; in contrast, the chromophore with DTT had a max at 450 nm and existed as a yellow colored solution. We then monitored formation of the chromophores with DTT using UV Nis spectroscopy. As a result, we found that diFDCL-2 has higher reactivity to DTT than both diF-DCL-l and natural-DCL. Furthermore, we monitored chromophore formation with symplectin by measuring its fluorescence spectra. We then found that the formation of chromophore with diFDCL-2 was a rapid reaction. Namely, diF-DCL-2 might also bind to apo-symplectin faster than diF-DCL-l. Here, a question again appeared why this diF-DCL-2 did not show any bioluminescence though a sufficient amount of chromophore with symplectin was produced. Chemical luminescence activity of diF-CLs. Di-fluoro-CLs were then tested for chemical luminescence activity. It proved that both diF-DCLs showed the same activities. We therefore concluded that diF-CL-2 has luminescence properties equivalent to those of d iF -CL-l. Working hypothesis. We now postulate that the absolute configuration of the chromophore in the symplectin active site might be important based on these results. Although both substrate (DCL) and product (amide compound and coelenteramine) have no chirality (Fig. 1), we now assume that dynamic chirality of the active site could play an important role in bioluminescence. We are now trying to analyze the reaction products in order to answer the question why diF-DCLs have different activity. ACKNOWLEGEMENTS We acknowledge the financial supports from Gant-in-Aid for Specially Promoted Research [16002007(2004)] from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We also special thanks to financial support to MK for Gant-in Aid for Young Scientists [19780087(2007)] from the MEXT. REFERENCES 1. Tsuji Fl, Leisman G. K+lNa+-triggered bioluminescence in the oceanic squid Symplectoteuthis oualaniensis. Proc Nat Acad Sci USA 1981 ;78:6719-23. 2. Takahashi H, Isobe M. Symplectoteuthis bioluminescence (1) --- structure and binding form of chromophore in photoprotein of a luminous squid. BioMed Chern Lett 1993;3:2647-52. 3. Takahashi H, Isobe M. Photoprotein of luminous squid, Symplectoteuthis oualaniensis and reconstruction of the luminous system. Chern Lett 1994;8436. 4. Usami K, Isobe M. Two luminescent intermediates of coelenterazine analog, peroxide and dioxetanone, prepared by direct photo-oxygenation at low temperature. Tetrahedron Lett 1995;36: 8613-6.

54

5.

Nakashima Yet al.

Usami K, Isobe M. Low-temperature photooxygenation of coelenterate luciferin analog --- synthes is and proof of 1,2-dioxetanone as lum inescence intermediate. Tetrahedron 1996;52: 12061-90. 6. Isobe M, Takahashi H, Usami K, Hattori M, Nishigohri Y. Bioluminescence mechanism on new systems. Pure Appl Chern 1994;66:765-72. 7. Isobe M, Fujii T, Swan S, Kuse M, Tsuboi K, Miyazaki A, Feng MC, Li J. Chemistry of photoproteins as interface between bioactive molecules and protein function. Pure Appl Chern 1998;70:2085-92. 8. Fujii T, Ahn JY, Kuse M, Mori H, Matsuda T, Isobe M. A novel 60 kDaphotoprotein from oceanic squid (Symplectoteuthis oualaniensis) with sequence similarity to mammalian carbon-nitrogen hydrolase domains. Biochem Biophys Res Commun 2002;293:874-9. 9. Isobe M, Kuse M, Yasuda Y, Takahashi H. Synthesis of 13C_ dehydrocoelenterazine and model studies on Symplectoteuthis squid bioluminescence. Bioorg Med Chern Lett 1998;8:2919-24. 10. Kuse M, lsobe M. Synthesis of 13 C-dehydrocoelenterazine and NMR studies on the bioluminescence of a Symplectoteuthis model. Tetrahedron 2000;56: 2629-39. 11. lsobe M, Fujii T, Kuse M, Miyamoto K, Koga K. 19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin. Tetrahedron 2002;58: 2117-26. 12. Kurahashi T, Miyazaki A, Suwan S, Isobe M. Extensive investigations on oxidized amino acid residues in H20 2-treated Cu, Zn-SOD protein with LCESI-Q-TOF-MS, MS/MS for the determination of the copper-binding site. J Am Chern Soc 2001 ;123:9268-78. 13. Kuse M, Kondo N, Ohyabu Y, Isobe M. Novel synthetic route of arylaminopyrazine. Tetrahedron 2004;60:835-40. 14. Kuse M, Doi I, Kondo N, Kageyama Y, Isobe M. Synthesis of azide-fluorodehydrocoelenterazine analog as a photoaffinity-Iabeling probe and photolysis of azide-fluoro-coelenterazine. Tetrahedron 2005;61 :5754-62.

MECHANISMS OF HEAVY ATOM EFFECT IN BIOLUMINESCENT REACTIONS EV NEMTSEV A, 1,2 TN KIRlLLOV A,2 TV BRUKHOVSKIH,I NS KUDRYASHEVA 1,2

Dept of Biophysics, Siberian Federal University, Krasnoyarsk 660041, Russia 2/nstitute of Biophysics SB RAS, Krasnoyarsk 660041, Russia Email: [email protected]

J

INTRODUCTION Studying the mechanism of the effects of heavy atoms on metabolic processes is an important task for developing methods to monitor the toxic effects of halides. Bioluminescent reactions are convenient models for such studies because of simple and prompt recording of the rate of enzymatic process. The heavy atom effect is traditionally studied by physicists as redistribution of electron transition rates in the presence of bromine or iodine atoms, In the presence of heavy atoms spin-orbit coupling in the molecules is substantiaL 1,2 The spin-orbit interaction fulfils the role of the disturbance factor that removes the multiplicity prohibition. Earlier, we found that heavy-atom effect can also be observed in bioluminescent systems: 3,4 bioluminescence inhibition coefficients were found to decrease in the series potassium halides: KCl, KBr, and KI. Two mechanisms can be responsible for the change of the intensity of bioluminescence in the presence of heavy ions: the physicochemical effect of external heavy atom mentioned above, and the biochemical effect, i.e. interactions with the enzymes resulting in changes in enzymatic activity. A series of model experiments was conducted to evaluate the contribution of the physicochemical mechanism, These involved the photoexcitation of model fluorescent compounds close to bioluminescence emitters in chemical nature and fluorescent properties - flavin mononucleotide, firefly luciferin and coelenteramide. These results are clear evidence of the smaller contribution of the physicochemical mechanism to the decrease in the bioluminescence intensity for the three bioluminescent systems under study.4 To estimate the contribution of the second mechanism the interaction of bioluminescent enzymes with halide-containing dyes was investigated. Two types of dyes were tested: xanthene and anthracene derivatives. The interaction of dyes with bacterial luciferase and apo-obelin was studied. MATERIALS AND METHODS Bioluminescent enzymes: bacterial luciferase from Photobacterium leiognathi and apo-obelin from the marine hydroid polyp Obelia longissima were purchased from the Photobiology Laboratory (Institute of Biophysics, SB RAS, Krasnoyarsk, Russia). Anthracene derivatives (2-chloranthracene (CA), 9-bromanthracene (BA)

55

56

Nemtseva EVet al.

and 9-iodanthracene (IA)) and xanthene dyes (fluorescein sodium salt (FS), eosin Y (EY), erythrosine B (EB)) were purchased from Sigma. Xanthene dyes were dissolved in water, anthracene derivatives were initially dissolved in ethanol and then were used as water-ethanol solutions. The fluorescent spectroscopy techniques were used to observe the interaction of 5 dyes with macromolecules. The fluorescent spectra and steady-state fluorescence anisotropy of dyes in the presence of enzymes were determined with an AmincoBowman Series 2 spectrofluorimeter (ThermoSpectronic, USA) equipped with polarizers. RESULTS Fluorescent anisotropy was studied for xanthene and anthracene dyes in the presence of bacterialluciferase and apo-obelin. Several effects were found.

r

A

... ...

0,057 0,047

~

....

.

.........•... ..-

........

r 0,30

.

...•.. --_ ....

0,29

... - .- EB

0,037

0,28 0,27

0,027 0,017

FS

• ·-e- ----e--

0,007

0,26 ____

._._.

__

•

__

°0

_."

."_

•••

__

••

__

•

•

0

•

0,25

~-----------.------------,------------+

0, I 0

0,05

0,00 0,05

0,15

B

0,16 0,14

0,04

0,12

I~

0,03

0,1

"" .. BA

1--

0,02

0,08

CA',_ --:t:~======-==============='"

l .......

0,0 I

It... ... _

o 0,00

0,06 0,04

IA

&-- - - . . - - - - - - .... - - - - - - - - - - - - ---,\.

0,02

0,05

0,10

0,15

apo-obelin concentration,mg/ml

Fig. 1. Steady-state fluorescent anisotropy r of xanthene (A) and anthracene (B) dyes in the presence of apo-obelin: 1 - FS, 2 - EY, 3- EB (plotted to the right ordinate), 4 - CA, 5 - BA, 6 - IA (plotted to the right ordinate).

Mechanisms of Heavy Atom Effect in Bioluminescent Reactions

57

I, rel.un.

537 50,0 ., 400 JI ,

-

without luciferase

- - . in the presence of bacterial luciferase

30,0 ~ 20,0 10,0

~

0,0 +i- - - - , - - - - - , - - - - - - - ,

500

550 600 wavelength, nm

650

Fig. 2. Fluorescence spectra of EY in phosphate buffer and in the presence of bacterial luciferase; EY concentration is 10-6 M. Firstly, the initial anisotropy ro of dyes (without proteins) and the total anisotropy change Ar increase with the weight of haloid atom (Fig. 1). Secondly, anisotropy of xanthene dyes goes up with protein concentration increasing, whereas for anthracene dyes it goes down (Fig. 1). Thirdly, fluorescent anisotropy change was accompanied by a spectral shift (-10 nm for EB) and small quantum yield perturbation (Fig. 2). Also, it was found that Ar is higher for dyes in the presence of bacterial luciferase than that in the presence of apo-obelin. It may be explained by the difference in the size of two proteins under study (bacterial luciferase is about 4-fold larger than obelin). It is known that solubility in water of xanthene dyes decreases in the order FS, EY, EB (the solubility is 50%, 40% and 11 % correspondingly). High anisotropy of EB (without proteins, Fig. lA) is caused by hydratation of this polarizable molecule. The anisotropy decrease for anthracene derivatives (Fig. IB) can be explained by destruction of their massive aggregates (or micelles) as a result of hydrophobic interaction with proteins. Similar results were obtained earlier for time-resolved fluorescence anisotropy of aromatic dyes in the presence ofbacterialluciferase. 6

CONCLUSION Thus, the interaction of bioluminescent enzymes with haloid-containing dyes was found and characterized. From results obtaines can be concluded that the contribution of the biochemical mechanism (i.e. the action of the heavy atom on the catalytic activity of the enzyme) is much greater than that of the physicochemical mechanism.

58

Nemtseva EVet al.

ACKNOWLEDGEMENTS The investigation was supported by Siberian Federal University (young scientists' grant N28, 2007) and Russian foundation for basic research (N07 -04-01340).

REFERENCES 1. McGlynn SP, Azumi T, Kasha M. External heavy-atom spin-orbital effect. 1. The nature of the interaction. J Chern Phys 1962;37: 1818-24. 2. Nag-Chaudhuri J, Stoessell L, McGlynn S.P. External heavy-atom spin-orbital coupling effect. II. Comments on the effect of ferric acetylacetonate on the spectra of polyacenes. J Chern Phys 1963;38:2027-8. 3. Gerasimova MA, Kudryasheva NS. Effects of potassium halides on bacterial bioluminescence. J Photochem Photobiol B 2002;66:218-22. 4. Kirillova TN, Kudryasheva NS. Effect of heavy atom in bioluminescent reactions. Anal Bioanal Chern 2007; 387:2009-16. 5. Lacowicz J R. Principles of fluorescence spectroscopy, Springer: New-York, 2006:353-82. 6. Kudryasheva NS, Nemtseva EV, Visser AJWG, van Hoek A. Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study. Luminescence 2003; 18: 156-61.

THEORETICAL ANALYSIS ON THE ABSORPTION SPECTRA OF INTERMEDIATES OF FIREFLY LUCIFERIN IN DEOXYGENATED DIMETHYL SULFOXIDE

HIRONORI SAKAI, NAOHISA WAOA Faculty o/Life Sciences. Toyo University. l-l-l.ltakura. Gunma. 374. Japan E-mail: [email protected] INTRODUCTION The proposed reaction scheme of the chemiluminescence (CL) of firefly luciferin (Ln) is shown in Fig.l. H0V:S

I~ .&

H N

NjCOOH

S

Ln

0 v : -S

I .& ~

H

N

t-BuOK,DMSO

r

0 v~: -s

N o-o S

Dioxetanone

-

0-

1.&

~

M440-----.,..

Nyo.

H.-J -

N

S

O2 M 420 -----"----

0 v~: -S

1.&

Oxyln*

Nyo

H..J

N

S

+ Photon

Oxyln

Fig. 1. The CL reaction scheme of luciferin.

The oxygenation of Ln occurs in dimethyl sulfoxide (OMSO) dissolved in a strong base such as tert-butoxide (t-BuOK) to form an unstable dioxetanone (OOX) via formation of two intermediates; an intermediate M420 is observed only after the formation of M440, where the absorption maxima are located at 420 nm and 440 nm, respectively. OOX decomposes to electronically excited oxyluciferin (Oxyln*), which decays to its ground state with the emission of visible light. It is well known that the concentration ofbase in the solvent affects the emission color of CL. J While the CL of Ln occurs on adding O 2 into the solution, M440 and M420 can be detected only in de-aerated DMSO; at low concentrations of t-BuOK (3.6mM), M440 can be detected, and M420 can be detected at high concentrations of t-BuOK (5mM). Although the ionized states of M440 and M420 were studied qualitatively by H-NMR,2 it is still necessary to clarify the electronic properties of these intermediates, successively leading to the Oxyln*. The purpose of this work was to determine the type of ion ized states of Ln that can be assigned to M440 and M420 judging from absorption peaks predicted by first-principles calculation. MATERIALS AND METHOD First, we modeled some ionized states of Ln and its related intermediates in order to reproduce the absorption spectra ofLn, M440 and M420. In addition, we also modeled Ln coordinated with K+. Since t-BuOK is indispensable to the CL of Ln, we assumed 59

60

Sakai H & Wada N

Ln coordinated with K+ participated in the CL reaction. All of compounds used in the calculation are shown in Table I, where "Anion-2" and "Anion-3" could be assigned to M440 and M420 by H-NMR study, respectively. Geometry optimization and absorption peak prediction were performed by the Gaussian 03 program;3 the geometries of all compounds were optimized by B3LYP/6-3J+G(d,p) level of theory, and absorption peaks were predicted by TD-B3LYP/6-3J+G(d,p). We also applied the Polarizable Continuum Model (PCM) to solvent effect of DMSO (E=46.7) on the geometry optimization and prediction of absorption peaks. Table 1. Compounds used in the calculation for prediction of observed absorption peaks. R3

RI'O)_(j-R2 h

N

S

Ionized states of Ln Neutral Anion-O

RI -OH -0'

-COOH -COOH

R3 -H -H

Anion-O+K+

-OK

-COOH

-H

Anion-COO

-OH

-COO-

-H

Anion-COO+K+

-OH

-COOK

-H

Anion-2

-0-

-COO-

-H

Anion-2+2K+

-OK

-COOK

-H

Anion-3

-0-

-COO-

Anion-3+ 3K+

-OK

-COOK

R2

-K

RESUL TS AND DISCUSSION The calculated absorption maxima are summarized in Table 2. In the case of neutral Ln, the calculated peaks are 323 nm, 338 nm and 339 nm, were nearly equal to the experimental value for Ln (335 nm). Calculated peaks of "Anion-2", which was supposed as M440 by H-NMR, are 423, 434 and 434 nm, i.e., nearly equal to 440 nm. Thus, the experimental absorption peaks of Ln and M440 were properly predicted by the first-principles calculation. However, each of the calculated absorption maxima of "Anion-3", which could be assigned to M420 by H-NMR, was at a much longer wavelength than the experimental value. Then we presumed the geometry of M420 consisted of "Anion-3" interacting with a DMSO molecule and K+ ion in t-BuOK. The optimized geometry is shown in Fig.2. The absorption peak of "Anion-3+DMSO+K+" was calculated at 417 nm in the

Theoretical Analysis on the Absorption Spectra of Firefly Luciferin Intermediates

61

which was shorter than that of "Anion-3" (457 nm). This result "U);;);;""'" that ionized state of M420 exists in "Anion-3" directly interacting with the DMSO molecule and it should be noted that Nakatani et al. 4 also modeled of interacting with the DMSO molecule and in order to predict the fluorescence energy. From their study, the DMSO molecule between causes a slight red-shift in emission energy in comparison to the isolated our calculations suggested that absorption energy is blue-shifted by the DMSO molecule between "Anion-3" and K+. Thus, it is concluded that the DMSO molecule and are crucial factors for generation of M420.

Absorption maxima (f*) Ionized states ofLn

Vac-vac a)

Vac-sol b)

Sol-sole)

Neutral Anion-O

323 (0.38) 475 (0.36)

338 (0.49) 453 (0.73)

339 (0.49) 451 (0.68)

403 (0.53)

430 (0.63)

434 (0.65)

492 (0.16)

380 (0.25)

319 (0.22)

313 (0.28)

329 (0.54)

331 (0.55)

423 (0.58)

434 (0.69)

434 (0.68)

404 (0.49)

425 (0.68)

429 (0.70)

Anion-COO Anion-2

440 (M440) 420 (M420)

Anion-3 were va,,,uoa,w b) Geometry optimization was performed the gas phase; absorption c) Geometry optimization and absorption peaks were calculated in 'Oscillator strength •• Experimental data

2. Optimized geometry for "Anion-3" interacting with DMSO and The absorption peaks of "Anion-COO" and "Anion-COO+K+" did not reproduce experimental values of Ln, M440 and M420. Therefore, "Anion-COO" and are likely not to be passed through in the CL of Ln.

62

Sakai H & Wada N

CONCLUSION The deprotonation pathway in the CL reaction of Ln is proposed as follows: Ln (Neutral) -> M440 ("Anion-2", "Anion-O+K+" or "Anion-2+2K+") -> M420 ("Anion-3+DMSO+K+"). The DMSO molecule between K+ and "Anion-3" makes absorption peaks shift to shorter wavelengths. Furthermore, we are going to study the ionized states ofM440 and M420 by experimental techniques ofIR and H-NMR. ACKNOWLEDGEMENT Results in this paper were partly obtained using supercomputing resources at Information Synergy Center, Tohoku University. REFFERENCES 1. White EH, Rapaport E, Seliger HH, Hopkins TA. Chemi- and bioluminescence of firefly luciferin. Efficient chemical production of electronically excited states. Bioorg Chern 1971;1:92-122. 2. Shibata R, Yoshida Y, Wada N. Filter-photometry of chemiluminescence from firefly luciferin intermediate M420 in deoxygenated dimethyl sulfoxide. J Photosci 2002;9:290-2. 3. Frisch MJ, Trucks GW, Schlegel HB et al. Gaussian 03, Revision D.DI Wallingford, CT, 2004. 4. Nakatani N, Hasegawa J, Nakatsuji H. Red light in chemiluminescence and yellow-green light in bioluminescence: Color-tuning mechanism of firefly, Photinus pyralis, studied by the symmetry-adapted cluster-configuration interaction method. J Am Chern Soc 2007;129:8756-65.

BIOPHOTON EMISSION OF BIOLOGICAL SYSTEMS IN TERMS OF ODD AND EVEN COHERENT STATES

Sl KUN, a CHUNLI LIU, b JlA HUN-YU a a

Laboratory of Quantum Optoelectronics, Southwest Jiao Tong University,

Chengdu 610031, PR. China; b College of Bioengineering, Southwest Jiao Tong University, Chengdu 610031, PR China INTRODUCTION Luminescence of biological systems was discovered by Gurwitsch. 1 Popp proposed that biophoton radiation was due to nonlinear interaction between a coherent electromagnetic field and living matter. 2 The existence of quantum coherence in living matter has been demonstrated by many experiments. 3 Also, the coherence of the biophoton field might play an important role in communications between cells and in self-modulation.

4

Characteristics of biophoton emission can be

partially understood in a coherent-state field.

Using a simple coherent states

approach, Popp et at. calculated the probabilistic distributions of various numbers of states5 and the decay of intensit/ of biophotons. However, obvious non-classical phenomenon, such as sub-Poissonian photo-count statistics (PCS),7 and high-step nonclasscal effect,2 could not be explained in such a simple coherent-state description. In principle, it should relate to various non-classical optical fields, e.g., the superpositions of coherent states,S and it might elucidate various non-classical effects in biophoton emissions. We assume two coherent states with opposite phases to form the so-called even and odd coherent states. Our results on the decays of intensities of biphotons agree with the experimental data.

BIOPHOTON EMISSION AS ODD AND EVEN COHERENT STATES The biophoton can be considered as the optical radiation from a changing electromagnetic filed relating to a homeostatic physical process. The general form of HamiItonian H(t) describing such a field takes the following form: 9 63

64

Kun Sf et al.

H(t) = w(t)a+(t)a(t) + J(t)a+(t) + J*(t)a(t) + [3(t). Here,

[3 (t)

wet)

Suppose t3(t)

!(t)

and

are

the

(1)

function

t

of

= - exp[ -(I I k)2] with k being a constant related to certain

parameter of biologic matter. a+ and a are the creation and annihilation operators, respectively. The Heisenberg equation corresponding to Hamiltonian (1) has the solution: 9

aCt) = exp[ -i'I/J (t)]a(O) - i exp[ -i'I/J (t)]1o J(t) exp[i'I/J (t) ]dt , with

'I/J (t)

=

(2)

10 w(t)dt .

Suppose that the quantum state of radiant field is the even and odd coherent states, (3) I

Ia

> is coherent state and Ce,o

coefficient,

d

-

dt

=

{2[1 ± exp( -21 a 12)]} -"2 is the unitary

We obtain the rate of energy' change: 2"

< ±,a 1 H(t) I a,± >= Ce [w(t)N + w(t)N + [3(t)«

'

I

'

A

+ F(t)[lf(t) - f(t)w(t)] + F(t)[ -If(t) - f ,

-a a > ±1)]

v

v

* (t)w(t)] , J

B

withF(t) = F;(t) = 2exp[i1jJ(t)](1+ < -a I a »!or(/)exp[-i1jJ(/)dt for the even coherent states and:

F(t) = F 2 (t) = i2exp[i1jJ(t)]« -a I a> -1)!or (t)exp[-i1jJ (t)dt for the odd coherent states (N =

(a+ a)

is the average of the photon number).

(4)

Biophoton Emission of Biological Systems in Terms of Odd and Even Coherent States

65

S.Oxl0' 4.5xl0'

4.0xl0' 35xlO'

\

.~

30xl0'

~

2.5xl0'

\V EXP

jg

2.0xl0

c--,~; , ...

5

f-

~occ

1.SX10'

(001)

! lOOt:

'~t'{):20$) 1.0xl0'

5.0xl0·

Time (s)

Fig.1 Radiant intensity curve of odd coherent state with time

The biological system is in homeostasis (at least in a sufficiently short time interval), which implies that its energy is a time-independent constant. Namely,

d < ±,a I H(t) Ia,± >= 00 dt

This requires that A

(5)

= B = O. For B = 0, Generally, wet) = }.. /(1 + At) ,6 hence

J(t)

=

J(O)exp[-iln(1 + At)]

(6)

0

This type of damping agrees with the hyperbolic-like relaxation function of DL, but also provides the frequency stability of every oscillation for time-independent

W(t).6

Furthermore, for the odd coherent state A = 0 yields (7), and

solution N (8).

ee

2

[w(t)N + w(t)N + ~(t)( < -a I a > -1)] I2

N = (l + At)· {3(t)(exp-2Ia -1)/ A + M

=

0,

(7)

(8)

Here, M is the photon number of background. There are tiny differences between the bio-photon emission for odd and even coherent states - u=2, A=O.5(see Fig. 1). It agrees with the experimental resu\t

decreases with the

10

.

The intensity of biophoton emission

increasing of parameter

k .

Using the method, the

66

Kun Sf et al.

solution N for the even coherent state can be written as, I2

N = (l + At)· f3(t)(exp-2 Ia + 1)/ A + M. CONCLUSIONS Biological luminescence is explained by quantum coherence theory assuming that the radiative field is an odd and even coherent field.

The radiant intensity curve

versus time of biophoton emission agrees with the experimental results. Hence, biophotons arise from a non-classical state radiant field, and the parameter

k is

also related to the biological characteristics: the decay time is shortened with an increasing in k, i.e., it can control the velocity ofbiophoton emission. ACKNOWLEDG EMENTS We sincerely thank Dr. LF Wei for useful discussions. REFERENCES I. Gurwitsh AG. Die natur des spezifischen erregers der zellteilung. Arch Entw Mech Org 1923;100:11-6. 2. Popp FA, Li KH, Gu Q. Recent advances in biophoton research and its applications. Singapore:World Scientific, 1992. 3. Sen X. The coherence ofbiophotons. Physics 1995;24:166-71. 4. Zhang JZ. Physical properties of biophotons and their biological functions. Physics 2005;34:123-30. 5. Popp FA, Chang JJ, Herzog A, Yan Z, Yan Y. Evidence of non-classical (squeezed) light in biological systems. Phys Lett A 2002;293:98-102. 6. Popp FA, Yan Y. Delayed luminescence of biological systems in terms of coherent states. Phys Lett A 2002;293:93-7. 7. Popp FA, Gu Q, Li KH. Biophoton emission: experimental background and theoretical approaches. Mod Phys Lett 1994;B8: 1269-96. 8. Gu Q. Quantum interference between coherent states, In: Beloussov LV, Popp FA, eds. Biophotonics. Moscow: Bioinform Services Co. 1995;115-35. 9. Mehta CL, Sudarshan ECG. The evolution of coherent states. Phys Lett, 1966;22:574-6. 10. Popp FA, Li KH, Gu Q. Recent advances in biophoton research and its applications. Singapore: World Scientific. 1992;26-42.

STUDY ON ATP-DEPENDENT LUMINESCENCE REACTION OF THE ARM LIGHT ORGANS OF THE LUMINOUS SQUID WATASENIA SCINTILLANS K TERANISHI,' 0 SHIMOMURA 2

lFaculty ofBioresources, Mie University, Tsu, Mie 514-8507, Japan E-mail: [email protected] 2Photoprotein Laboratory, 324 Sippewissett Road, Falmouth, MA 02540-2210, USA E-mail: [email protected] INTRODUCTION The mollusk Watasenia scintillans is a small bioluminescent deep-sea squid. The squid has three black-pigmented light organs « I mm diameter) on the tip ofthe fourth pair of ventral arms and the light emission from the arm light organs is extremely bright when stimulated. Inoue et al. isolated coelenterazine disulfate from the arm light organs.' In 1985, Tsuji reported that the luminescence of the homogenate of the arm light organs is an ATP-dependent reaction that requires a soluble luciferin, an insoluble membrane-bound luciferase, ATP, and Mg2+ as the essential factors,2 and 17 years later he reported that the spectrum of the A TP-dependent luminescence shows a maximum at 470 nm, and that an addition of coelenterazine disulfate to luminescing reaction mixtures enhances 3 the luminescence. He proposed a mechanism for the A TP-dependent luminescence reaction as shown in Fig. 1. 3,4 However, no experimental evidence was presented to validate the proposed mechanism, and moreover, the membrane-bound luciferase has not been isolated and purified from the homogenates of the arm light organs. Tsuji reported the membrane-bound luciferase in the homogenates of the arm light organs was highly unstable and lost its activity in about 3 h in an ice-bath; the loss at -40°C was 35% in 24 h, and no activity was found after storage at -80°C for several days.4 Such a high instability and the insolubility of a substance essential for the luminescence would make the study of this luminescence system extremely difficult. In efforts to understand the mechanism involved in the ATP-dependent luminescence, we investigated the conditions for the stabilization, extraction and partial purification of the active insoluble luciferase existing in the arm light organs, and then studied the characteristics of the ATP-dependent luminescence reaction of coelenterazine disulfate in the presence of the active insoluble luciferase obtained.

°UCa

e

d 'N1

0 3 50

~ I

H

r

oso,

I

'"

Adenyl coelenterazlne disulfate

Coelenterazine disulfate

0, H,O Coelenteramide disulfate

Fig. 1. The reaction scheme proposed by Tsuji for the Watasenia bioluminescence 67

68

Teranishi K & Shimomura 0

METHODS Materials. Specimens of Watasenia scintillans were caught with trap nets set offshore in Toyama Bay, Japan. In about 8 h the living squids were frozen in liquid nitrogen. Immediately before use, arm light organs were picked off from the arms using ceramic tweezers. Measurement of luciferase activity. The luciferase activity was measured by the following methods (standard assay method): To 0.4 mL of 20 mM phosphate120 mM Tris (pH 8.3) containing I mM MgCI2' 40 ~L of 0.1 mM coelenterazine disulfate (in water) containing I mM MgCI2' 50 ~L of 5 mM A TP containing I mM MgCI2' and 20 ~L of the extract were added in this order at 0 °C, then luminescence intensity was measured with an Aloka luminescence reader BLR-301 (Aloka, Co., Ltd., Tokyo, Japan). The luminescence reader was calibrated with the luminol method;S I light unit (LU) on the Aloka instrument corresponded to 5.2 x 109 photons. Spectral measurement of luminescence and fluorescence. Spectrum of the ATP-dependent luminescence was measured at about O°C using an FP-750DS spectrotluorometer (JASCO, Tokyo) with its excitation lamp turned off. Chemiluminescence spectra of coelenterazine disulfate (I ~M) in DMSO containing 0.01 M NaOH and 3.5% water and in 20 mM phosphate/20 mM Tris (pH 8.3) in the presence ofH 20 2 and peroxidase (horseradish, type VI-A) were measured at 25°C with the same instrumental setup. Fluorescence spectra of coelenteramide disulfate were measured at 25°C with the same instrument. Bioluminescence spectrum of the arm light organs of a live specimen was measured using LumiFISpectroCapture AB 1850 (Atto Corporation, Tokyo).

RESULTS Stabilization, extraction, and partial purification of active luciferase existing in the arm light organs. We found that high concentrations of sucrose can markedly stabilize and also solubilize (at least in appearance) active luciferase in the homogenates of the arm light organs. Dark-red homogenate made with 2 M sucrose was centrifuged, and then the supernatant containing active luciferase was diluted with 20 mM phosphate buffer (pH 7.0) to precipitate the active luciferase. After centrifugation, the pellet containing active luciferase was extracted with 1.7 M sucrose/5.7 mM phosphate/0.29 mM MgCI2 and centrifuged. The colorless supernatant containing the active luciferase was able to be stored at -80°C for a long period of time (at least for 6 months) and was used as an "arm-organ extract". Characteristics of ATP-dependent luminescence catalyzed by the arm-organ extract. The concentrations of coelenterazine disulfate, ATP, and MgCl 2 have strong effects on the light intensity of luminescence reaction catalyzed by the arm-organ extract. About 8 ~M coelenterazine disulfate, 0.5 mM ATP, and 0.8 mM Mg2+ are needed to have maximum levels of luminescence intensity (Fig. 2). The optimum pH of the luminescence reaction catalyzed by a homogenate of the arm light organs was reported at 8.8 in a Tris buffer by Tsuji. 3 In our study using the arm-organ extract, the light intensity varied widely by the buffer solution chosen, but

Study onATP-Dependent Luminescence Reaction ofW scintillans

69

we found that luminescence was equally intense in 4 buffer systems: 40 mM phosphate, 20 mM phosphate/20 mM Tris, 80 mM phosphate, and 40 mM phosphate/40 mM Tris. The pH optima were broad in these buffer systems, but the optima in the first two were at 8.75, in good agreement with Tsuji's data. The optimum temperature of luminescence reaction appears to be about 5 °c (Fig. 2). The light intensity sharply decreased by raising temperature, giving only 25% of the light intensity at 20 DC. The light intensity at 0 DC was 85% of that at 5 DC. The emission spectrum of the luminescence reaction of coelenterazine disulfate under the conditions of standard assay (Amax 470 nm) was identical with the luminescence 3 spectrum of homogenate and also with the bioluminescence spectrum of the arm light organs. A light emitter in the luminescence of coelenterazine disulfate catalyzed by the arm-organ extract. Coelenterazine disulfate in DMSO under alkaline conditions emited chemiluminescence with a peak around 470 nm, and yielded coelenteramide disulfate quantitatively. Coelenteramide disulfate showed its fluorescence emission maximum at 390 nm in DMSO and at 400 nm in 20 mM phosphate120 mM Tris buffer, pH 8.3, indicating that in both cases fluorescence is emitted from the excited state of uncharged amide form and the chemiluminescence of coelenterazine disulfate in DMSO under alkaline conditions is emitted from the excited state of amide anion form of coelenteramide disulfate. In aqueous solution, coelenterazine disulfate emited weak luminescence in the presence of H 20 2 plus horseradish peroxidase (Amax around 400 nm), which is apparently emitted from the excited state of the uncharged amide form. When luminescing mixtures of coelenterazine disulfate under the standard assay condition was analyzed by HPLC, the amount of coelenteramide disulfate increased with reaction time, and the rate of the increase paralleled that of the total light emission. Thus, in the A TP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the arm-organ extract, the light emitter must be the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate catalyzed by arm-organ extract under the condition of the standard assay was calculated to be 0.36.

20

§

18

d

16

,

~ 14 ~

12

1l

]0

~

8

!i : 2

.~-------------~ iO 20 IS ConcentratIon of coclcnterazme disulfate (j..lM)

concentration of MgCl z or ATP (mM)

Time (mull

Fig. 2. Effect of the concentration of coelenterazine disulfate (left), ATP (solid line), and MgCl 2 (dotted line) (center) on the initial luminescence intensity, measured under the conditions of standard assay, and time-course of the luminescence intensity at various temperature under the conditions of standard assay (right).

70

Teranishi K & Shimomura 0

Function of A TP in the ATP-dependent luminescence reaction catalyzed by the arm-organ extract. The amounts of coelenteramide disulfate, AMP and ADP produced in luminescence reaction under the conditions of standard assay were measured by HPLC. In this experiment, the luminescence reaction mixture had initially contained 4 nmol of coelenterazine disulfate and 250 nmol of ATP, and after 10 min of luminescence reaction, 0.58 nmol ofcoelenteramide disulfate, 0.1 nmol or less of AMP, and 15 nmol of ADP were found. Thus the molar amount of AMP produced is much less than that of coelenteramide disulfate, indicating that the production of AMP is unrelated to the luminescence reaction. Therefore, the mechanism of A TP-dependent luminescence reaction suggested by Tsuji that involved the formation of adenyl coelenterazine disulfate as a key intermediate, must be incorrect. In connection with the unexpectedly large amount of ADP produced, we discovered that this compound was produced in nearly the same amount even when coelenterazine disulfate was omitted from the reaction mixture. Thus, the formation of ADP from A TP is mostly unrelated to the luminescence reaction, although we can not exclude a possibility that a small part of the ADP (corresponding 0.58 nmol coelenteramide disulfate formed) was produced from A TP by an unknown reaction that was linked to the light emitting reaction. In any event, it would be highly probable that most of the ADP was produced from ATP in phosphorylation reactions. Considering that ATP can activate multi-enzyme processes such as the respiratory burst that involves phosphorylation reactions, A TP might indirectly result in the oxidative activation of this bioluminescence system. ACKNOWLEDGEMENTS We thank Mr. Tsutomu Irie (Atto Corporation, Tokyo, Japan) for the measurement of the bioluminescence spectrum of the arm light organs. One of authors (K.T.) thanks Grant-in-Aid for Scientific Research (C; No. 19510215) from the Ministry of Education, Science, and Culture, Japan. REFERENCES I. Inoue S, Kakoi H, Goto T. Squid bioluminescence III. Isolation and structure of Watasenia luciferin. Tetrahedron Lett 1976; 17:2971-4. 2. Tsuji F 1. ATP-dependent bioluminescence in the firefly squid, Watasenia scintillans. Proc Natl Acad Sci USA 1985; 82:4629-32. 3. Tsuji F 1. Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid," Watasenia scintillans. Biochim Biophys Acta 2002; 1564: 189-97. 4. Tsuji F 1. Role of molecular oxygen in the bioluminescence of the firefly squid, Watasenia scintillans. Biochem Biophys Res Commun 2005; 338:250-53. 5. Lee J, Wesley A S, Ferguson III J F, Seliger H H. The use ofluminol as a standard of photo emission. In: Johnson F H. Haneda Y. eds. Bioluminescence in Progress. Princeton University Press, Princeton, NJ. 1966;35-43.

MECHANISM OF BACTERIAL LUCIFERASE: ENERGETIC AND QUANTUM YIELD CONSIDERATIONS S-CTU Dept of Biology and Biochemistry, University of Houston, Houston, Texas, 772045001, USA; Email: [email protected]

INTRODUCTION The reaction mechanism of bacterial luciferase has been studied extensively, 1,2 An electron-exchange reaction mechanism (Scheme I )3,4 has been postulated and gained considerable acceptance, II

III

(HF.QO-)

- O2

FMNH -

~~-O r

CH3=(XX' CH3

'"

I

H

(HF.QOCH(OH)R) R'

,

R'

g0

RCHO+

H+

N

N

CH3~·y·'f0

CH3~~NH

NH

?0

--

o I

H--C-OH

I

R

, IV

Iv*

(HF.QH) R'

+

H2 0

R' ,

N

FMN ___ C H 3 f ' y

CH3~

N

Y'yO.-----,-/

~

(HF.QH*)

-{yNH

0 0

R-COOH +

CH3

*

r"yyN"r O

CH3~~NH

Light

H

H

~

0

Scheme 1. Reaction mechanism for bacterialluciferase Following this mechanism, luciferase-bound reduced FMN (FMNH-; intermediate I) reacts with oxygen to form the 4a-hydroperoxy-4a,5-dihydroFMN intermediate II (HF-OO-), The addition of a long-chain aliphatic aldehyde generates the 4aperoxyhemiacetal-4a,5-dihydroFMN intermediate III (HF-OOCH(OH)R), which is subsequently converted to a radical pair of 4a-hydroxy-4a,5-dihydroFMN radical cation (IV+'; HF-OH+') and a carboxylic acid radical anion RC(OH)O-', A I-e transfer from RC(OH)O-' to IV+' produces the excited 4a-hydoxy-4a,5-dihydroFMN intermediate IV*, Relaxation of IV* to the ground state produces bioluminescence (Amax ~490 nm) with a quantum yield of about 0,16, Finally, IV decays to form FMN and water. The present report addresses several key issues of this mechanism with respect to the identity of the proposed excited emitter HF-OH*, the energetics of its formation, and the requirement of a hydrophobic luciferase active site for a high quantum yield of the emitter. 71

72

Tu SoC

RESULTS AND DISCUSSION The energy of photon for 490-nm emission is 59 kcallmol. The relaxation of the singlet excited HF-OH* to the ground state can yield 68 kcallmol,5 more than sufficient for producing the 490-nm bioluminescence. However, when Scheme 1 was first formulated, HF-OH+' was a purely hypothetical species. Critical questions as to whether HF-OH+' can be formed and whether I-e transfer from RC(OH)O-' to HF-OH+' is sufficiently energetic for forming HF-OH* must be addressed. Using 5ethyl-4a-hydroxy-3-methyl-4a,5-dihydroflavin (EtF-OH) as a model for HF-OH, the formation of EtF-OH+' was demonstrated and the absorption and redox potential of 3 this flavin radical cation were determined. Moreover, the energy level of the RC(OH)O-' and HF-OH+' radical pair has been estimated to be 90 kcallmol higher 5 than that of R-COOH plus HF-OH. Therefore, I-e transfer radical annihilation of RC(OH)O-' and HF-OH+' is sufficiently energetic for forming the HF-OH* emitter. A-'

LUMO

c"

-.L-Jt....-JL®

HOMO

A

--

.JL

c'

.-L .-L

r

~

~

A*

--L -.L

C

--

.JL

Fig. 1. Chemiexcitation by I-e transfer radical annihilation

Another mechanistic issue arises from the following considerations. One-e transfer from a radical an ion (A _.) to a radical cation (C+') can generate either the excited C* (pathway I) or A * (pathway 2) as shown in Fig. I. For Scheme I, theoretically either RCOOH* or HF-OH* could be formed as the primary excited species. If RCOOH* is first formed, a subsequent energy transfer to HF-OH could generate HF-OH* for the 490-nm emission. Which one is the primary excited species? We synthesized and used a-parinaraldehyde (cis, trans, trans, cis-9, II, 13, 15octadecatetraenoaldehyde abbreviated a-PAD) and ~-parinaraldehyde (trans, trans, trans, trans-9, I I, 13, 15-octadecatetraenoaldehyde abbreviated ~-PAD) to replace the saturated aliphatic aldehyde substrate in the luciferase reaction. 6 Both a-PAD and ~-PAD are active substrates for the luciferase bioluminescence reaction to produce, respectively, the a-parinaric acid (a-PAC) and ~-parinaric acid (P-PAC) products. a-PAC and P-PAC each binds to the luciferase active site in competition with the normal aldehyde substrate and, in the luciferase-bound form, emits strong fluorescence (Amax 420 nm), which overlaps with the absorption of HF-OH. We

Mechanism of Bacterial Luciferase: Energetic and Quantum Yield Considerations

73

found that the bioluminescence spectrum using a-PAD or ~-PAD was identical to the normal 490-nm emission without any detectable 420-nm component from aPAC* or ~-PAC*. This indicates that either HF-OH* is directly generated as the primary excited emitter or the efficiency of energy transfer from a-PAC* or ~ PAC*, if first formed as the primary emitter, to HF-OH is 100%. Resonance energy transfer is sensitive to the relative orientation of donor and acceptor. Since a-PAC and ~-P AC are distinct in their stereo structures, it is quite unlikely that luciferasebound a-PAC* and ~-PAC* can both transfer energy to HF-OH with the same 100% efficiency. Moreover, HF-OO- intermediate II emits fluorescence similar to the 490-nm bioluminescence from HF-OH*. Excitation of II-bound a-PAC or ~-PAC at 312 nm led to 420-nm fluorescence without any significant increase in 490-nm emission. This is again inconsistent with 100% efficiency of energy transfer from aPAC* or ~-PAC* as a donor. Taking all findings together, we conclude that HFOH* is the primary excited emitter in the luciferase reaction. The proposed structure of 4a-hydroxy-4a,5-dihydroFMN for IV has long been questioned on the basis of a huge difference between the luciferase bioluminescence quantum yield (~0.16) and the extremely low fluorescence quantum yields of flavin model compounds 5-ethyl-4a-ethoxy-3-methyl-4a,5-dihydroflavin (EtF-OEt) and EtF-OH ($ :s 10. 5).7.8 Neither EtF-OH nor EtF-OEt shows any detectable binding to luciferase active site. Therefore, the possibility that luciferase binding site environment can greatly enhance the emission efficiency of 4a-hydroxyflavin cannot be tested with EtF-OH or EtF-OEt. We have synthesized 5-decyl-4a,-hydroxy-4a,5dihydroFMN (DF-OH) as a new model for the proposed HF-OH intermediate IV.9 Both the wild-type Vibrio harveyi luciferase and the catalytically active aCI06A variant bind DF-OH at a 1:1 molar ratio, and the binding is inhibitory to luciferase bioluminescence activity. These findings suggest that DF-OH binds specifically to the luciferase active site. aCI06A was used in addition to the wild-type luciferase because the aCI06A-bound DF-OH was more stable than that bound by the native luciferase. Importantly, the fluorescence of free DF-OH was barely detectable at room temperature but DF-OH bound to wild-type luciferase or aCI06A showed strong fluorescence with quantum yields increased markedly to about 50% of that for the luciferase bioluminescence. These results provide a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN for intermediate IV. Finally, we have also shown that the ability of luciferase to greatly enhance the emission quantum yield of the bound HF-OH* (
74

Tu S-C

that the emission quantum yields (cI>IV') of their excited emitter IY* were one to two orders of magnitude lower than that of the native luciferase. Therefore, both this mutagenesis study and the DF-OH model study described above indicate that the hydrophobic luciferase active site is capable of enhancing the quantum efficiency of the HF-OH* emitter.

ACKNOWLEDGEMENTS Supported by Robert A. Welch Foundation grant E-I030. REFERENCES 1. Hastings JW, Potrikus CJ, Gupta SC, Kurflirst M, Makemson Je. Biochemistry and physiology of bioluminescent bacteria. Adv Microb Physiol 1985 ;26:23S-91. 2. Tu S-C. Bacterial bioluminescence: biochemistry. In: Horspool WM, Lenci F, editors. CRC Handbook of Organic Photochemistry and Photobiology. 2nd ed. Boca Raton, FL: CRC Press, 2004: 136/1-17. 3. Mager HIX, Sazou D, Liu YH, Tu S-C, Kadish KM. Reversible one-electron generation of 4 a ,S-substituted flavin radical cations: Models for a postulated key intermediate in bacterial bioluminescence. J Am Chern Soc 1988; 11 0:37S9-62. 4. Eckstein JW, Hastings JW, Ghisla S. Mechanism of bacterial bioluminescence: 4a,S-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substitutions at the 8-position of flavin on luciferase kinetics. Biochemistry 1993;32:404-11. S. Merenyi G, Lind J, Mager HlX, Tu S-e. Properties of 4a-hydroxy-4a,Sdihydroflavin radicals in relation to bacterial bioluminescence. J Phys Chern 1992;96: 1OS28-33. 6. Cho KW, Tu S-C, Shao R. Fluorescent polyene aliphatics as spectroscopic and mechanistic probes for bacterial luciferase: Evidence against carbonyl product from aldehyde as the primary excited species. Photochem Photobiol 1993;S7(2):396-402. 7. Ghisla S, Massey Y, Lhoste J-M, Mayhew SG. Fluorescence and optical characteristics of reduced flav ins and flavoproteins. B iochem istry 1974; 14: S89-

97. 8. Kaaret TW, Bruice Te. Electrochemical luminescence with N(S)-ethyl-4ahydroxy-3-methyl-4a,S-dihydrolumiflavin. The mechanism of bacterial luciferase. Photochem Photobiol 1990;SI :629-33. 9. Lei B, Ding Q, Tu S-C. Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of Sdecyl-4a-hydroxy-4a,S-dihydroriboflavin-S'-phosphate as a model. Biochemistry 2004;43: IS97S-82.

10. Li CH, Tu S-c. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase. Biochemistry 200S ;44:12970-7.

MECHANISM RESPONSIBLE FOR THE SPECTRAL DIFFERENCES IN FIREFLY BIOLUMINESCENCE NN UGAROVA Dept of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia Email: [email protected] Several mechanisms have been proposed to explain the vanatlOns of the bioluminescence color for native and mutant luciferases. According to White at all changes in bioluminescence spectra are the result of keto-enol tautomerization of oxyluciferin (LO). The efficiency of this process depends on a correct location of the Bl and B2 bases in proximity of the thiazole ring for effective transformation of the ketone form of LO (LO=O) to the enol (LO-OH) which can interact with the B3 base to form enolate-ion (LO-O-). When the Bl base is absent or protonated (e.g. at pH :::: 6.0) bioluminescence of the LO=O will be observed with Amax in the red region of the spectrum. At intermediate pH all forms: LO=O, LO-OH and (LO-O-) - will be observed in the bioluminescence spectra. 2

()

Il

In::

(LO=O)

(LO-OH)

(LO-D")

Scheme 1 3

Branchini et al proposed that LO=O is the only emitter, and luciferase modulates bioluminescence spectra by controlling the resonance-based charge de localization of the anionic keto form of the oxyluciferin excited state. A mechanism 4 involving 'twisted' conformation to explain color changes in the firefly bioluminescence is not feasible as was shown by experimental 5 and theoretical studies. 6 Besides, independently of the specific molecular structure of "red" and "green" emitters it is the properties of the emitter microenvironment that are responsible for the observed differences in bioluminescence spectra and their pH sensitivity. According to the accepted photo-physical concepts on the influence of the medium on emission spectra, the effects observed may be divided into general (non-specific) and specific ones. 7 General effects result in changes in orientation polarizability of the emitter microenvironment whose value depends on dielectric constant and refractive index of the medium. Maximum spectral shift is observed for the emitters with maximum changes in dipole moments upon excitation and in the medium with

75

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Ugarova NN

maximum orientation polarizability. The emission maximum (Amax) shifts to longer wavelengths with increasing polarizability, but the shape of the spectrum remains constant. 8 As an example of such effect, emission spectra of two red mutants of Luciola cruciata luciferase (I288A and S286N) may be considered that have the Amax values at 605 and 613 nm, respectively.5 It is important to underline that general effects of the emitter microenvironment result in spectral shifts that, as a rule, do not exceed ~ 12 nanometers. Specific effects are due to the formation of hydrogen and/or acid-base bonds, or charge-transfer interactions between emitter and surrounding groups of the enzyme active site, it is due to formation of new emitter species. They are usually characterized by larger Stokes shifts and the appearance of new spectral bands. In many cases the bio lum inescence spectra for firefly luciferases become nonsymmetric with, for example, changing pH, or upon mutation. This is typical of systems where light is emitted from several electronically excited species rather than from a single one? How can luciferase modify microenvironment of the emitter in the active site? Usually an enzyme molecule exists in solution in various protein conformers which are in equilibrium:

EI

+-+

E2

+-+

E3

Depending on external conditions (pH, temperature, composition of solution) and molecular structure (changes of protein structure upon natural or artificial mutations), equilibrium between different conformers can be dynamic. During enzymatic reaction the excited LO (LO*) is generated in the active site of each conformer. Initially there is a mixture of complexes: E1(LO*)

+-+

E 2 (LO*)

+-+

E 3 (LO*)

Due to general and specific interactions between (LO*) with the microenvironment of the enzyme conformer, new forms of the complexes appear: E1(LO=O*)

+-+

E 2 (LO-OH*)

+-+

E 3 (LO-O-*)

They contain different emitters and generate light with different Amax The more rigid the conformer structure around (LO*), the higher its emission energy, and the lower Amax. When the pH value gradually drops, the equilibrium is shifted to the conformer with more flexible structure, the emitter energy decreases, and a new form of the emitter can be appear with lower energy, and new bands appear in the bioluminescence spectra. As a result, we observe a superposition of at least two different emitters that differ in energy level and in structure. The majority of firefly luciferases demonstrate a pH-dependence of bioluminescence spectra. Analyzing bioluminescence spectra over a large pH interval, the relative contribution of different (LO*) forms to the total bioluminescence spectra of native and some mutant luciferases was characterized quantitatively.2,9 For example, the point mutation H433Y in Luciola mingrelica firefly luciferase resulted in 40 nm

Mechanism Responsible for the Spectral Differences in Firefly Bioluminescence

77

shift of bioluminescence AMax from 566 nm for WT to 606 nm for mutant at pH 7.8 and changed noticeably its pH-dependence. 2 Gaussian mUlti-peak fitting of the bioluminescence spectra revealed three forms of oxyluciferin, i.e., (LO-O-*) with Amax = 556 nm, (LO-OH*) with Amax = 587 nm, and (LO=O*) with Amax = 618 nm with the correlation coefficient 0.999 (Fig 1). 1,0/0 100

50 - 587 nm ~"~

f

,

orange" --'-...

'~ __-=~

Of-dd~~--==~----~--~~----~

500

550

600

650 A,nm

Fig. 1. Superposition of different forms of oxyluciferins for WT (at pH 6.4) and mutant H433Y (at pH 7.6) L. mingrelica firefly luciferase The spectra at Fig. 1 show that mutation provoked the same changes in the relative content of different emitter forms at pH 7.6 as the decrease in pH down to 6.4 for WT luciferase. In both cases we observed changes of both Amax and the shape of the bioluminescence spectrum. Computer modeling of the luciferase-oxyluciferin-AMP complex 2 showed that the mutation H433Y increases flexibility of the polypeptide loop binding the Nand C-domains of luciferase and impedes rigid fixation of several amino acid residues of C-domain in vicinity of (LO*) which is necessary for effective tautomerization process (Scheme 1). The increase of flexibility of the protein structure in the vicinity of the thiazole ring results in an increase in the content of the E1(LO=O*). Substitutions Y35H, N in L. mingrelica luciferase resulted in mutants which had nearly pH-insensitive bioluminescence spectra. IO Analysis of the crystal structures described 5 allowed us to propose a possible explanation of the striking effect of these substitutions in L. mingrelica luciferase. A significant difference was found in the orientation of the loop 233-237. In the red-emitting enzyme the residues Y35 and P235 are separated by 3.9 A. In the green-emitting mutant the residue P235 contacts closely with Y35 and forms a 'closed' conformation of the active site. Probably such loop conformation is necessary for the increase of E3 (LO-O-*) conformer. The substitution of a bulky Y35 to a smaller N or H residues stabilizes the close packing of P235 and 35 th residue thus sustaining enzyme in the rigid conformation and compensating deteriorative effects of low pH.

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We conclude that there are no strictly specific residues affecting the equilibrium between different luciferase conformers demonstrating various pH-dependences of bioluminescence spectra. Many different mutations result in local changes in enzyme conformer structure and shifting equilibrium between different luciferase conformers to the red 2,3,8 or green bioluminescence.ID,11

ACKNOWLEDGEMENTS The research was supported by Russian Foundation for Basic Researches (Project 08-04-00624-a).

REFERENCES White E, Rapaport E, Seliger H, Hopkins T. The chern i- and bioluminescence of firefly luciferin: an efficient chemical production of electronically excited states. BioorgChem 1971;1:92-122. 2. Ugarova N, Maloshenok L, Uporov I, Koksharov M. Bioluminescence spectra of native and mutant firefly luciferase as a function of pH. Biochemistry (Moscow) 2005:70:1262-7. 3. Branchini B, Southworth T, Murtiashaw M, Magyar R, Gonzalez S, Ruggiero M, Stroh 1. An alternative mechanism of bioluminescence color determination in firefly luciferase. Biochemistry. 2004;43 :7255-62. 4. McCapra F, Gilfoyle D, Young D, Church N, Spencer P. The chemical origin of colour differences in beetle bioluminescence. In Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects. Campbell AK, Kricka LJ, Stanley PE (eds). Chichester:Wiley, 1994;387-91. 5. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature 2006;440:372-6. 6. Orlova G, Goddard J, Brovko L. Theoretical study of the amazing firefly bioluminescence: the formation and structures of the light emitters. J Am Chern Soc 2003; 125 :6962-71. 7. Lakowich J. Principles of fluorescence spectroscopy. New York Kluwer Academic/Plenum Press, 1999. 8. Ugarova N, Brovko L. Protein structure and bioluminescent spectra for firefly bioluminescence. Luminescence 2002;17:321-30. 9. Ando Y, Niwa K, Yamada N, Enomot T, Irie T, Kubota H, Ohmiya Y, Akiyama H. Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. Nature Photonics 2008;2:44-7. 10. Koksharov M, Ugarova N. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes whose bioluminescence spectra show low pHsensitivity. Biochemistry (Moscow) 2008;73: in press. 11. Law G, Gandelman 0, Tisi L, Lowe C, Murray J. Mutagenesis of solventexposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance. Biochem J 2006;397:305-12. 1.

LUMINOUS MUSHROOMS GA VYDRYAKOVA,I NV PSURTSEVA,2 NV BELOVA,2 AA GUSEV,I NV PASHENOVA/ SE MEDVEDEVA,I EK RODICHEVA,I 11 GITELSON 1 iInstitute of Biophysics SB RAS, Krasnoyarsk, Russia; 2 Komarov Botanical Institute Russian Academy of Sciences, St. Petersburg, Russia; 3Institute of Forest SB RAS, Krasnoyarsk, Russia; E-mail: [email protected]

INTRODUCTION There is a great variety of living things able to emit light. The luminous bacteria (17 species) are most investigated and most used for bioluminescent analysis. At the same time about 70 species of luminous fungi are known, but they are not widely used in basic and applied investigations until now. Most are found in the tropics. Armillaria mellea is one of the most widely distributed of the bioluminescent fungi. It is a common root rot and wood decay fungus found across North America, Europe and Asia. Luminous fungi emit light during different stages of their life cycle. Armillaria's mycelium and rhizomorphs are luminous, I in number of Mycena species luminescence presents in mycelia, Mycena rorida produces only luminous spores, Panellus stipticus and Omphalotus olearius (syn. Clitocibe illudens) have luminous mycelia and fruiting bodies,z- 4 Collybia tuberosa produces only luminescent sclerotia, 5 while Omphalotus af illudent, 6 able to emit light in filament and fruitbody form (its whole body - pili, stem, gill, spore - emit light). All known species of luminous fungi produce bluish-green light with emission maximum close to 530 nm. The mechanism of fungal luminescence is still unknown. It was supposed that it was due to a lucifer inluciferase reaction. However, structure of natural luciferins was not determined and a luciferase was not detectedY Nevertheless, naturally bioluminescent fungi, A. mellea and Micena citricolor were used to develop bioluminescence-based bioassays for toxicity testing. 8 ,9 Genetically modified bioluminescent fungi Aspergillus awamori with recombinant aequorin gene were used to monitor [Ca2+] changes in living fungal cells in the presence of toxicant. 10 It should be noted that on agar media that the luminescence of mushrooms can last for 2 weeks, in wood for 2 months, which makes it and interesting prospect for use in biotesting. An estimation of possibility to use Russian strains of luminous mushrooms for bioluminescence-based toxicity testing was the aim of our investigation. There is little data about luminous mushrooms in Russia.

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MATERIALS AND METHODS We tested 26 strains from the Mushrooms Collection of Komarov Botanical Institute of Russian Academy of Sciences (Mushrooms Collection of BIN) and found that 6 of them were luminous: 1043 Armillaria gallica - Central Ural, Perm region, Russia 0350 A. mellea - Leningrad region, Russia 0356 A. mellea - Minsk region, Belarus 0358 A. mellea - Bohemia, Czech Republic 0918 A. mellea - Irkutsk region, Russia 0491 Lampteromyces japonicus - United Kingdom Half of these mushrooms were collected in different Russian regions: Leningrad region, Central Ural region and Irkutsk (Baikal) region. We also used in our study, a culture of Armillarea borealis which was isolated in the Krasnoyarsk region. Strains of luminous mushrooms were cultivated on Saburo medium. Intensity of luminescence was measured with using multimode microplate reader Luminoscan Ascent (Thermo Electron Co, Finland). Peaces of Saburo agar containing luminous mycelium were put in wells of plate and intensity of luminescence was estimated. After adding of 200 flL of toxicant in well with fungal mycelium the intensity of luminescence was measured again after 5 and 30 min. The normalized values of light emission were expected in relation to an initial luminescence. RESULTS AND DISCUSSION Intensity of luminescence of cultures A. isolated in Krasnoyarsk area in 2007 was higher in comparison with the cultures from the Mushrooms Collection of BIN. We studied the effect of different concentrations (from 10-6 mg/mL to 1 mg/mL) of organic and inorganic toxicants on culture luminescence of 3 species of luminous fungi A. borealis, A. mellea and L. japonicus. It was shown that the mushroom A. mellea were more sensitive to action of toxicants than A. borealis and L. japonicus. The minimal concentration of benzoquinone resulted in a 21 % decrease in luminescence in comparison with the initial value, and a decrease in luminescence on 73 % was found at higher concentrations. The lowest concentration of copper ions which could be determined using this system was 10-5 4 mg/mL (19 % decrease in luminescence and a concentration 10- mg/mL caused a 33 % reduction ofluminescence in comparison with the initial value) (Fig. 1,2). Lyophilized bioluminescent bacterial biosensors are popular nowadays because they are very sensitive even to micro-quantities of toxicants, and are very simple to

Luminous Mushrooms

1. The normalized values of luminescence of Armillaria borealis after 30 min action of toxicants.

2. The normalized values of luminescence of Armillaria mellea after 30 min action of toxicants.

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use. Bioluminescent analysis is the simplest rapid method for biological monitoring of environment and detecting chemical toxicants, including organic carbon compounds and heavy metals. Our results show that the sensitivity of mushrooms-based assays for toxicants are not as sensitive as bacterial-based assays, but they could be used to develop bioassays.

REFERENCES 1. Coder KD. Foxfire: Bioluminescence In the forest, 1999. www.forestry.uga.eduiefr 2. Weitz, HJ. Naturally bioluminescent fungi. Mycologist 2004;18:4-5. 3. Desjardin DE, Capelari M, Stevani CV. Bioluminescent Mycena species from Sao Paulo, Brazil. MycoI2007;99:317-31. 4. Wassink Ee. Luminescence in fungi. In: Herring PJ, ed. Bioluminescence in action. London:Academic Press, 1978:171-97. 5. Bioluminescence fungi: living light, 1999. www.psms.org/sporeptints/sp359.htm I 6. Van DT. The successful cultivation of a new luminous mushroom Omphalotus aJ Illudent. http://malJuires.com/research/externaliomphalotlis .afi Iludent.pd fl. 7. Shimomura O. Bioluminescence: chemical principles and methods. Singapore:World Scientific. 2006:470 p. 8. Weitz HJ, Colin D, Campbell CD, Killham K. Development of a novel, bioluminescence-based, fungal bioassay for toxicity testing. Environ MicrobioI2002;4:422-429. 9. Horswell J, Weitz HJ, Percival HJ, Speir TW. Impact of heavy metal amended sewage sludge on forest soils as assessed by bacterial and fungal biosensors. BioI Fertj] Soils 2005;42:569-76. 10. Kozlova 0, Zwinderman M, Christofi N. A new short term toxicity assay using Aspergillus awamori with recombinant aequorin gene. BMC Microbiol 2005;5:40-2.

USE OF CYPRJDINA LUCIFERIN ANALOG FOR ASSESSING THE MONOAMINE OXIDASE-LIKE SUPEROXIDE-GENERATING ACTIVITIES OF TWO PEPTIDE SEQUENCES CORRESPONDING TO THE HELICAL COPPER-BINDING MOTIF IN HUMAN PRION PROTEIN AND ITS MODEL ANALOG K YOKAWA, TKAGENISHI, TKAWANO Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan Email: [email protected].)p

INTRODUCTION Experimental evidence for involvement of reactive oxygen species (ROS) such as HzO z, superoxide anion radicals (Oz') and hydroxyl radicals (HO") in the toxic mechanism of prion protein (PrP) have been documented, suggesting that fundamental molecular mechanisms underlying the pathogenesis in neurodegenerative diseases could be attributed to the production of ROS that stimulates the formation of abnormal protein aggregates. 1,z The likely sites of action involved in the key redox reactions in mammalian PrPs are six to seven putative Cu-binding sites consisted of 4 distinct sequences. Recently, Kawano 3 has reported that the generation of Oz" is catalyzed by Cu-binding PrP fragments in the presence of certain co-factors such as HzO z and aromatic monoamines known to behave as precursors and/or analogs of catecholamine-type neurotransmitters highly rich in brains. Among the aromatic monoamines tested, tyramine shows the highest activity. The tyramine-dependent oxidative burst catalyzed by the model PrP helical peptide sequence was notably robust and long lasting (over 10-20 min) while those measured in the presence of benzylamine or phenylethylamine were merely minor spikes lasting only for some seconds. 3 In this study, we compared the Oz"-generating activity of two peptide sequences corresponding to the PrP helical Cu-binding site namely the model analog sequence often employed in the biochemical studies,3.4 and the original sequence found in the native PrP, using Cypridina luciferin analog (CLA) as a chemiluminescent probe for Oz". MATERIALS The model sequence for PrP helical Cu-binding region (VNITKQHTVTTTT) proposed by Brown et al. 4 was used since this sequence was shown to be active as the catalyst for generation of Oz" in presence of some aromatic monoamines. 3 Additionally, a peptide corresponding to the natural helical sequence, VNITIKQHTVTTTT, was also prepared (Fig. I). Two peptides were chemically synthesized and purified on HPLC (purity, 99.20%, 99.10%, respectively) by Sigma Genosis Japan (Ishikari, Hokkaido). 83

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Yokawa K et al.

Cypridina luciferin analog (CLA; 2-Methyl-6-phenyl-3,7-dihydroimidazo[I,2-a] pyrazin-3-one), a chemiluminescence reagent specific to O 2'-, was obtained from Kasei Kogyo Co. (Tokyo, Japan). Tyramine HCI, tyrosine, salicylic acid, benzoic acid and its derivatives were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Other chemicals used in this study were of reagent grade purchased from Sigma (St. Louis, MO, USA). The peptides and other chemicals were dissolved in phosphate buffered saline and generation of O 2'- was monitored by chemiluminescence of CLA with a Luminescensor PSN AB-2200-R (Atto Corp, Tokyo, Japan) and expressed as relative luminescence units (rlu) as previously described. 3•5 CLA-chemiluminescence specifically indicates the generation of O 2'(and 10 2 to a lesser extent) but not that ofH 2 0 2 or HO·.6 According to our previous study, the signal for 10 2 can be minimized by avoiding the use of high concentration of organic solvents such as ethanol over 2% (v/v) in the reaction mixture. 7 Thus the chemiluminescence recorded here surely reflects the generation of O 2 '- rather than 10 2 .

A Generalized PrP structure

NH2-l~@@@

rB

~1-----8--COOH

(PHGGGWGQ)x4 GGGTH KTNMKHMA * • KTNMKHVA KTNLKt.tVA Octarepeats

B

neurotoxic region

VNITIKQHTVTTTT VNITIKEHTVTTTT VNITtKQHTVTTTT VNITIKQt.tTTTTTT helical region

Peptides synthesized (1) VNITKQHTVTTTT (well studied PrP helix 2 analog) (2) VNITIKQHTVTTTT (natural helical sequence in PrP)

Fig. 1. Cu-binding sites in PrPs. (A) Generalized PrP structure, (8) peptides tested.

RESULTS AND DISSCUSION Oxidative burst catalyzed by two peptides. Direct interactions between Cu-Ioaded PrP-derived peptides and some aromatic monoamines leading to generation of O 2'- has been examined in phosphate buffered saline containing CLA, CUS04, H 2 0 2, and PrP-derived Cu-binding peptides, by measuring the 02'--specific chemiluminescence. 3 Since requirements for two co-factors, copper and H20 2 in addition to substrates were suggested in the previous study, we tested the effects of substrates on the reaction catalyzed two helical peptides in the presence of standardized compositions (known to be optimal for aromatic mono amine oxidation). The molar ratios among the components in the reaction mixture (totally 200 ilL), namely, peptides, Cu 2+, H 20 2, and substrates were approximately I :3:3:3 (i.e., each reaction mixture contained 0.15 mM peptide, 0.5 mM CUS04, 0.5 mM H20 2 and 0.5 mM substrate.

Use of Cypridina Luciferin Analog for Assessing of Two Peptide Sequences

85

generation catalyzed by two PrP helical sequences in the presence of various putative substrates.

OH

R=COOH, CH2CH(NH2)COOH, CHzCH(NHAc)COOH

3. Possible chemical structure as novel substrate for reaction. to aromatic monoamines, aromatic amino acids and phenolics salicylic acid, benzoic acid and its derivatives were tested as putative substrates. 2 shows the comparison of the peptides' O 2'. activities in the presence of various substrates. Reaction was in itiated and assessed by and substrates (aromatic monoamines, amino acids, or phenolics) to buffered saline in this order. The extent of CLA-chemiluminescence was the relative luminescence unit (rlu). that the extent of phenolic-dependent reactions catalyzed by the model were shown to be greater rather than that catalyzed by native sequence 2;

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Yokawa K et at.

exception with 2,3- and 2,5-dihydroxybenzoic acids). Interestingly, difference with single amino acid (isoleucine) between the model and native sequences resulted in drastic changes in generation of O 2'". Required positioning of the functional groups on the aromatic ring. In general, the compounds lacking -OH group are not utilized in the reaction. Phenolics having only -OH(s) in ortho-orientation with -R showed none or negligible activities. We found that the peptides likely react with the substrates with common chemical structure showing R-Ph-OH with arrangements of -R and -OH with meta or para but not ortho orientations as summarized in Fig. 3.

ACKNOWLEDG EMENTS This work was partly supported by a grant of Knowledge Cluster Initiative implemented by Ministry of Education, Culture, Sports, Science and Technology (MEXT). REFERENCES I. Tabner BJ, Turnbull S, EI-Agnaf 0, Allsop D. Production of reactive oxygen species from aggregating proteins implicated in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. Curr Top Med Chern 2001;1:507-17. 2. Tabner BJ, EI-Agnaf OMA, Turnbull S, et al. Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer's disease and familial British dementia. J BioI Chern 2005;280:35789-92. 3. Kawano T: Prion-derived copper-binding peptide fragments catalyze the generation of superoxide anion in the presence of aromatic monoamines. Int J BioI Sci 2007;3:57-63. 4. Brown DR, Guantieri V, Grasso G, Impellizzeri G, Pappalardo G, Rizzarelli E: Copper(IJ) complexes of peptide fragments of the prion protein. Conformation changes induced by copper(Il) and the binding motif in C-terminal protein region. J Inorg Biochem 2004;98:133-43. 5. Kawano T, Kawano N, Hosoya H, Lapeyrie F. Fungal auxin antagonist hypaphorine competitively inhibits indole-3-acetic acid-dependent superoxide generation by horseradish peroxidase. Biochem Biophys Res Commun 2001 ;288:546-51. 6. Nakano M, Sugioka K, Ushijima Y, Goto T. Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[I,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O 2". Anal Biochem 1986;159:363-9. 7. Yokawa K, Suzuki N, Kawano T. Ethanol-enhanced singlet oxygen-dependent chemiluminescence interferes with the monitoring of biochemical superoxide generation with a chemiluminescence probe. Cypridina luciferin analog. ITE Lett Batter New Technol Medic 2004;5:49-52.

PART 2 APPLIED BIOLUMINESCENCE

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BIOLUMINESCENT ASSAY OF ANTIBIOTIC SUSCEPTIBILITY OF CLINICAL SAMPLES VG FRUNDZHY AN, NN UGAROV A Dept of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia Email: [email protected]

INTRODUCTION It is very important to select promptly the most effective antibiotic for successful therapy of infectious diseases, and wound and post-surgical infections. The duration of standard microbiology assays applied in clinical practice exceeds 3-5 days since preliminary isolation of the pathogen from the clinical sample is required. In the present study we optimized a rapid bioluminescent antibiotic susceptibility assay based on comparison of bacterial ATP concentrations (bioluminescent signals) in a control (aliquot of the sample, free of the antibiotic) and probe (aliquot of the sample, supplied with antibiotic examined) after short-time incubation. For validation of the proposed assay, bacteria strains isolated from clinical samples were analyzed in parallel by the Bioluminescent Assay and Standard Microbiology Assay - Disk Method or Serial Dilutions Method.

MATERIALS AND METHODS Reagents and Instrumentation. The lyophilized A TP-reagent 1 based on recombinant Luciola mingrelica luciferase was from Lumtek LLC (Russia). Before use the ATPreagent was reconstituted with sterile deionized water obtained on MilliQ, Millipore (France). Dimethylsulphoxide (DMSO) was from Reakhim (Russia). The following antibiotics (on standard disks or lyophilized) were from Sensidise Becton Dickinson (USA) or Bio Rad (Switzerland): Amikacin, Amoxicillin, Ampicillin Azlocillin, Carbenicillin, Cefaxime, Cefepime, Cefoperazone, Ceftazidime, Ceftriaxon, Cefotaxime, Cefoxitin, Ciprotloxacin, Clindamycin. Doksiciklin, Erythromycin, Fosfomycin, Gentamicin, Imipenem, Levotloxacin, Linezolide, Meropenem, Metronidazole, Moxitloxacin, Oxacillin, Penicillin, Piperacillin, Sulperason, Tarivid, Tienam, Tetracycline, Ticarcillin, Tobramycin, Vancomycin. Bacteria strains Burkholederia cepacia (I), Enterococcus durans (1), Enterococcus faecium (I), Enterococcus faecalis (2), Enterobacter aerogenes (I) Enterobacter spp. (1), Escherichia coli (4), Klebsiella pneumoniae (4), Klebsiella oxitoca (I), Lactobacillus sp. (1), Proteus mirabilis (1), Pseudomonas aeruginosa (5), Staphylococcus aureus (6), Staphylococcus epidermidis (2), Staphylococcus spp. (2)

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were Isolated from clinical samples. The number of each strain examined in different hospitals is indicated in the brackets. Portable PMT-based luminometer LUM-l used for bioluminescence measurement was from Lumtek LLC (Russia). The 48- and 96-well multidishes were from Nuclon TM. The 96-well strip plates were from Nunc. Bioluminescent Assay (BA) of antibiotic susceptibility. Bacteria stains were isolated from clinical sample and pure cultures of pathogens were obtained. Suspension of the each pure culture in saline was prepared and bacteria titer was established by the McFarland density standard. Bacteria suspension was diluted with rich nutritive media 10 2 _10 4 times. Aliquots (0.2 or 1 mL) were pipetted into the cells of multidish supplied with disks of antibiotics examined (probes) and free of antibiotic (control). The multidish was incubated at 37°C up to 12 h. After incubation 0.02 mL of probes and control were transferred using multi-channel pipette into the cells of 96-well strip plate filled with 0.18 mL of DMSO. The content of cells was mixed well and bacterial ATPextracts were obtained. Microcuvette was placed into the measuring chamber of luminometer LUM-l. 0.10 mL ATP-reagent and 0.02 mL ATP-extract were pipetted into microcuvette, mixed rapidly, and bioluminescent signal, (/probe) or (/controJ, was recorded. All measurements were performed in triplicate. The average values were figured out and the index of relative inhibition of bacteria growth under the action of antibiotic, U (%), was calculated using equation (1) U (%) = 100 x [1- (IprobJI(IcontroJI

(I)

Standard Microbiology Assay of antibiotic susceptibility. Bacteria suspensions in saline (~1 0 8 cell/mL) were assayed by Disk Method (DM) or Serial Dilutions Method (SDM) using automated analyzer Vitek (bio Meriuex, France). RESULTS Antibiotic susceptibility assay based on measurement of bioluminescent signals from probes and control treated with DMSO was developed in our laboratory earlier. 2,3 In the present study we simplified the BA protocol and performed validation of this protocol in five hospitals in Moscow city. First, we optimized incubation of probes and control. It was shown that the initial bacteria titer in probes and control should not exceed ~ 10 4 celllmL, otherwise for several strains, the inhibition of bacteria growth in probes with active antibiotics was not observed during the short incubation time. The volume of probes and control, 1 mL, was selected (48-well multidish). The lower volume, 0.2 mL, resulted in 5-fold higher concentration of antibiotic in the probes. The following duration of incubation was selected: 5 h - for aerobic strains, 10 h - for anaerobic or micro aerophilic strains.

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Under the conditions selected, 184 probes comprising 33 aerobic and micro aerophilic clinical strains (5-7 antibiotics per strain) were examined both by optimized BA and OM or SOM. The following criteria for assessment of antibiotic susceptibility of pathogen using BA were determined (Table I). Table 1. Criteria of antibiotic susceptibility of pathogen, U(%), by bioluminescent assay Antibiotic susceptibility of pathogen determined by standard microbiology assay S (susceptible) I (intermediate) R (resistant)

U(%) determined by Bioluminescent Assay U(%);;, 70% 60% s U(%) < 70% U(%) < 60%

The analysis of data obtained by BA and Standard Microbiology Assay was performed (see Table 2). Table 2. Comparison of results obtained by standard microbiology assay and bioluminescent assay Hospital (Standard Microbiology Assay applied) NI (SOM) N2 (OM) N3 (OM) N4 (OM) N5 (OM) Total

Number of probes examined 30 35 35 35 49 184

Number of probes agreed with BA (%) 28 (93.4) 31 (88.6) 30 (85.7) 30 (85.7) 42 (85.7) 161 (87.5)

Number of probes Number of probes mismatched mismatched less than 5%, more than 5%, (%) (%) I (3.3) I (3.3) I (2.9) 3 (8.5) I (2.9) 4(11.4) I (2.9) 4(11.4) 2 (4.1) 5 (10.2) 6 (3.3) 17 (9.2)

A good correlation (total and within the hospitals) between BA and OM/SOM was obtained. The best correlation (93.4%) was achieved when the SOM was applied as a Standard Microbiology Assay (Hospital NI). For 20 clinical strains, complete agreement of both assays was observed for all antibiotics examined. Other 13 clinical strains comprising 23 probes could be divided into two groups. First group consisted of 6 probes which had a discrepancy with OM of not more than 5 %. For second group, BA showed a higher discrepancy with OM. It is

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important to underline that only two probes demonstrated lower antibiotic susceptibility in comparison with DM. The other 15 probes showed higher antibiotic susceptibility in comparison with DM. Probably this phenomenon could be explained as follows: 1) peculiarities of bacteria growth on solid (DM) and in liquid (BA) nutritive media resulting in various resistance of bacteria to the same antibiotic; 2) significantly different initial titer of bacteria suspensions obtained for assays: 104 cell/mL and 10 8 celllmL for BA and DM, respectively. As an example the P.aeruginosa can be considered. It was assayed in five hospitals using 14 antibiotics. For 8 antibiotics BA showed higher antibiotic susceptibility than DM. In conclusion one can state that the optimized BA protocol proposed is simple and rapid (up to 5-10 h). Validation ofBA in 5 hospitals demonstrated high accuracy and potential applicability of the assay in clinical practice.

REFERENCES I. Ugarova NN, Maloshenok LG. Reagent for determination of adenosine 5'triphosphate. Russian Patent 2004:N2268944. 2. Titov A, Romanov·a N, Danilova T, Brovko L, Ugarova N. Bioluminescent method for assessment of microflora susceptibility to antibiotics. Laboratornoye Delo (Rus) 1990; 10:61-6. 3. Brovko L, Ugarova N, Dukhovich A. Bioluminescent reagents and "Rapid microbiology" methods. In: Stanley PE, Kricka LJ. eds. Bioluminescence and chemiluminescence. Current status. Chichester:Wiley, 1991: 433-6.

BART: SMART BIOCHEMISTRY, BRIGHT BIOLUMINESCENCE, LOW-COST HARDWARE OA GANDELMAN, G KIDDLE, CJ MCELGUNN, M RlZZOLl, JAH MURRA yl, LC TISI Lumora Ltd., Denmark House, Cambridgeshire Business Park, Angel Drove, Ely Cambs CB7 4ET, UK; I Institute of Biotechnology, Cambridge University, Tennis Court Road, Cambridge CB2 IQT, UK [email protected]

INTRODUCTION Bioluminescent Assay in Real-Time (BART) is a new bright reporter system for molecular diagnostics. BART produces light resulting from an enzymatic conversion of inorganic pyrophosphate (PP j ), a by-product in any nucleic acid amplification reaction, using an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) (Fig. I). BART does not utilise any fluorescent probes and relies only on light coming straight from the assay. This distinguishes it from qPCR which relies on irradiation of flu oro ph ores in the sample. The high levels of light produced in the course of the assay significantly simplify the hardware requirements for BART, in particular when compared to traditional bioluminescent assays. As such BART enables molecular diagnostics to be performed with simple robust low-cost hardware unlike qPCR which requires bulky, capricious and expensive hardware which is affordable for big laboratories only. (DNA}n + dNTP PP j + APS A TP + LHz

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Fig. 1. Biochemistry of BART The requirement for fast and accurate thermocycling adds to the complexity of the qPCR hardware. Thermocycling becomes unnecessary when using isothermal nucleic acid amplification technologies (iNAA Ts), which rely on the use of strand-displacing polymerases instead of high temperatures and are carried out at constant temperature (mostly below 65°C). A wide range of iNAATs has been developed and implemented over the last decade. iNAATs exemplify all of the features of PCR but advantageously can be run on any heating block. iNAA Ts have been steadily gaining popularity due to the simple hardware requirements. However, until now iNAA Ts have been monitored mostly fluorimetrically using real-time PCR machines. BART is an ideal reporter system for iNAATs as the combination of the two with the bespoke simple, robust and low-cost hardware will open new avenues for in vitro 93

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Gandelman OA et al.

molecular diagnostics. We present here the exemplification of BART along its unique features and describe two prototypes of the relevant hardware. RESULTS AND DISCUSSION Instrumentation for BART. Two design concepts were developed for building robust and low-cost hardware for BART: one based on CCD-cameras and another one based on photodiodes. Prototypes of two bespoke machines named Byson and (photodiode quantification system) were designed and built. Byson is a CCD-based machine with a small foot-print, suitable for the simultaneous screening of 96- or 384in commercially available PCR plates in a laboratory setting (Fig. 2a). In this is measured from the top and it is operated by user-friendly PCsoftware available in two modes: research and diagnostic. Research mode allows a user to vary such parameters as temperature, integration time, total of the run, etc. The diagnostic mode offers a choice of tests in which all parameters are pre-set and the resulting data are automatically processed by a simple algorithm a answer for each sample: Positive, Negative, Blank or Inspect (Fig. a b

2.

CCD-based machine for high-throughput BART applications (a) and a snap-shot of the resulting screen in diagnostic mode (b)

photodiode device for simultaneous analysis of up to eight uses commercially available 0.2 ml PCR tubes or 8-well PCR light is detected from the bottom of tubes. PDQ can be used in standalone mode or when connected to a PC through a USB-port. Power low for the machine to be run off a car battery if used in a field. cost distributed device suitable for point-of-care applications. Dedicated software offers a choice of tests with pre-set parameters (Fig. 3b) and analyses the data final result for each sample at the end of the run, when used in the BART. BART kinetics is very different from that observed in 4a). Negative samples typically display gradually while any positive reaction is characterised by a bright flash followed a switch off to almost zero level. The combination of both i.e. flash and switch is required for the distinctive qualitative result. We have previously shown that the flash reflects the exponential production of PP i resulting from an accumulation of DNA through amplification, and Peak time is a quantitative measure

BART: Smart Biochemistry, Bright Bioluminescence, Low-Cost Hardware

95

of the amount of nucleic acid target present in the assay, similar to the Ct value in b

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BART curves from (a) a positive and a negative sample; (b) BART assay run in different volumes showing the same peaking time; (c) dilution series of target DNA amplified by LAMP; (d) linear calibration for DNA amplified by dilution series of target DNA amplified by RDC; (f) linear calibration DNA amplified by ROC. We used BART for quantification of DNA in two different iNAATs: Lamp-Mediated (LAMP)2 and chimeric primers amplification (RDC)3. Both amplifications show sensitivity down to 10 copies of DNA and the BART has no adverse effect on the sensitivity. Good linear correlation between Peak time and DNA copy number is observed in both cases independent of the length of the assay BART reagent retained high activity for 4 hours at 60°C.

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Gandelman OA et at.

An essential feature of BART assays is that it is the rate of change of light intensity, which is truly important, rather than the absolute intensity of BART signals. Therefore, quantification in BART can only rely on Peak time. Very bright flashes make it possible to carry out assays in volumes as small as 1 Ill. Neither Peak time nor signal to background ratio are dependent on the assay volume (Figure 4b). Reduction in volume is a huge benefit for high-throughput screening and an easy way to bring down the costs, particularly taking into account that assays in such small volumes can be successfully performed using the same hardware. In qPCR followed fluorimetrically such small volumes are challenging and decrease of assay volume inevitably causes huge increases in hardware costs. We demonstrated that BART accurately quantifies the amount of DNA amplified by LAMP or RDC and reliably reports on amplification in several other iNAATs. BART has no no adverse effect on either sensitivity or specificity of the amplification method used. It allows testing in a closed-tube format preventing amplicon carry-over. It can be exemplified in submicroliter volumes reducing assay costs and allowing multiple repeats and a thorough statistical analysis of data. It opens up wide prospects for highthroughput screening in 96-, 384-, 1536- and higher formats. We reported here smart biochemistry resulting in bright light output combined with low-cost robust hardware which makes BART a Brilliant Alternative to Real-Time qPCR.

REFERENCES I.

2. 3.

Gandelman OA, Church VL, Moore CA, Carne C, lalal H, Murray lAH, Tisi LC. BART - bioluminescent alternative to real-time PCR. In: Szalay AA, Hill Pl, Kricka LJ, Stanley PE. eds. Bioluminescence and Chemiluminescence. Chemistry, Biology and Applications. Singapore:World Scientific, 2007:95-8. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cel Probes 2002;16:223-9. Cleuziat P, Mandrand B. Method for amplifying nucleic acid sequences by strand displacement using DNA/RNA chimeric primers. PCTIFR96/01166.

BART APPLICATIONS IN MEDICAL AND FOOD DIAGNOSTICS OA GANDELMAN, G KIDDLE, M RIZZOL!, JAH MURRAY!, LC TIS! Lumora Ltd., Denmark House, Cambridgeshire Business Park, Angel Drove, Ely Cambs CB7 4ET, UK; 1 Institute oj Biotechnology, Cambridge University, Tennis Court Road, Cambridge CB2 IQT, UK [email protected]

INTRODUCTION Bioluminescent Assay in Real-Time (BART) is an exciting alternative to real-time PCR.! It is a bioluminescence-based reporter system for monitoring isothermal nucleic acid amplification technologies (iNAA Ts) in real-time. BART assays can be performed using simple, robust and low-cost hardware which has significant implications for the molecular diagnostic market. We demonstrated that BART is compatib Ie with a number of iNAA Ts and has no adverse effect on either specificity or sensitivity of the amplification method used. BART gives both quantitative and qualitative results. It can be run in very small volumes, significantly reducing assay costs and offering great potential for higher throughput screening. It is carried out in a closed-tube format, which is essential for containing the contamination. We showed that BART is tolerant to different impurities and contaminants and is therefore compatible with crude, fast and simple sample preparations. We report here the use of BART in diagnosis of Chlamydia trachomatis from human urine specimens, detection of an RNA virus and screening test for GM contamination in maize. BART is a way forward for molecular in vitro diagnostics with great potential for bringing it into any setting including small laboratories, point-of-care or even into the field. RESUL TS AND DISCUSSION Detection of Chlamydia trachomatis using BART. A fragment of Chlamydia trachomatis (CT) cryptic plasmid was targeted and amplified by loop-mediated amplification (LAMP) and assayed by BART. 14 different strains of CT were tested in triplicate and all samples came strongly positive proving wide microbial range of the test. 32 strains of pathogenic bacteria and commensals of the oropharynx and genital tract, including Neisseria gonorrhea, Mycoplasma Jermentans, Mycoplasma hyorhinis, Clostridium difficile and others, were tested in triplicate for exclusivity. All 96 samples came negative, demonstrating 100% specificity of the test. BART reliably reported all cases of successful amplification and did not result in any false positives where amplification did not occur. RNA detection and quantification with BART. Purified RNA from classical swine fever virus (obtained from FL!, Germany) was amplified in a one-step format which included reverse-transcription with AMY and LAMP. As with any DNA target for a wide dilution series of RNA samples BART resulted in a sequence of light peaks

97

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Gandelman OA et al.

observed down to 1,000 RNA copies (Fig. la) with Peak time showing reverse linear proportionality to RNA target copy number (Fig. 1b). BART was shown to accurately report the detection and quantification of RNA in reverse-transcription amplification without affecting its specificity or sensitivity. a

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Development of screening test for GM maize using BART. Screening tests for GM contamination are a significant challenge for molecular methods as they need to be highly sensitive, quantitative, tolerant to high levels of genomic DNA and at the same time retain the possibility to be carried out with crude sample preparations, preferably in a field. BART has a good potential to address all of the above requirements. We tested the performance of BART on a model plasmid DNA system in the presence and absence of high concentrations of carrier DNA (500 ng salmon sperm DNA per 20-111 reaction which exceeded the levels expected in real GM tests). Neither sensitivity nor speed of the assay were affected by the presence of carrier DNA. In both cases BART resulted in good linear correlation between the Peak time and DNA copy number and reliably and accurately detected 10 copies of target DNA in less than 40 minutes (Fig. 2). Genomic DNA from CRM maize (Sigma) with different GM content (0%, 0.1 % and 1%) was purified. Neat and a few consecutive 10-times dilutions of prepared samples were tested in LAMP-BART with either alcohol dehydrogenase 1 (ADHl) primers or 35S primers. ADHI is a reference gene and its copy number is 100-1000 times higher in all samples than that of 35S, which is an indicator of a GM event. ADHI was reliably detected even after 100-fold dilution and in two out of three samples after a lOOO-fold (Fig. 3a). 35S was detectable in both 0.1 % and 1% GM maize only after a 10-fold dilution truly reflecting the expected low copy number of the sequence in analysed samples (Fig. 3b). The possibility to quantify such low copy number in less than 45 minutes places BART in a highly competitive position along the other methods and provides a solid proof-of-principle for BART's applicability and suitability for GM screening tests.

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We have exemplified BART applications in molecular IVDs and demonstrated its compatibility with various amplification methods, its ability to detect and quantify different targets, to cope with crude sample preparations and to provide rapid results under challenging conditions. Overall, BART is a universal reporter system for any molecular in vitro diagnostic tests based on isothermal nucleic acid amplification techniques.

REFERENCES 1.

Gandelman OA, KiddIe G, McElgunn CJ, Rizzoli M, Murray JAH, Tisi LC. BART: smart biochemistry, bright bioluminescence, low-cost hardware. In: Shen X, Yang X-L, Zhang X-R, Cui Z-J, Kricka LJ, Stanley PE, eds. Bioluminescence and chemiluminescence. Light emission: Biology and scientific applications. Singapore:World Scientific, 2009:97-100.

CHANGE OF EXPRESSION EFFICIENCY OF NATURAL AND CLONED LUX-OPERON IN CONDITIONS OF FAMINE AAGUSEV Institute of Biophysics of the Siberian Branch of the Russian Academy of Science,660036, Krasnoyarsk, Russia [email protected]

INTRODUCTION The maintenance of a high level of expression of cloned lux-genes is an important strategic problem which justifies cloning of alien genes in new host cells. In order to find approaches to an estimation of efficiency and consequences of wide use of the cloned fragments of DNA, in particular lux-operon from luminous bacteria, it is necessary to develop appropriate experimental methodology. The expression of bioluminescent operons is the parameter which depends on structure of operon and metabolic activity of a host cells. 1 The purpose of the present work is to study the influence of famine on expression efficiency of lux-operon (natural and cloned in plasmid) from marine luminous bacteria Photobacterium leiognathi. MATERIALS AND METHODS For our research we have used a marine luminous bacteria, Photobacterium leiognathi, strains 54, 1210 and variants of transgenic strain Escherichia coli Z905/pPHL7 from a Culture Collection CCIBSO 836. 2 Characteristics of places of isolation of the strains investigated are presented in Table I. Natural luminous bacteria were grown up on full and minimal semi-synthetic media with glycerin in Erlenmeyer flasks on temperature-contro lied shake-flask propagator at 28°C. 3 Variants of transgenic strains were grown up in microcosms. Optical density of cell suspension was measured by photocolorimeter CPC-2MP (the Optic-Mechanical Factory, Russia) in 0.5 cm quartz cuvette at 540 nanometers of wave length (the green filter). The error of measurement of optical density did not exceed II %. Intensity of a luminescence was measured by bioluminometer designed in SCTS "Science" (Krasnoyarsk). The present equipment allows measurement of intensity of luminescence in a range from 10-4 up to 10 2 mkA (1 mkA == 4xl0 9 quantum/s). The error of measurement of a luminescence did not exceed 5 %. Quantitative criteria of expression efficiency of bioluminescence were: copy number of lux-operon (level of a replication) in a cell, duration of the latent period of induction of bioluminescence (level of a transcription), the maximal intensity of luminescence (level of a translation). The following quantitative criteria and coefficients are used: fl - number of copies of lux-operons in a cell (for natural marine bacteria - 1 operon/chromosome, for transgenic luminous bacteria the parameter varies depending on strain). Number of copies of plasmid was determined on electrophoregrams with program Scion Image; f2 == lit - coefficient (5- 1), which reflects influence of the latent period in dynamics of bioluminescence on expression 101

102

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efficiency of lux-operon. The latent period is a time (t) from inoculation to the beginning of a phase of linear growth of intensity of a luminescence, if the latent period is absent f2 = 1 S·I. f3 - the maximal value ofbioluminescence (quantals). Efficiency expression of bioluminescence on a population of cells: Fpop = f2xf3 (quantals 2). Efficiency expression of bioluminescence on one cell: Fcell = FpoplN (quantals 2xnumber of cells). Efficiency expression of bioluminescence on one copy of lux-operon: Fcopy = Fcell/ fl (quantals 2xnumber of cellsxnumber of copy of lux-operon). The basic quantitative characteristics necessary for the calculation of expression efficiency of lux-operon have been obtained during study of the dynamics of bioluminescence and growth of different strains of cells on different nutrient media (Table 1). Coefficients of efficiency of bioluminescence at the molecular, cellular and population levels have been calculated. As it can be seen from data in Table 2, the Photobacterium leiognathi 54 strain is more adapted for conditions of famine, as expression efficiency of lux-operon is higher than on the full medium. On both media the coordination of an induction of growth with an induction of a bioluminescence was observed for strain 54. For the Photobacterium leiognathi 1210 strain, famine was stressful, and it affected expression efficiency of lux-operon at a cellular level in comparison with the full medium and resulted in to heterogeneous population level. Therefore, the strain P. Ie iognathi 1210 is less adapted to conditions of a life in an oligotrophic water ecosystem. Reduction of the latent period of bioluminescence of strain P. leiognathi 54 and its increase in strain P. leiognathi 1210 (Table 1) shows adaptable capabilities of these strains to famine as well as to stress. The expression efficiency of lux-operon at the cellular and molecular levels was equal (one lux-operon/cell). A different situation exists for transgenic strains, since there are many copies of lux-operon/cell and this produces an increased loading on a metabolism of a cell. With increase of time of adaptation in water microcosm (Table 2), expression efficiency of bioluminescence of transgenic microorganisms increased in conditions of famine. At the same time at all variants obtained from the microcosm it was lower than at initial laboratory strain (Tables 1, 2). It is clearly visible for strain Escherichia coli Z905-2 which had the least time of adaptation in the oligotrophic microcosm. Already at a population level, the expression efficiency of lux-operon of strain Escherichia coli Z905-2 in conditions of famine was more than on 400 times lower than on the full medium. It was a unique strain which at cellular and molecular levels had expression efficiency on the minimal medium lower than on the full medium. At a molecular level expression efficiency of lux-operon of all adapted transgenic strains in conditions of famine was higher than on the full medium. The increase of the latent period of bioluminescence in transgenic microorganisms (Table 1) promoted their survival

Table 1. Characteristics of variants of natural and transgenic luminous bacteria Characteristics

Species ofluminous bacteria Escherichia coli

Photobacterium leiognathi

Extraction

Number of luxoperons on cell The latent period in dynamics of a bioluminescence (h) the maximal value of bioluminescence, IgI Number of cells in a point of the maximal luminescence, IgN

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therefore the substratum was not spent for a bioluminescence reaction. During the stay in the microcosm the copy number of recombinant plasm ids was restored, that as a whole lead to increase of expression efficiency of cloned lux-operon at all levels including population. Thus the maintenance of super-expression of bioluminescence is possible only for natural luminous bacteria. It is not to be expected that cloned lux-operon can provide super-expression or full loss of expression. At purposeful introduction of microorganisms in the ecosystem there will be no super-expression. At casual introduction of microorganisms in the ecosystem (owing to accident) the incomplete loss of expression will be undesirable.

REFERENCES 1.

2. 3.

Bainton Nl, Lynch 1M, Naseby D, Way lA. Survival and ecological fitness of Pseudomonas fluorescens genetically engineered with dual biocontrol mechanisms. Microb Ecol 2004;48:349-57. Rodicheva EK, Vydryakova GA, Medvedeva SE. Catalogue of luminous bacteria cultures. Novosibirsk:Nauka, 1997. Popova LYu, Kargatova TV, Ganusova EE, et al. Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures. Adv Space Res 2005;35:1573-8.

CONSTRUCTION OF RECOMBINANT LUMINESCENCE BACTERIA VECTOR TO EVALUATE GENETOXIC ENVIRONMENTAL POLLUTANTS XIN-XIN HUANG, MIAO HE, HAN-CHANG SHI, QIANG CAl Division of water environment, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China

INTRODUCTION The luminescent bacteria test, a quick, sensitive and convenient method for the acute toxicity evaluation of environmental pollutants, is widely utilized all over the World.) Luminescent bacteria are mostly marine in origin, and this might affect the sensitivity, and stability of the test for complex water samples, many researchers have studied the construction and application of recombinant bioluminescent bacteria. Engineered bacteria have some advantages over traditional ones in more moderate test conditions, including greater response and sensitivity, as well as identifying diverse types of toxicity. The inducible system can respond to a specific stress or chemical, which leads to a capability for measuring toxicity or stress induced, as well as recognition of a single or a group of compounds. In this study, two inducible recombinant bioluminescent strains sensitive to DNA-damage stress were constructed. Genetoxic pollutants have attracted attention because of their great potential for harm to human beings and the environment. In this study, we fused promoters of uvrA or alkA to the promoterless Vibrio fischeri luxCDABE operon present within the broad host range, multicopy plasmid pUCD6IS. 2 The engineered strains were induced to emit luminescence in the presence of DNA-damage stress, so they can be utilized in the evaluation and monitoring of the genetic toxicity of environmental pollutants. MATERIALS AND METHODS Bacterial strains and growth conditions. Escherichia coli DHSa, E. coli 1Ml 09 were purchased from TaKaRa company and used in this study, E. coli W311 0 was kindly presented by professor Zhou of Academy of Military Medical Sciences of China. Strains were routinely grown in Luria-Bertani media at 37°C for 24 h. DNA extraction and design of the primer set. DNA was extracted from pure E. coli W3110 culture employing the Insta-Gene matrix (Bio-Rad, Madrid, Spain) according to the manufacture's protocol. Purified DNA was stored at -20°C until use. For the PCR assay two pairs of primers were designed for the uvrA gene and alkA gene] that contained recognition sequences for BamH I or EcoR I restriction enzymes (underlined). The uvrA primers used were: S'-ACTTTTGGATCCGTGTAAACGCGCGATTG-3' and

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, the aiM and

used were

of amplicons. The amplified products were cloned into vector (Prom ega) and used to transform competent E. coli DH5a. Transformed E. coli DH5 was then plated onto Luria-Bertani agar with 50 USA) and the recombinant cloned were screened by colony PCR primers. The clones were taken for sequencing and the correct were cloned into the PUCD615 vector. The recombinant electrotransformed into E. coli JMI09 cells. DNA sequencing was done Prism 377 automated DNA sequencer. The GenBank database was searched using the NCBI BLAST program. Consensus sequences were generated and analysed using the programs of the DNASTAR. alignments of nucleotide were performed with the Clustal X software package. '

electrophoresis gel of PCR products revealed of uvrA and aiM were 237 bp and 326 bp respectively 1). 2

M

the

bp ....- 2000

""-]000

326bp 237bp

n.

+-500 .--250 +-100

Amplified product of uvrA and aikA by PCR (I: uvrA: 2: atkA: M: Marker)

Identification of recombinant vectors Each eight clones were selected for PCR from containing pT-uvrA and pT-alM vectors, all the clones were clone 3 of the pT-uvrA vector (Fig. 2.). Each two clones ofPUCD-uvrA and PUCD-aIM recombinant vectors were and clones were confirmed by isolation of the plasmid. Amplification ofthe insert and all the PCR products yielded the correct size offragments (Fig. 3.).

Construction of Recombinant Luminescence Bacteria Vector

107

bp 2000 1000 500

100

Determination ofpT-uvrA and pT-alkA recombinat vectors by PCR ~8: clones ofpT-uvrA; 9-16: clones ofpT-alkA; M: marker

12M34

bp

1000 500

237bp

326 100

Determination ofPUCD-uvrA and PUCD-alkA recombinants 3,4: clones ofPUCD-alkA; M: marker vectors by PCR.

The sequencing results of uvrA, alkA products were 3495.1) alkA (K02498.l) of GenBank: they matched with 99% and so the amplification results were correct. results of the fragments inserted into PUCD615 vector also revealed alkA genes had been inserted into the mUltiple clone sites the insert direction and the reading frame were also correct. So the recombinant PUCD-uvrA and PUCD-alkA vectors were constructed successfully.

DISCUSSION The increase in poisonous pollutants threatens the health of human beings and the so determination of universal bio-toxicity based on biological effects has become an important monitoring method for human protection and environmental safety. We constructed an engineered strain that responds to

108

Huang X-X et al.

genotoxic substances in water that could be utilized for water quality assessment in the geographic area surrounding China. The lux gene utilized in a recombinant luminescent organism can be either luxAB or luxCDABE. The former requires aliphatic aldehydes as one of the substrates for the bioluminescence. The length of luxCDABE genes is about 6kb while that of luxAB genes is not more than 3kb, and the pUCD6I5 containing luxCDABE genes is 17.55kb long. This brings great difficulty for gene construction, so most researchers in China have focused on the utilization of luxAB genes. In this study, we used the whole gene cassette of luxCDABE to construct the recombinant, thus this fusion in E. coli allows visualization of the transcriptional responses induced by DNA damage, without the need to perform enzyme assays or to add luciferase substrates exogenously. It makes the recombinant-based assay simpler and more convenient and suitable for the insitu monitoring with biosensors or detection kits. Since the PUCD615 vector is large while the inserted fragments were about 300bp, this made it difficult to construct the recombinant vector. By optimizing the ligation conditions and electroporation treatment for transformation, we could construct the PUCD-uvrA ':PUCD-alkA recombinant vectors successfully. In the future research, we will investigate its response to DNA-damage stress, compare its performance with Photobacterium phosphoreum and V. fischeri, optimize test condition and utilize it in the genetic toxicity evaluation of water samples.

ACKNOWLEDGEMENTS The plasmid pUCD615 was kindly presented by Prof Sayler from the University of Tennessee. REFERENCES I. Hu HY, Wei DB, Dong C H. Assessment and management on water quality safety of sewage. Environ Assess 2002; 11 :37-41. 2. Rogowsky PM, Close TJ, Chimera lA. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. 1 BacterioI1987;I69:5IOI-12. 3. Vollmer AC, Belkin S, Smulski DR. Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids. Appl Environ Microbiol 1997;63 :2566-71. 4. Thompson JD, Gibson TJ, Plewniak F. The Clustal-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 1997;25:4876 -82.

DEVELOPMENT OF A NOVEL BIOLUMINESCENT ASSAY FOR NITRIC OXIDE BY USING SOLUBLE GUANYLATE CYCLASE YOSHIHIRO SANO,l MASA YUKI SEKI,l SHIGEY A SUZUKI,2 SEIJI ABE,l KA TSUTOSHI ITO,l HIDETOSHI ARAKAWA 1 1School of Pharmaceutical Science, Showa University, Tokyo 142-8555, Japan. 2Research & Development Division, Kikkoman Corporation, Chiba 278-0037, Japan, Email: [email protected]

INTRODUCTION Nitric oxide (NO) has been known to be involved in many biological processes including vasodilatation and neuronal communication. Soluble guanylate cyclase (sGC), which is a well-known "NO-sensor", plays an essential role in signal transduction with respect to enzyme initiation for the conversion ofGTP to cGMP in the NO signaling cascade, leading to the concomitant production of pyrophosphate (PPi).l Reportedly, cyclase activity is activated by as much as 200-fold upon NO binding to sGc. We previously described a highly sensitive bioluminescent assay for PPi utilizing the PPDK-luciferin/luciferase reaction. In this study, we developed a novel bioluminescent assay for NO utilizing sGC based on the PPDKluciferin/luciferase reaction. The measurable range of NO obtained by the proposed method is 200-20,000 nM; the detection limit is 200 fmol/assay. Moreover, this method is sensitive and specific for NO. MATERIAL AND METHODS Materials. PPDK-luciferin/luciferase solution, PPDK from Microbispora rosea subsp. Aerata (EC 2.7.9.1) and thermostable Luciola cruciata firefly luciferase (EC 1.13.12.7) were obtained from Kikkoman Co. (Chiba, Japan). Tricine and NOC7 (3(2-hydroxy-l-methyl-2-nitrosohydrazino)-N-methyl-l-propanamine), which functioned as NO donors for NO detection or measurement, were purchased from Dojin Laboratories (Kumamoto, Japan). Dithiothreitol (DTT), MgCI 2 .6H 2 0, MnCI 2 .4H 2 0, NaOH and soluble guanylate cyclase from calf lung were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Guanine 5 '-triphosphate (GTP) was purchased from Roche Diagnostics, Inc. (Basel, Switzerland). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Co. (St. Louis, USA). Other reagents were analytical grade. Reagents. The reaction buffer was prepared with the following reagents: 20 mM Tricine, 2 mM MgCh, 1 mM DTT and 0.1% BSA. GTP or sGC solution was prepared and diluted with the reaction buffer. NOC7 was dissolved with 0.01 N NaOH (NOC7 solution). Methods. A novel bioluminescent method for NO utilizing the sGC/PPDKluciferin/luciferase system is as follows. GTP solution (8 ilL) (10-5 mol/L), sGC solution (1 ilL) (10 ng/IlL) and NOC7 solution (1 ilL) were added into a sample 109

110

Sana Y et at.

tube; subsequently, the mixture was incubated at 37°C. The sample tube was cooled for several minutes on ice in order to terminate the enzymatic reaction. Next, the reaction product (PPi) in 10 JlL of the aforementioned sample solution was measured by addition of 10 JlL of PPDK-Iuciferin/luciferase solution (PPDK reagent). After lOs, the light emission intensity was measured at lOs intervals for a period of3 min with the luminescent reader (Aloka). RESULTS AND DISCUSSION Principle of the novel bioluminescent assay for NO utilizing PPDKluciferin/luciferase. In this study, we developed a novel bioluminescent assay for NO utilizing the activity of soluble guanylate cyclase; the scheme is presented in Fig. l. The cyclase activity of sGC is enhanced upon NO binding of the beta subunit of sGC containing the heme region. GTP is converted to cGMP; concomitantly, PPi is produced. PPi is measured with a highly sensitive bioluminescent assay for PPi utilizing the PPDK-Iuciferin/luciferase reaction. 2 Measurement conditions of the novel bioluminescent assay for NO utilizing sGC/ PPDK-luciferinlIuciferase. To determine the optimum measurement conditions for this assay, the effects of substrate concentration (sGC and GTP), temperature, reaction time and metal ion (Mn 2+ or Mg 2+) were examined. The effects of various conditions were estimated based on signal/noise ratio and light emission intensity. The optimum concentrations of sGC and GTP were found to be 10 ng//JL (10- 6 g/assay) and 10-5 mollL (lO- IO mol/assay), respectively. In addition, optimum temperature and reaction time were 3rC and 10 minutes, respectively. In terms of the metal ion, light emission of Mg2+ was 5 times greater than that of Mn2+ (Fig. 2). It is weB-known that the luciferin-Iuciferase reaction requires Mg2+. Therefore, use of a common metal ion would exert a positive effect in this assay with respect to sGC and the luciferin-Iuciferase reaction. Standard curve for NOC7. Based on the aforementioned optimum measurement conditions, NOC7 (10- 13 to 10- 11 mol/assay) was examined. The standard curve for NOC7 is shown in Fig. 3. The detection limit ofNOC7 was 10- 13 mol/assay. This result was lower than the expected sensitivity of the PPDK-Iuciferase bioluminescent system; the PPi detection limit was 10- 15 mol/assay for this system. 2 The reason for the reduced limit is attributable to the non-specific reaction of GTP and luciferase. The non-specific reaction of luciferin-Iuciferase was reflected in the presence of excess GTP; consequently, the light emission intensity of the background was increased to measure NOC7 at less than 10- 14 mol/assay.

Development of a Novel Bioluminescent Assay for Nitric Oxide

III

NO

D

PEP:pll()~l·lI()tlloll)Yl1lyatt

Fig. 1. Principle of the novel bioluminescent assay for NO utilizing sGC. (X 10;)

L5.---------------.

...
-§

2.0

0'----'----1------'----

10. 3

10-"

NOC- (mol L)

Fig. 2. Comparison between Mn2+ and Mg2+ in the novel bioluminescent assay for NO utilizing the PPDK-luciferin/luciferase system. CONCLUSIONS This assay facilitated the detection of NOC7; the sensitIVIty was 10- 13 mol/assay. Two mol of NO are released from I mol of NOC7; as a result, the detection limit of NO was 200 fmol/assay. Furthermore, this bioluminescent assay could specifically detect NO released from NOC7, and could not detect nitrate anhydride, N0 2- or N0 3 -, unlike the fluorescent assay utilizing DAN (diaminonaphthalene) (data not shown).

112

Sana Yet at.

lO-r--------------,

10z~--~--~--~--~

10' u

10. 13

1(I'u

10. 11

10. 10

NOC7 (mol/assay) Fig. 3. Standard curve for NOC7. Briefly, this assay, which is based on biological enzyme activity, namely, the cyclase activity of sGC, and which is a highly sensitive bioluminescent assay for PPi, is a novel bioluminescent assay for NO. Optimum conditions of this assay will be modified in order to detect NO release from nitrite drugs, e.g., isosorbide nitrite, nicorandil and glyceryl nitrite, etc.

ACKNOWLEDGMENTS This work was partially supported by Showa University Research Fund and "HighTech Research Center" Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 20072009.

REFERENCES 1. 2.

Denninger JW, Marietta MA. Guanylate cyclase and the .NO/cGMP signaling pathway. Biochim Biophys Acta 1999;1411:334-50. Arakawa H, Karasawa K, Igarashi T, Suzuki S, Goto N, Maeda M. Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reaction and a novel bioluminescent pyrophosphate assay. Anal Biochem 2004;333:296-302.

PART 3

BASIC CHEMILUMINESCENCE

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MASS SPECTROMETRIC APPROACH TO ELUCIDATION OF CHEMIEXCITATION OF DIOXETANES HK IJUlN, M OHASHI, M TANIMURA, N WATANABE, M MATSUMOTO Department a/Chemistry, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa,259-1293,Japan Email: [email protected]

INTRODUCTION A dioxetane bearing an aromatic electron donor, such as a phenoxide anion, displays intramolecular charge-transfer-induced chemiluminescence (CTICL),' in which charge transfer (CT) occurs from the electron donor to dioxetane 0-0 bond to induce its decomposition, producing excited species. On the other hand, we have very recently reported that electron transfer ionization takes place for dioxetanes bearing a hydroxyphenyl moiety substituted further with an aromatic ring that acted as an antenna to capture an electron from the matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).' Herein, we disclose that dioxetane Ib undergoes intramolecular CT-induced decomposition to give excited keto ester 2b through such ionization, while 2b gives a species in the vibrational excited state in MS/MS. MATERIALS AND METHOD Dioxetanes la-c were prepared by sensitized photooxygenation of the corresponding dihydrofuran, and their related keto esters 2a-c were synthesized by thermolysis of the corresponding dioxetanes (Fig. 1). MALDI measurements of dioxetanes la-c and keto esters 2a-c were performed with a reflectron TOF mass spectrometer (AXIMA-CFR, Shimadzu / Kratos, Kyoto, Japan) that was equipped with a pulsed nitrogen laser at 337 nm. It was operated in the negative ion mode with an accelerating voltage of -20kY. Negative ion mode, MALDI low-energy cm MS n (n = 2,3) measurements were carried out on an AXIMA-QIT instrument (Shimadzu / Kratos, Kyoto, Japan). Helium gas was used for col1isional cooling according to the instrument system's procedure. A matrix ofpoly(3-octylthiophene-2,5-diyl) (POT) was prepared as a dilute solution in benzene at a concentration of 1 mg/mL. Analytes 1 and 2 were dissolved in an appropriate solvent

a: R J = R2 = H b: R J = H, R2 = benzo[d]thiazol-2-yl c: R J = Me, R2 = benzo[d]thiazol-2-yl

1

2 Fig. 1. Structures of dioxetanes and related keto esters 115

116

Jjuin HK et at.

at a concentration of 1 mg/mL. Samples were prepared by mixing the matrix and the analyte in a ratio of 1: 1 (v/v). A sample solution (1.0 JlL) was deposited onto the sample plate and allowed to air-dry at room temperature. RESULTS AND DISCUSSION Dioxetane la gave deprotonated anion [M-H] - as expected, when POT was used as a matrix under the negative mode. In contrast to dioxetane la, both dioxetanes Ib and Ie gave radical anion [M] -' in addition to deprotonated anion [M-H] - or demethylated anion [M-Me] -. The difference in electron affinity of the aromatic substituent hydroxyphenyl and benzothiazolylhydroxyphenyl in dioxetanes was presumably reflected in the formation of [M] -' or [M-H] - as molecule-related ions. Thus, an aromatic substituent acts most likely as an antenna to catch an electron from the matrix. Furthermore, in the case of dioxetane I b, two pairs of characteristic fragment ions {[M-56] -' or [M-57] -} and {[M-86] -' or [M-87] -} were observed (Fig. 2a). Considering the structure of dioxetane Ib, the fragment ions [M-56] -' and [M-86] -' should be produced by the elimination of 2-methyl-l-propene (56 u) or pivalaldehyde (86 u) from radical anion [M] -', respectively. For dioxetane Ie, two pairs of fragment ions {[M-56] -', [M-86] -'} and {[M-Me-56] - and a trace amount of [M-Me-86] -} were also observed in addition to radical anion [M] -' and demethylated ion [M-Me] -. These fragment ions could not be produced directly from dioxetanes while retaining the four-membered ring framework. One strong candidate species that could cause such fragmentation is a keto ester, which is generated by the CT-induced decomposition of dioxetanes. Next, the ionization of authentic keto esters 2a-e was investigated. Features of the formation of molecule-related ions from keto esters 2a-e resembled those of the corresponding dioxetanes. However, none of the expected fragment ions could be detected as shown in Fig. 2b. These results in the full-scan spectra strongly suggested that keto ester produced from the highly strained dioxetane through ionization in MS would be energetically different from authentic keto esters 2. Thus, we performed further MS/MS measurements focusing on deprotonated ion [M-H] -for dioxetane lb. In the MS/MS spectrum of the precursor ion [M-H] - from dioxetane I b at mlz 410, product ions were observed at mlz 354,324,282,241,228 and 225 (Fig. 3a). Thus, the fragment ion at mlz 354 ([M-57] - ) from dioxetane Ib was concluded to be most likely produced by the loss of2-methyl-l-propene (56 u) from [M-H] -. Similarly, the fragment ion at mlz 324 ([M-87] - ) from dioxetane 1b was apparently generated by the loss of pival aldehyde (86 u) from [M-H] -. On the other hand, when we carried out MS/MS measurement of the precursor ion [M-Hr from keto ester 2b at mlz 410, from which the charactristic fragment ions could not be detected in a full-scan spectrum, the product ions were observed at mlz 354, 324,282,241, 228 and 225 (Fig. 3b). High energy dioxetane molecules give an electronically excited carbonyl fragment by thermal decomposition or by intramolecular charge-transfer induced decomposition. Thus, dioxetane Ib is presumed to decompose similarly into the corresponding excited

Mass Spectrometric Approach to Elucidation of Chemiexcitation of Dioxetanes

117

keto ester which releases 2-methyl-l-propene (56 u) or pivalaldehyde (86 u) by Norrish-type cleavage under negative-mode MALDI-TOF-MS conditions. The result of MS/MS measurements of keto ester 2b described earlier agree completely with this fragmentation of dioxetane lb. Considering that such fragment ions observed in MS/MS measurements of keto ester 2b could be hardly detected in full-scan spectrum, authentic keto ester 2b received most likely under MS/MS conditions collisional energy enough to generate 2b in the vibrationally excited state which caused fragmentation same as that observed for dioxetane lb under MALDI-TOF-MS condition (Fig. 4). It should be noted that the chemiexcitation of dioxetane lb apparently led to 2b in which a pivaloyl moiety was excited in addition to 2b in which aromatic ester moiety was excited in a gas phase. In contrast, only the formation of the latter excited species was observed through emission of bright light, but Norrish-type decomposition was little observed for the base-induced decomposition of lb in solution. The discrepancy is due to the fact that the energy of an excited pivaloyl moiety would be quickly lost by thermal relaxation processes induced by collision with solvent molecules in solution, while such relaxation does not occur in a gas phase at low-pressure. In conclusion, the present study has shown that intramolecular CT-induced decomposition of dioxetanes I b and Ie takes place to afford excited species, while keto ester 2b is excited vibrationally in MS/MS. (a)

(b)

T Fig. 2. (a) Full-scan MALDI-TOF mass spectrum of dioxetane I b (b) Full-scan MALDI-TOF mass spectrum of keto ester 2b (b) MS/MS : 410

(a) MSIMS : 410

~,

I

""

'"

J ~ J .,

1_

I

Fig. 3. (a) MS/MS spectrum of precursor ion at mlz 410 of dioxetane I b; (b) MS/MS spectrum of precursor ion at mlz 410 of keto ester 2 b

118

/juin HK et al.

-",*

°o~

- - - - -. w Not detected in MS spectrometry

Fragment ions

dioxetane Electronic excited state

'-------------------0--

o

o~ em

NOl'ri...h Type cleavage

-----o---~*

o~:

Fragment ions Norrish Type cleavage

ground state keto ester

Vibration ally excited state

~~--------------

No fragment ions

Fig. 4, Decomposition of dioxetane producing keto ester in the excited state and vibrational excitation of keto ester (CID: collision-induced dissociation)

ACKNOWLEDGEMENT We greatly thank Dr. Masaki Yamada in Life Science Laboratory of Shimadzu Corporation for measurement ofMALDI-TOF-MS/MS,

REFERENCES l. Matsumoto M, Watanabe N. Structural aspects of 1,2-dioxetanes active toward intramolecular charge-trans fer-induced chemiluminescent decomposition. Bull Chern Soc Jpn 2005;78:1899-1920. 2. Ijuin HK, Yamada M, Ohashi M, Watanabe N, Matsumoto M. Electron-transferinduced decomposition of 1,2-dioxetanes in negative-mode MALDI-TOF-MS. Eur J Mass Spectrom in press.

THEORETICAL CONSIDERATIONS ON THE ROLES OF HYDROGEN BONDING IN THERMAL DECOMPOSITION OF PEROXIDES

H ISO BE, S YAMANAKA, M OKUMURA, K YAMAGUCHI Dept of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan Email: [email protected] INTRODUCTION A recent study has clarified the roles of spin-orbit coupling (SOC) in chemiluminescence from the viewpoints of (i) the activation mechanism for the 302 reaction and (ii) the regulation mechanism of SOC in the thermolysis of a resulting peroxide.' Pursuing the latter problem, we can get a glimpse of the mechanism of the high efficiency reaction. As summarized in Table 1, the extent of SOC is closely related with the topicity1.2 created in the decomposition of a peroxide. The identity of the protonation state plays a decisive role in determining the number and label of the topicity and hence the extent of SOC. The optimal suppression of SOC is achieved when the charge-transfer (CT) transition structure (TS) occurs early in the reaction, which is the case when an electron-donating (deprotonated OH) substituent is present (category III). As a straightforward continuation, it is very interesting to examine perturbative influence such as substituent and hydrogen-bonding effects on SOC, which enable us to investigate the behavior of SOC in the "intermediate region" among categories I, II, and III. In this paper, we report a simple theoretical analysis of the roles of hydrogen bonding in the thermolysis ofdioxetanes (lH, r + nH 2 0, and 4-) and dioxetanones (2H, 2- + nH 20, and 5-).

Table 1. Classification of decomposition mechanisms ofperoxl'd es I I II III Charge and Spin Homolytic Diradical Charge-Transfer Diradical (CT) Distributions (HD) Orbital Forbidden-Radical Symmetry-Stability Allowed-Radical (AR) (FR) Relation (a*, n+, n_)h (a" n+, n_)a2 a*, h Topicity2 Tetratopic Tetratopic Bitopic Late Position ofTS Intermediate Early Strong Intermediate Extent of SOC Weak . .. In the case of dioxetanes, additional n± should be added to the toplcltles of I and II . COMPUTATIONAL METHOD The decomposition mechanisms were studied by using B3LYP/6-311+G(d,p)IIB3LYP/6-31+G(d).3 The SOC matrix elements between singlet and triplet states were estimated by using CASCI wave functions based on Boys localized orbitals with respect to the full Pauli-Breit SOC operator; see ref 1 for details. 119

120

lsobe H et al.

RESULTS AND DISCUSSIONS Let us first consider the thermolysis of dioxetanes (IH and 1~ + nH 20). The formation of hydrogen bond(s) with each water at the oxido anion moiety of 1~ has an effect that makes the position of a TS later on a reaction coordinate; i.e., the free energy of activation (~Gt), the 0-0 bond length (r(){}), and the diradical character (JiHOMOt ) for the TS increase monotonically with increasing the number of water molecule(s) considered (n) (Table 2). The important point to note is the change in the charge and spin density distributions to the TS. We can see the monotonous decrease in the extent of CT from the phenoxide anion group to the peroxide bond (~PD t) (Table 2). Judging from spin density distributions (Fig. IA), the thermolysis of anionic I~ belongs to the CT diradical mechanism,' in which up and down spins reside at the electron-donor and electron-acceptor parts. With the presence of one or two water molecule(s) at the anion site ofC however, the reaction cannot be directly assigned to a single category in Table 1, but rather can be characterized as an intermediate between the CT and homolytic diradical (HD) mechanisms. When three waters are added, the reaction falls into the HD mechanism (up and down spins are localized at the peroxide oxygen atoms),' which is the same category as the thermolysis ofneutralIH. The reason for this can be attributed to the fact that the presence of strong electronic interaction at the oxido anion moiety decreases the electron-donating ability of the phenoxide anion group, which can be easily assessed by calculating the gas-phase adiabatic ionization potentials of the clusters PhO~(H20)n (2.22, 2.66, 2.91, and 3.20 V for n = 0, 1, 2, and 3). As is expected from Table 1, the SOC values for the HD mechanism (24.0 and 19.6 cm~' for IH and C .. 3H2 0) are about 3.7-4.5 times larger than that for the CT mechanism (5.3 cm~' for I~). To our surprise, the SOC values for the HD/CT intermediate mechanism is found to be very small (0.5 and 0.6 cm~' for C .. IH 20 and C .. 2H 20). We may recall 2 2 that there is a So(n n)/S,(nn ) near-degeneracy region before the C-C dissociation for the 4 thermolysis of dioxetane C which is absent from the thermolysis of dioxetanone 2~. The SOC between the S, and T, states, in turn, yields very large values (95.3 and 95.4 cm~' for C .. 1H20 and C .. 2H 2 0). At the present stage, we are not competent to discuss nonadiabatic phenomena and related dynamics associated with intersystem crossing, but reflection on these results indicates that the conformational dynamics in actual aqueous solution, may bring about a charge and spin fluctuation and therefore variable SOC. Our models presented here are, of course, too simple to treat such a complex chemical event. Turning now to the thermolysis of dioxetanones (2H and 2~ + nH2 0). The tendency for a TS to move toward a later position under the influence of hydrogen bond(s) can also be observed, as shown in Table 2, but is rather insensitive, as compared with that for dioxetanes. The point that deserves explicit emphasis is that there is almost no change in the spin distribution, as depicted in Fig. 1B. The CT mechanism, 0*, h bitopic in more detail, applies to the thermolysis of anionic dioxetanones, irrespective of the number of water molecules considered, leading to a very small SOC value (0.2-0.3 cm~'). Only one expectation is the case in which the oxido anion moiety is protonated (2H), for which a large SOC value onO.6 cm~' results from the HD mechanism, (0" n+, n~) 02 tetratopic in more detail. For dioxetanones, the strong electrostatic interaction makes only a minor contribution to SOC, in contrast to the identity of the protonation state, which is a dominant factor controlling SOC. The dioxetanone functionality is,

Theoretical Considerations on the Roles of Hydrogen

DUJ"U"JK

121

advantageous to the suppression of SOC as far as the optimal prcltOlJatlon state is realized.

[%]

[kcal mor']

2.008 1.839 1.915 1.954 1.973 2.034 1.667 1.715 1.748

22.4 3.9 7.5 10.0 10.6 21.4 0.5 1.1 1.5

1

0.00 -0.25 -0.26 -0.22 -0.13 -0.07 -0.13 -0.17 -0.19 -0.21

HD CT HD/CT HD/CT HD HD

24.0 5.3

T 1 states near a was used for and the {lO,6} model {6,4} + two additional oxygen ",-"nn.no orbitals) for dioxetanes. bSOC values between SI and states. (A) Dioxetane (1-) + nH 20

1-

1- .. , H20

CT Oiradical

1- '" 2H 20

iii

CTIHomolytic Diradical

(8) Dioxetanone (2-) + nH 20

2- "H2 0

2H

Homolytic Diradical

density distributions of 0-0 elongated peroxides (roo

2.0

results have a potential importance to bioluminescence, since a recent of aequorin has revealed that a luciferin is buried at a hydrophobic 5 and is bonded to sUITounding amino acid residues. To take a let us consider a model complex for the substrate-binding site of that consists of a dioxetane (4-) or dioxetanone (5-) substrate hydrogen bonded form), and Trp. 4-- and 5- can be generated from the attack of on the 5- and 2-positions of coelenterazine ;r. It turns out that 4- leads to the HD while the CT mechanism arises from 5- as far as the substrate is de~)r01tonate,d, as shown in Fig. 2. This example makes it clear that the reQ'IQ(;nem control in the 01'3- and the pK. regulation of the resulting nPr'f'\vllip

122

Isobe H et al.

important to the suppression of intersystem crossing in the product formation. In conclusion, the dioxetane functionality is highly subject to the in the and distributions on thermolysis via the hydrogen bonding, while this is not the case when the dioxetanone This conclusion will be of great significance in bioluminescence. (A) Dioxetane (4"") + His16W (protonated) + Tyr82 + Trp86

*

3-

" Atoms

with
5-

coelenterazine

distributions of 0-0 elongated

REFERENCES 1.

Yamanaka S, Kuramitsu S, Yamaguchi K. coupling in charge-transfer-induced luminescence of derivatives. J Am Chern Soc 2008;130:132-49. Dauben Salem L, Turro NJ. A classification Chern Res 1975;8:41-54. Frisch MJ, et al. Gaussian 98 and 03, Gaussian Inc. 2004. Matsumoto M. Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence. J Photochem Photobiol :27-53. Head Inoue S, Teranishi K, Shimomura O. The structure photoprotein aequorin at 2.3 A resolution. Nature 2000;405 <:rl!in-"()rhlt

2. 3. 4.

5.

A NEW BRIGHT CHEMILUMINESCENT REACTION: INTERACTION OF ACETONE WITH SOLID-PHASE POTASSIUM MONOPEROXYSULFATE IN THE COMPLEX OF EUROPIUM NITRATE DV KAZAKOV,*' FE SAFAROV,' R SCHMIDT,2 VP KAZAKOV' }Institute of Organic Chemistry, Ufa Scientific Center of the RAS, 71 Pro Oktyabrya, 450054 Ufa, Russia, E-mail:[email protected] 2Institut fvr Physikalische und Theoretische Chemie, Johann Wolfgang Goethe-Universitat Frankfurt, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany, E-mail: [email protected]

INTRODUCTION The ketone-catalyzed decomposition of monoperoxysulfuric acid (Caro's acid) in solution' has attracted attention of many research groups.2-4 One of the reasons for such a long-standing interest is that this reaction is a main synthetic route to dioxiranes,3 highly efficient and selective oxidants.5.6 Moreover, dioxiranes appeared to be a new class of hyperenergetic peroxides which produce chemiluminescence (CL) during rearrangement to the corresponding esters or oxidation of organic and inorganic 6 substrates. -s Apart from dioxirane involvement, the Caro's acid/acetone system is a highly efficient source of singlet excited oxygen ('0 2),9.10 the importance of which in environmental and biomedical sciences, chemi- and bioluminescence as well as in organic synthesis is well recognized.",'2 Notably it is the reaction of intermediary dioxirane with monopersulfate ion that leads to the formation of '02. Scheme 1.

o H2 S0 5 RftR

~

[:Xb] -03S00-~

10

2

Thus, the study of excited states generation in the acetone- catalyzed decomposition of monoperoxysulfuric acid could provide promising interdisciplinary perspectives. But the CL, observed in visible spectral region, in the reaction of HS0 5- with acetone in aqueous solution is very weak with a CL yield as low as 10- 11 Einstein mor' even in the presence of the CL activator europium nitrate. However, we discovered that a simple change of the reaction conditions, namely going from the liquid to the solid phase, results in a drastic increase of the CL efficiency by several orders of magnitude, such that under appropriate conditions in the presence of EU(N03)3 the luminescence may be observed in a slightly darkened room even by naked eye.

MA TERIALS AND METHODS Chemiluminescence measurements were performed at 90°C with a photomultiplier 123

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sensitive between 330 and 800 nm. KHS0 5 was the triple salt 2KHS0 5xKHS0 4 xK 2S04 (Curox) and was used as received. Dimethyldioxirane solution in acetone was prepared as described. 3 Identification of the reaction products was performed as follows: after the CL reaction was completed, the crude reaction mixture was diluted in H 20 and extracted with CDCI 3 with subsequent analyses by lH_ and 13 C_NMR spectroscopy. Alternatively, reaction mixture was diluted in D 20 and directly analyzed by NMR spectroscopy (Bruker AM-300) in this solvent. RESULTS AND DISCUSSION After rapid (ca. 1 s) injection of 0.3 mL of liquid acetone to a cuvette containing a finely pounded mixture of 20 mg Curox (= 5.8xlO'5 mol of KHS0 5) and 17 mg Eu(N03)3 (= 3.8xIO,5 mol) powder, preheated for 5 min at 90°C, the appearance of a strong luminescence signal was observed. CL reached maximum intensity after 5 min and decayed then slowly to zero in about 5.6 h at 90°C. During that time 4.5x 10,5 mol of KHS0 5 have been consumed as revealed by iodometric analysis of the reaction mixture. Notably, when the interaction of acetone with the powder was performed at higher temperature (200°C) and with 50 mg of each Curox and Eu(N03)3 a bright red emission could be observed even by naked eye in slightly darkened room. The red color of the CL is caused by the emission of Eu 3+, as shown by the high temperature solid phase CL spectrum (Fig. 1) with its double maximum at 614 and 628 nm which correlates well with emission maximum of the europium photoluminescence between 610 and 630 nm. 13 The maximum intensity of CL and the total amount of light evolved in the reaction at 90°C were determined by actinometry to ICL = 1.7xlO,l5 Einstein's,l and S = 4.3xlO,12 Einstein, respectively. These data allow the calculation of the CL yield to 'f/CL = l.OxIO,7 Einstein mor l (based on the consumed peroxide). Because the reaction temperature of 90°C is much higher than the boiling point of acetone (56°C), the ketone was immediately evaporated after addition to the mixture of Curox and Eu(N0 3)3 powders. It is therefore obvious that the overall CL, lasting for several hours, is caused by the interaction of Eu(N0 3hIKHS0 5 with vaporous but not with liquid acetone. The following experiment was conducted to confirm this assumption. 3 mL acetone have been transferred to a room temperature cuvette, which was connected by a tube with a second cuvette placed above the photocathode of the photomultiplier and containing the mixture of 20 mg Curox and 17 mg Eu(N0 3)3 powders. This cuvette was thermostatted at 90°C for 5 minutes, and then a gentle argon stream entering the first cuvette captured the acetone vapor and entered the second one with the powder mixture. A rise of the CL signal was instantly observed which reached its maximum after 20 min and decayed during seven hours at 90°C. Chemiluminescent characteristics were found to be comparable with those obtained with the above described injection experiment: ICL = 1.7xlO,l5 Einstein S'l, S = 6.7xlO'12 Einstein, 'f/CL = 2.0xIO,7 Einstein mol'l.

A New BriJ?ht Chemiluminescent Reaction

125

-

-~_~-,r-'--.-.-'~.

5')0

5711

f>J(J

610

I,. ""

Fig. 1. Chemiluminescence spectrum taken during the reaction of 0.1 mL acetone with a solid mixture of20 mg Curox and 17 mg Eu(N0 3)3 at 90°C. We propose that excitation of Eu 3+ occurs via involvement of the intermediary dioxirane as shown in Scheme 2 (pathway a): rearrangement of the dimethyldioxirane may lead to the excited methylacetate6 followed by energy transfer to the Eu ion. Indeed, we have found that interaction of the vapor of isolated dimethyldioxirane with 17 mg pure EU(N03h powder at 90°C is accompanied by CL whose efficiency was found with S = 1.1xl0- 11 Einstein and 1]CL 2.2xI0- 7 Einstein-mor 1 to be even higher than that obtained in acetoneIKHSOs/Eu(N0 3)3 system. Scheme 2.

~

H3 C

H3C CH 3 ~ a HC

X'0 .....

+

KHSO S

0

o·

-.JL H3C

3 (b)

1 1 O2 + O 2 - - -

OCH~. J ~

EU(N03)V Eu*(N0 3h

(10) " 2 2

1 ,

hv (610-630 nm)

Furthermore, NMR analysis, taken after the CL reaction (injection experiment with KHSOsIEu(N0 3)3 powders) revealed the presence of acetone and methyl acetate - a product of dimethyldioxirane rearrangement. Methylacetate and acetone were not evaporated at 90°C because they were bound in a complex with europium so that interaction of acetone with KHSO s takes place in the metal coordination sphere. However, considering involvement of the intermediary dioxirane we cannot exclude the alternative pathway b of Scheme 2, in which europium nitrate may be excited by

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energy transfer from the singlet-oxygen dimol species e02)2, which are known to be formed during the acetone-catalyzed decomposition ofKHSOs in solution. 14

ACKNOWLEDGEMENTS The research in Ufa was supported by the Russian Foundation for Basic Research (grants No 08-03-00147-a), the PNSh (grant No 5486.2006.3), and OKHIM (N21OKh). RS thanks the Adolf-Messer-Stiftung for fmancial support. REFERENCES 1. Montgomery RE. Catalysis of peroxymonosulfate reactions by ketones. J Am Chern Soc 1974;96:7820-1. 2. Edwards JO, Pater RH, Curci R, Di Furia F. On the formation and reactivity of dioxirane intermediates in the reaction of peroxoanions with organic substrates. Photochem Photobiol 1979;30:63-70. 3. Murray RW, Jeyaraman R. Dioxiranes: synthesis and reactions of methyldioxiranes. J Org Chern 1985;50:2847-53. 4. Shi Y. Organocatalytic asymmetric epoxidation of olefins by chiral ketones. Acc Chern Res 2004;37:488-96. 5. Adam W, Saha-Miiller CR, Zhao C-G. Dioxirane epoxidation of olefins. Org React 2002;61 :219-516. 6. Kazakov VP, Voloshin AI, Kazakov DV. Dioxiranes: from oxidative transformations to chemiluminescence. Russ Chern Rev 1999;68: 253-86. 7. Kazakov DV, Barzilova AB, Kazakov V.P. A novel chemiluminescence from the reaction of dioxiranes with alkanes. Proposed mechanism of «oxygentransfer» chemiluminescence. Chern Commun 2001: 191-2. 8. Adam W, Kazakov DV, Kazakov VP, Kiefer W, Latypova RR, Schliicker S. Singlet-oxygen generation in the catalytic reaction of dioxiranes with nucleophilic anions. Photochem Photobiol Sci 2004;3:182-8. 9. Evans OF, Upton MW. Studies on singlet oxygen in aqueous solution. Part 3. The decomposition of peroxy-acids J Chern Soc Dalton Trans 1985: 1151. 10. Lange A, Brauer H-D. On the formation of dioxiranes and singlet oxygen by the ketone-catalysed decomposition of Caro's acid J Chern Soc, Perkin Trans 2 1996:805-11. 11. Adam W, Kazakov DV, Kazakov V.P. Singlet-oxygen chemiluminescence in peroxide reactions. Chern Rev 2005; 105: 3371-87. 12. Schweitzer C. Schmidt R. Physical mechanisms of generation and deactivation of singlet oxygen. Chern Rev 2003; 103: 1685-1757. 13. Lochhead MJ, Wamsley PR, Bray KL. Luminescence spectroscopy of europium(II1) nitrate, chloride, and perchlorate in mixed ethanol- water solutions. Inorg Chern 1994;33:2000-3. 14. Kazakov DV, Kazakov VP, Maistrenko GYa, Mal'zev DV, Schmidt R. On the effect of 1,4-diazabicyclo[2.2.2]octane on the singlet-oxygen dimol emission: chemical generation of C02)z in peroxide reactions. J Phys Chern A 2007;111: 4267-73.

STUDY ON NOVEL ARYLOXALATE CHEMILUMINESCENCE REACTION WITHOUT ADDITION OF HYDROGEN PEROXIDE N KISHIKA WA, K OHY AMA, K NAKASHIMA, N KURODA Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Email: [email protected].)p

INTRODUCTION Peroxyoxalate chemiluminescence (PO-CL) is based on the reaction between hydrogen peroxide and aryloxalate, which produces strong luminescence in the presence of a f1uorophore through a chemically initiated electron exchange luminescence mechanism. The PO-CL is easily combined with HPLC and applied to the determination of fluorescent compounds. However, the addition of high concentrations of hydrogen peroxide causes an increase in background noise and also the instability of hydrogen peroxide may affect the repeatability of the CL reaction. In this work, we found that CL was generated when specific f1uorophores, which have 2-phenyl-6-dimethylaminobenzofuran (DBP) structures (Fig. 1), were mixed with aryloxalate without addition of hydrogen peroxide. It was observed that there is a direct relation between the concentration of the DBP derivative and CL intensity. It is thought that the CL reaction without addition of hydrogen peroxide has several advantages in respect to sensitivity, selectivity and simplicity. Therefore, we investigated the characteristic of the proposed CL reaction system by using 2-( 4-aminopheny 1)-6-dimethy lam inobenzofuran (0 BP-NH 2 ). Moreover, we combined the proposed CL reaction with HPLC for the determination of biological thiols such as cysteine, homocysteine, N-acetylcysteine and glutathione by using N-[4-(dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) as a chemiluminescent labeling reagent. The labeled thiols were separated and detected by CL after mixing with just the aryloxalate. Finally, the method was applied to the determination of biological thiols in human serum.

m-o-

NH2

........... ,

~

0

-

DBP-NH 2

,~ i ~ DBPM

Fig. 1. Structures ofDBP-NH 2 and OBPM 127

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EXPERIMENTAL Chemicals and reagents. OBP-NH 2 and OBPM were synthesized in our laboratory as described previously. I Bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl] oxalate (TOPO) was obtained from Wako (Osaka, Japan). Acetonitrile (HPLC grade) was from Kanto Chemical (Tokyo, Japan). Water was distilled and passed through a Pure Line WL21P system (Yamato, Tokyo). Chemiluminescence measurement. In a small test tube, 100 f!L of OBP-NH 2 in acetonitrile and 800 f!L of buffer solution (phosphate buffer, Tris-HN0 3 buffer or imidazole-HN0 3 buffer) were mixed. The test tube was placed in Lumatag Analyzer Auto-250 (Berthold, Germany) and then 300 f!L of aryloxalate including TOPO in acetonitrile was automatically injected, and the produced CL was measured for 60 s. Derivatization of thiols with DBPM. The derivatization reaction was carried out 2 as follows: to a 50 f!L sample solution, 150 f!L of 200 f!M OBPM in acetonitrile and 300 f!L of 50 mM borate buffer (pH 8.5) were added. The reaction mixture was heated at 60°C for 30 min. A 5 f!L of the solution was injected into the HPLC system. HPLC system and chromatographic conditions. The HPLC-CL system (Fig. 2) consisted of three LC-I0AS liquid chromatographic pumps (Shimadzu, Kyoto, Japan), a Rheodyne 7125 injector (Cotati, CA, USA) with a 5 f!L of sample loop, a CLO-I0A CL detector (Shimadzu), Chromatocorder 12 integrator (Sic, Tokyo) and Cosmosil 5CI8-AR-I1 column (Nacalai, Osaka; 250 x 4.6 mm, i.d.) as stationary phase. The OBP labeled thiols were separated by a mixture of acetonitrilelI mM phosphate buffer (pH 5.8) (=40:60, v/v) containing 3 mM NaPF 6 as a mobile phase. The eluent was mixed with 5 mM phosphate buffer (pH 8.5) to adjust the suitable pH for proposed CL reaction and then mixed with 0.5 mM TOPO in acetonitrile as a CL reagent solution.

Column

Buffer 1.0 mL/min

CL reagent 1.2 mLlmin

Fig. 2. HPLC-CL system for the determination ofOBP labeled thiols P, pump; I, injector; 0, CL detector; R, integrator

Study of Novel Aryloxalate Chemiluminescence Reaction

129

RESUL TS AND DISCUSSION Characteristics of proposed CL reaction. In the preliminary study, CL intensities of several types of fluorophores were measured after mixing with only aryloxalate and without addition of hydrogen peroxide. DBP derivatives and pyrimidopyrimidine derivatives showed CL, but the CL was not observed with typical fluorophores that are frequently used in conventional PO-CL reactions such as dansyl, benzoxazole, lophine and fluorescein derivatives. Therefore, it is proposed that this CL reaction can detect DBP derivatives selectively in the presence of other fluorophores. The effects of aryloxalate and buffer on CL intensity were investigated by using DBP-NH 2 . The effect of aryloxalate was examined with 11 kinds of aryloxalates. Among them, the aryloxalates such as TDPO with strongly electron-withdrawing substituents gave the strongest CL intensity. Therefore, TDPO was selected for subsequent work. The effect of buffer types was examined with phosphate buffer, imidazole-HN0 3 buffer and Tris-HN0 3 buffer. When TDPO was used as the aryloxalate, phosphate buffer gave the highest CL intensity. This result was different from that of a conventional PO-CL reaction with addition of hydrogen peroxide, which requires the presence of an imidazole buffer. HPLC system for determination of thiols based on the proposed CL reaction. Based on the proposed CL reaction, an HPLC-CL system for the determination of biological thiols was developed. For this purpose, DBPM, which is known as a fluorescent labeling reagent for thiols, was employed for the conversion of thiols to DBP derivatives that can be detected by the proposed CL reaction. TDPO and phosphate buffer were selected as aryloxalate and buffer, respectively. Because the thiol derivatives could not be retained on the ODS column due to their hydrophilicity, a mixture of acidic phosphate buffer and acetonitrile containing NaPF 6 as counter ion was used for the separation. However, because the CL intensity decreased under acidic conditions, an alkaline phosphate buffer was mixed with the eluent to modify the pH, and mixed with TDPO and then the CL generated was detected by CL detector. The effect of phosphate buffer concentration for modifying pH on CL intensity was examined. The CL intensities increased with the increase of phosphate buffer concentration up to 5 mM and then decreased. The effect of buffer pH was also examined, the CL intensities increased with increasing pH, but in consideration of the buffer capacity, 5 mM phosphate buffer (pH 8.5) was chosen. The effect of TDPO concentration was also investigated. It was found that CL intensity increased with increasing TDPO concentration and 0.5 mM of TDPO was selected based on the cost and solubility ofTDPO. Fig. 3(A) shows a typical chromatogram of standard thiols after the derivatization reaction. The derivatives of four thiols and 2-mercaptoethanol (internal standard) were separated and detected within 20 min. The derivatives of cysteine gave two peaks (due to hydrolysis during the reaction).

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Calibration curves were prepared with a standard mixture of thiols according to the labeling procedure and good correlations were observed between the CL intensity as peak height ratio and concentration ofthiols up to 500 pmol/injection. The detection limits of standard thiols ranged from 0.23 to 0.30 pmol/injection at a signal-to-noise ratio (SIN) of 3. The repeatability of the proposed method was examined at the 50 pmol/injection level for thiols. The relative standard deviations (RSD) for within-day (n = 3) and between-day (n = 3) runs were < 4.6 and 4.9%, respectively. This method was successfully applied to the determination ofthiols in human serum. A typical chromatogram of the extract from human serum determined by the proposed HPLC-CL method is shown in Fig. 3(B).

(A)

(B) 3 2

IS

.e.;;; = .s

-

IS

4

4

.e.;;;

-=

CI.I

CI.I

.s

~

5

u

5

~i""~"""~""~""~i

o

20 Retention time, min

....~i......~....~....~i

~i

0

5

20

Retention time, min

Fig. 3. Chromatograms of standard thiol (A) and human serum (B) Peaks: 1=glutathione, 2=homocysteine, 3=N-acetyl-L-cysteine, 4=L-cysteine, 5=L-cysteine, IS=2-mercaptoethanol (internal standard)

REFERENCES 1.

2.

Akiyama S, Akimoto H, Nakatsuji S, Nakashima K. Development and application of organic reagents for analysis VI. Synthesis and fluorescence spectral properties of 2-( 4-substituted phenyl)benzofurans. Bull Chern Soc Jpn 1985;58:2192-96. Nakashima K, Umekawa C, Yoshida H, Nakatsuji S, Akiyama S. High-performance liquid chromatography-fluorometry for the determination thiols in biological samples using N-[ 4-( 6-dimethy lamino-2-benzofurany I)pheny I]maleim ide. J Chromatogr 1987;414:11-7.

NUCLEOPHILIC ACYLATION CATALYSTS EFFECT ON LUMINOL CHEMILUMINESCENCE

E MARZOCCHI,! S GRILLI,! L DELLA CIANA,! M MIRASOLI/ P SIMONI,3 L PRODI,4 A RODA 2 I Cyanagen srI. via Stradelli Guelfi 401c. 40138 Bologna. Italy of Pharmaceutical Sciences. Univ. of Bologna. Via Belmeloro 6. 40126 Bologna. Italy ofinternal Medicine and Gastroenterology. Univ. of Bologna. via Massarenti 9. 40138 Bologna. Italy "Dept. of Chemistry "G. Ciamician ". Univ. of Bologna. Via Selmi 2.40126 Bologna. Italy e-mail: [email protected]

2Dept.

3Dept.

INTRODUCTION The HRP-catalyzed chemiluminescent oxidation of luminol is widely used in many molecular biology based assays such as Western blots, dot blots, ELISA and in immunohistochemistry. Much effort has been made to improve the efficiency and the analytical performance of this reaction. In particular, addition of enhancers to the reaction substrate greatly increases light output and duration kinetics.! Molecules with enhancer properties are essentially redox mediators 2 capable of exchanging electrons between the peroxidase enzyme and luminol. Enhancers include substituted phenols,3 substituted boronic acids,4 indophenols and N-alkyl phenothiazine derivatives. 5 The enhanced HRP-catalyzed oxidation of luminol is a complex, multi-step reaction. While redox enhancers are useful in improving enzyme turnover and increasing the equilibrium concentration of a key intermediate, luminol radical anion, a second bottleneck may need to be removed in order to obtain a further increase in light output. According to a well-established mechanism,6 luminol radical anion, once formed, is subject to rapid dismutation to luminol and diazaquinone. In turn this species is attacked by hydroperoxide anion and converted into reactive peroxides (Fig. 1).

« 0

I

yYN

II N

#

NH,

~~;H

N

NH,

0

L (diazaquinone)

0

L hydro peroxide

Fig. 1. Reaction scheme of luminol diazaquinone and hydrogen peroxide.

This type of reaction is often accelerated by nucleophilic acylation catalysts,7 especially 4-aminopyridines. This study investigates the effect of these compounds on chemiluminescence light output when used in association with redox enhancers and their use in substrates for HRP chemiluminescent assays with the goal to achieve a higher detectability ofHRP. 131

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METHODS Luminol sodium salt was obtained by recrystallization of the commercially available luminol (5-amino-2,3-dihydro-I,4-phthalazinedione) from sodium hydroxide. All the other compounds were used as purchased. A Cary Eclipse (Varian) fluorescence spectrophotometer was used for kinetic measurements using the following setup: kinetic mode, Aem = 425 nm, gate time 5 ms, emission slits properly adjusted, Savitzky-Golay smoothing. Luminoskan (Ascent) luminometer was used for microtiter well readings (1 sec integration per well). A NightOwl LB98 I (Berthold Technologies) luminograph was used to read the luminescence from Western blot membranes (120 seconds total integration time). The following working solutions (chemiluminescence substrates) were freshly prepared: NoMORP, containing 5 mM luminol; 1.5 mM 3-(10'-phenothiazinyl)propane-I-sulfonate (SPTZ); 4 mM sodium perborate in 0.125 M TrislHCI pH 9.0. MORP, contaning 5 mM luminol; I.5 mM SPTZ; 1.5 mM 4-morpholinopyridine (MORP); 4 mM sodium perborate in 0.125 M TrislHCI pH 9.0. RESULTS AND DISCUSSION The enhancer SPTZ was synthesized in high yield and extreme purity, using a new one-pot procedure from phenothiazine and 1,3-propanesultone. Its performance and that of other commonly used enhancers, p-coumaric acid, p-iodophenylboronic acid and p-iodophenol was evaluated under similar conditions. The best performance, both in terms of luminosity and duration was obtained with SPTZ. In the next experiment, 4-dimethylaminopyridine (DMAP), a powerful acylation catalyst, was added to each substrate solution. While a very slight signal increase was observed for p-iodophenol and 4-iodophenylboronic acid (about 10%), the effect was much more significant for SPTZ (>800%). Therefore, all further studies were focused on the SPTZ system. Reference

Triethylamine (TEA) 4-morpholinopyridine (MORP)

F=-C~~~-=-~~~

4-dimethylaminopyridine (DMAP) ~==~~~~~~. 4-Pyrrolidinopyridine (PPY) i=;"==:--~~~~~ 4~Picoline

Pyridine _'*'--.~~~~~~~~ 1000

2000

3000

4000

5000

Integral value (a.u.)

Fig_ 2_ Total light output of 15-minute signal acquisitions for solutions containing different DMAP analogues. Reference is the cocktail without catalyst addition. Pyridines, and especially 4-dialkylaminopyrines, are well-known nucleophilic acylation catalysts and are widely used in organic synthesis. 7 When they are added to the SPTZ substrate the reported 7 overall reactivity is observed:

133

Nucleophilic Acylation Catalysts Effect on Luminal Chemiluminescence

MORP>DMAP>PPY»4-picoline>pyridine, as shown in Fig. 2. The small differences between the expected and the observed behaviour of PPY, DMAP and MORP is likely to be at least partially due to their differences in pK a . In order to understand the effect of these catalysts, it is important to observe that they do not act themselves as enhancers (no effect on light output is observed, when used alone). An explanation is proposed by considering in more detail the reaction steps from diazaquinone (L) to the luminol peroxide intermediate. This reaction involves nucleophilic attack on one of the diazaquinone carbonyls by hydrogen peroxide monoanion. A hydroperoxide species is formed, which can rearrange to the endoperoxide. Either compound is very unstable and collapses to 3-amino-phthalate in its excited state. Perhaps pyridine acylation catalysts could facilitate hydrogen peroxide attack by converting L into a more reactive intermediate. For analytical applications, the non-toxic acylation catalyst MORP is preferred over the highly toxic DMAP and PPY. The analytical performances of MORP and NoMORP working solutions were explored by performing a dose-response assay for HRP in microtiter wells. The detection limits for HRP resulted 0.35 pg (8 amol)!well for the reaction containing MORP compared to 2.20 pg (50 amol)!well for the NoMORP substrate, as shown in Fig. 3 (left panel). 200 , - - - - - _ - - - - - - - - , •

o

NaMORP MORP

10r--------~-----~ •

o

NoMORP MORP

150

4

o

2

4

6

10

HRP Amount (pg/well)

12

14

o

2

4

6

10

12

14

16

Bacteria Concentration x 10' (cell/ml)

Fig. 3. Left: dose-response curves for HRP. Right: calibration curves obtained from a sandwich chemiluminescent enzyme immunoassay for Yersinia. enterocolitica. Next, a sandwich chemiluminescent enzyme immunoassay for the determination of Yersinia enterocolitica was carried out to compare the performance of MORP and NoMORP chemiluminescent reagents. The detection cocktail containing MORP produced for each bacteria concentration a signal about 1 order of magnitude higher than that obtained with NoMORP, thus providing a steeper calibration curve. Indeed, MORP reagent allowed reaching a limit of detection of 0.6 x 10 6 cell/ml, as compared with that of NoMORP cocktail, which was 5.0 x 10 6 , as shown in Fig. 3 (right panel). Finally, Western blots for chicken egg albumin were performed using the NoMORP

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and MORP chemiluminescent substrates. The much stronger signal produced by the MORP catalyzed substrate (Fig. 4) allowed the detection of more diluted protein bands, with a clear advantage in the Western blot technique.

12000

•

o

NoMORP MORP

10000 =>

8000

i

6000

Albumin amount (pg) 2.5 1.7 1.1 NOMORP_ MORP2 ; 8 ,

~

~

4000

•

•

2000 0 2

3

4

6

Albumin Amount (pg)

Fig. 4. Western blots for chicken egg albumin. Left: calibration curves. Right: acquired image from a membrane. In conclusion, the incorporation of an acy lation catalyst in enhancer/lum inol/oxidant HRP substrates is highly advantageous. Although the exact mechanism of action of acylation catalysts has not yet been fully investigated, it is clear from the results of this study that the very significant increase in light output observed in their presence can be translated into a corresponding improvement in sensitivity of chemiluminescent assays.

REFERENCES 1. 2.

3. 4.

5. 6. 7.

Kricka LJ, Voyta JC, Bronstein I. Chemiluminescent methods for detecting and quantitating enzyme activity. Methods EnzymoI2000;305:370-90. Easton PM, Simmonds AC, Rakishev A, Egorov AM, Candeias LP. Quantitative model of the enhancement of peroxidase-induced luminol luminescence. J Am Chern Soc 1996; 118:6619-24. Thorpe GHG, Kricka LJ, Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods EnzymoI1986;133:331-53. Kricka LJ, Cooper M, J i X, Synthesis and characterization of 4-iodopheny 1boronic acid: a new enhancer for the horseradish peroxidase-catalyzed chemiluminescent oxidation of luminol. Anal Biochem 1996;240: 119-25. Sugiyama M., Method of the chemiluminescence assays of the activity of peroxidase. US Patent No. 5,171,668 (1992). McCapra F. Chemical generation of excited states: the basis of chemiluminescence and bioluminescence. Methods EnzymoI2000;305:3-47. Hatle G, Steglich W, Vorbrilggen H. 4-Dialkylaminopyridines a highly active acylation catalyst. Angew Chern lnt Ed EngI1978;17:569-83.

EFFECT OF SURFACTANTS ON PEROXYOXALATE CHEMILUMINESCENCE REACTION K NAKASHIMA,I K ABE,I S NAKAMURA,I M WADA,I S HARADA,2 N KURODA 1 I Department

of Clinical Pharmacy, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan 2 Lumica Co., 651togaura, Koga-city, Fukuoka 811-3136, Japan E-mail: [email protected]

INTRODUCTION The peroxyoxalate chemiluminescence (PO-CL) method has been widely utilized in pharmaceutical and biomedical analysis due to its high sensitivity and a use of simple instrumentations without a light source. I.2 In a PO-CL system, oxalates or oxamides react with H 20 2 in the presence of a fluorophore to produce a light emission. The reaction has been thought to follow a chemically initiated electron exchange luminescence (CIEEL) mechanism via an high energy intermediate (1,2dioxetane derivative, 1) which forms a charge complex with the co-existing fluorophore. The electron is transferred to the fluorophore which is raised to an excited state and the energy emitted as a light. It has also been reported that surfactant can enhance PO-CL intensity in aqueous conditions via improvement of the fluorescence yield. 3 In order to establish a detection method for a synthetic surfactant by PO-CL, the effects of surfactants on PO-CL were examined using the bis(2,4,6trichlorophenyl)oxalate (TCPO)/rhodamine B/H 2 0 2/imidazoie system.

o

0 II II Ar-O~C~C~O-Ar

Aryloxalates

1 + F _ .........--=0 ..

F*

OH 0 I II

0 II

0 II

0-0

0-0

+ Hz02 -='+' Ar-O~T~T or T~T 1,2-Dioxetane derivatives,

1lo~o H- F~J

-~~..

F* + 2CO 2

--F+ hv

F=fluorophore Fig. 1. Reaction scheme ofPO-CL 135

136

Nakashima K et at.

METHODS Surfactant samples. Fifteen surfactants (a-o) used in this study were commercially available and involved anion-, cation-, zwitterionic- and non-ionic types. Each of surfactant samples at 0.5 or 2 % (w/v) prepared in water or methanol was used. The surfactants examined in this study are listed in Table I. Procedure for CL measurement. Twenty five micro litter of surfactant solution in a test tube were evaporated under N2 gas. To the residue, 25 f!L of 0.5 mM rhodamine B in CH3CN, 5 mM imidazole-HN0 3 (pH 6.5) and 120 mM H 20 2 in CH 3CN were added and mixed for lOs. After adding 25 f!L of 0.25 m M TCPO in CH3CN, CL measurement was performed at room temperature for 120 s in a Luminescencer PSN AB-nOO (Atto Co., Tokyo, Japan).

Sample

a b c

d e f g

h

k I

m n

o

Table 1. Surfactant samples examined in this study Compound Type Alkylsulfate/triethanolamine Sodium poly( oxyethylenetridecylether) laurylsulfate Disodium poly(oxyethylene) laurylsulfosuccinate Anionic Sodium diocty Isulfosuccinate Sodium poly( oxyethylene laurylether)acetate Poly(oxyethylene laurylether)phosphoric acid Cetyl trimethylammonium chloride Cationic Benzalkonium chloride Lauryl dimethylamino acetic betaine Zwitterionic Lauryl dimethylamine oxide Mixture ofpoly(oxyalkylene) and poly(alkylether) Poly( oxyethylene )tridecylether Poly( oxyethylene)tridecylether aqueous solution Non-ionic Fatty acid diethanolamide from Palm Poly( oxyethylene)sorbitane monooleic acid ether

RESULTS The effects of surfactants on the PO-CL reaction were examined. The surfactant solutions were spiked into (I) TCPOlrhodamine BIH 20 2/imidazole-HN0 3 buffer system, (2) TCPO/rhodamine B/H 20 2 system and (3) TCPOIH 2 0z/imidazole-HN0 3 system, respectively. In system (I), the CL intensity of the blank sample was taken as 100. The effect of surfactant was shown as relative CL intensity (RCI) to the CL intensity without surfactant (Fig. 2). Surfactants quenched CL at a 2% concentration and their RCIs ranged from 0.6 to 93.5. One of the reasons for this may be notable change of pH that occurred on adding the surfactant. In contrast, in the system (2), RCIs spiked with several surfactants (B-E, H-L) at 0.5% and CL was enhanced compared to that of the blank (Fig. 3). The RCIs were in range 124-472. This result suggested that several surfactants might playa role as a

Effect oj SurJactants on Peroxyoxalate Chemiluminescence Reaction

137

catalyst. However, their CL intensities were lower than that of the positive control (RCI=700) spiked with 5 mM imidazole-HN0 3 (pH 6.5), and no CL intensity enhancement properties could be observed among the surfactants tested. In system (3), CL intensities were also enhanced by adding surfactants, which might be caused by the fluorescent impurities present in the surfactants. In conclusion, the TCPO/rhodamine BIH 20 2 system was suitable for detection of synthetic surfactants, and the proposed method will be applicable to detect natural detergents.

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138

Nakashima K et al.

REFERENCES I.

Tsunoda M, Imai K. Analysis applications of peroxyoxalate chemiluminescence. Anal Chim Acta 2005;541: 13-23. 2. Li F, Zhang C, Guo X, Feng W. Chemiluminescence detection in HPLC and CE for pharmaceutical and biomedical analysis. Biomed Chromatogr 2003; 17:96-1 05. 3. Dan N, Lau ML, Grayeski ML. Micellar-enhanced aqueous peroxyoxalate chemiluminescence. Anal Chern 1991; 63: 1766-71.

SOLVENT-PROMOTED CHEMILUMINESCENT DECOMPOSITION OF BICYCLIC DIOXETANES BEARING A 4-(BENZOTHIAZOL-2-YL)-3- HYDROXYPHENYL M TANIMURA, N WATANABE, HK. IJUIN, M MATSUMOTO Dept a/Chemistry, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan

INTRODUCTION Dioxetanes with an hydroxyaryl group undergo base-induced decomposition (BID) to effectively give a singlet-excited carbonyl that emits light by intramolecular charge-transfer (CT) mechanism.'" On the other hand, uncatalyzed thermal decomposition (TD) of dioxetanes gives mainly a triplet-excited species, so that bright light emission is unexpected. In addition to these two types of decomposition, we report here that aprotic polar solvent promoted decomposition of dioxetane bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety 1 to give bright light, and that this solvent-promoted dexomposition (SPD) resembled the BID of 1, which proceeds through unstable dioxetane 2 to keto ester 3 (Fig. 1), however, SPD was found to be an entropy-controlled reaction, which was in sharp contrast to BID.

08

OH

-0 ~ ~ ;a6 o

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_

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I

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0

~ t-BU--\"

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\

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2

()

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08

~

Ar

~o 3

OH

t.Bu=l}-O" 4

Fig. 1. Chemiluminescent decomposition of dioxetane 1

RESUL TS AND DISCUSSION First, we examined BID of 1. When 1 was treated with tetrabutylammonium fluoride (TBAF) in N-methylpyrrolidone (NMP) at 45°C, chemiluminescence occurred effectively with Ama/ID = 498 nm, qJ3ID = 0.29, and rate constant J!'ID = In21t1l2 BID = 2.5 X 10.4 s'\ (vide infra). The BID of 1 also proceeded to give bright light in acetonitrile,' DMF and DMSO. The chemiluminescence spectra for BID of 1 are 139

140

Tanimura M et al.

illustrated in Fig. 2. Next, the SPD of 1 was investigated in NMP. When 1 was heated without any strong base in NMP at 100°C, 1 decomposed to exclusively give 4 accompanied by the emission of green light with Amax SPD = 498 nm, qfPD = 0.25, and 3 !!,PD = 8.0 X 10. s. Thus, the chemiluminescence spectrum coincided with that for the BID of 1, as illustrated in Fig. 3, and qfPD was comparable to that for BID. These results suggested that the SPD of 1 in NMP should proceed through dioxetane bearing an oxidophenyl anion 2 to give anionic keto ester 3 in the excited state or a closely related species such as a solvated anion by a mechanism similar to BID. This effective SPD chemiluminescence of 1 was also observed in aprotic polar solvents, such as DMF, N,N-dimethylpropyleneurea (DMPU), and propylene carbonate (PPC) at 100°C. These chemiluminescence spectra are illustrated in Fig. 3.

(a)

(a)

350

450

550

wavelength / nm

Fig. 2. CL spectra for the BID of 1 in (a) NMP, (b) DMF, (c) acetonitrile, and (d) DMSO.

650

350

450

550

650

wavelength / nm

Fig. 3. CL spectra for the SPD of 1 in (a) NMP, (b) DMF, (c) DMPU, (d) PPC, and (e) acetonitrile, and for the TD in (f) p-xylene and (g) DGM.

p-Xylene is a typical nonpolar solvent that is used often to examine the uncatalyzed TD of dioxetanes.' Thus, we carried out the TD of 1 in p-xylene to compare the behavior with that in an aprotic polar solvent. In p-xylene at 100°C, dioxetane 1 decomposed to afford 4 with the emission of yellow light (Ama/ D = 536 nm), though the efficiency, c[>TD, was only 8 x 10.4 and the rate was very slow (rate constant kTD = 1.2 X 10.5 S·I) (Fig. 3). The small value for kTD indicates that 1 underwent typical uncatalyzed TD in p-xylene, as reported for various analogs of dioxetane 1, in contrast to SPD. However, the chemiluminescence for the TD of 1 was observed at a longer wavelength than those for BID and SPD (Fig. 2). The emission of yellow light for the TD of 1 in p-xylene is presumably attributed to ESIPT (excited state intramolecular proton transfer) in which quinone methide 5 was produced as an emitter (Fig. 4). This idea was supported by the fact that methoxyphenyl-analog 6 of 1 underwent TD in p-xylene at 100°C to give weak violet light with Amax TD = 390 nm, c[>TD = 9.2 X 10.4 , and kTD = 4.3 X 10.4 S·1 (Fig. 4).

Solvent-Promoted Chemiluminescent Decomposition of Bicyclic Dioxetanes

141

Fig. 4. Thermal decomposition (TD) of 1 emitting light due to ESIPT and TD of 6

The effects of solvent on the thermal chemiluminescent decomposition of 1 described above are summarized as follows. First, SPD proceeded in an aprotic polar solvent to afford bright light with AmaxSPD = 493-498 nm similar to BID by aCT-induced decomposition mechanism. Second, 1 underwent uncatalyzed TD to give excited 5 which emitted yellow light with Amax TO = 536 nm due to ESIPT in nonpolar p-xylene. It was important to elucidate whether or not SPD is mechanistically different from BID. Thus, the kinetics of the SPD of 1 was investigated in NMP as a representative solvent. SPD should be a consecutive reaction, which consists of the reversible formation of dioxetane bearing an oxidophenyl anion 2 or its solvated species that undergo CT-induced decomposition to give light. However, the reaction proceeded following practically first-order kinetics at 60-100 dc. On the other hand, both AmaxSPD and qJ>PD changed scarcely with the SPD reaction temperature. These results suggested that the thermodynamic profile of the reaction could be simply but meaningfully analyzed in terms of pseudo-first order kinetics. Based on the thus-measured rate constants, 0 PD s, at 60-100 DC, the thermodynamic parameters for the SPD of 1 in NMP were estimated from the Arrhenius plots to be free energy of activation ~G~ = 99.3 kJ mor l, enthalpy of activation ~H~ = 82.4 kJ mor l, and entropy of activation M~ = -56.8 J Klmor'. BID using TBAF in an aprotic polar solvent has been known to proceed following pseudo-first order kinetics independent of the concentration of TBAF when a large excess of TBAF is used. Thus, rate constants J(l1D for 1 were measured using a large excess of TBAF in NMP at 35-55 dc. The Arrhenius plots revealed that the activation parameters for BID were ~G~ = 97.3 kJ mor', ~H+ = 99.8 kJ mor~, and M~ = 8.2 J Klmor'. If we compare these activation parameters with those for SPD, we see that the entropy term of SPD has a large negative value in contrast to that of the BID. These results reveal that SPD proceeded through a transition state with considerably less disorder than that for BID. This is presumably attributed to the participation of hydrogen-bonding between NMP molecule(s) and a phenolic OH of 1. Thus, an intermediary anionic dioxetane 7 was produced through hydrogen bonding as an ion pair with protonated NMP molecule(s) to cause SPD. A model scheme is

142

Tanimura M et al.

illustrated in 5. On the other hand, the intermediary anion 2 should be slightly solvated or naked as an extreme for BID in an aprotic polar solvent such as NMP and DMSO especially with the use ofTBAF.

Solvated 1

7 Ar=

5.

Solvent-promoted decomposition (SPD)

In conclusion, a variety of aprotic polar solvents were shown to promote the (SPD) of bicyclic dioxetane bearing 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety 1 to emit light as effectively as the in an aprotic polar solvent. SPD caused intramolecular CT-induced BID of chemiluminescence, similar to BID, though the SPD reaction proceeded through a with a large negative entropy of activation in contrast to BID. Furthermore, decomposition of dioxetane 1 was found to occur to light due to ESIPT in ACKNOWLEDGEMENTS The authors (NW and MM) gratefully acknowledge financial assistance provided Grants-in aid (No. 1550043, and No. 17550050) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. REFERENCES 1. Beck S, Koster H. Applications of dioxetane chemiluminescent probes to molecular biology. Anal Chem 2000; 45: 2258-70. 2. Matsumoto M. Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence. 1. Photochem. Photobiol. C: Photochem Rev 2004; 5: 27-53. 3. Matsumoto M, Akimoto T, Matsumoto Y, Watanabe N. Bicyclic dioxetanes a 4-(benzoazol-2-yl)-3-hydroxyphenyl moiety: chemiluminescence for base-induced decomposition in aprotic medium and in aqueous medium. Tetrahedron Lett 2005; 46: 6075-8. 4. Baumstark AL. The 1,2-dioxetane ring system: preparation, thermolysis, and insertion reactions. In: Frimer AA ed. Singlet O 2 Vol II. Boca Raton:CRC, 1985: 1-35.

SYNTHESIS AND CHARACTERIZATION OF NEAR-INFRARED CHEMILUMINESCENT PROBES K. TERANISHI Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan Email: [email protected]';p

INTRODUCTION Imidazopyrazinone compounds such as 2-methyl-6-phenylimidazo[1,2-a] and 2-methyl-6-( 4-methoxyphenyl)imidazo[1 ,2-a] pyrazin-3(7 H)-one (CLA) I pyrazin-3(7 H)-one (MCLA)2 have been widly used as chemiluminescent probes for detecting superoxide anions in biological systems (Fig. 1). These compounds presumably react with superoxide anions to form singlet-excited amides to generate amide compounds and light. I have recently developed Red-CLA (Fig. 1) that emits red light (emission A.max 610 nm) by reaction with superoxide anions. 3 Red-CLA emits light with the longest wavelength known for chemiluminescent probes. Detecting luminescence in the near-infrared region (700-900 nm) is advantageous as there is minimal absorption by other biomolecules. Thus, a near-infrared chemiluminescent probe should be important as a reporter molecule for many analytical applications. Herein, I present the synthesis and luminescence properties of chemiluminescent probes that emit the near-infrared light by reaction with superoxide anions.

°nr

d I'"

HlCO

&

1 1 N H

d

CHJ

•

NrN 9"

~

1

so,

~

H

02S"N~N~O H

MetA

1

0'T-i(CHJ N

r

N

N H

,4

0 Red-CLA

Figure 1. Chemical structures of MCLA and Red-CLA

METHODS General measurement of chemiluminescence intensities and spectra. To a mixture of20 mmollL Mops / 0.2 mollL KCl (pH 7.2, 0.1 mL), 0.3 mmollL hypoxanthine (0.1 mL), and probe solution was added xanthine oxidase (XOD) at 20°C. Luminescence intensity and spectra were measured for 1 min using a LumiFlSpectroCapture AB 1850 (Atto Corporation, Tokyo). RESULTS Synthesis. In my system the singlet-excitation energy is generated by the reaction between the MCLA moiety and superoxide anions, followed by transfer of the energy to the indocyanine moiety that in turn emits the near-infrared light via an resonance 143

144

Teranishi K

I MCLA moiety I o

h r N N

R'

---

R,i)" H

R~R 14®....::#~~,/.;'/ N

(6H3)4S0~

o

*1'

o£

N I

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trNH R'

N

' ) Energ y

R~R IgffilA,'lC"-:::'''--;:'

N

N

Id

I

(CH3)4S0~

~ndocyanjne moietyJ

+

Near-infrared light

Figure 2. Strategy for the near-infrared chemiluminescence energy transfer (CRET) mechanism (Fig. 2). As shown in Scheme 1, two chemiluminescent compounds 8 and 9 were successfully synthesized. Compounds 3 and 4 were prepared from compounds 1 and 2,4 respectively, followed by amide-coupling with amino compound 7 prepared from 5. 5 Chemical structures of compounds 8 and 9 were confirmed by IH NMR and LC/MS (ESI, positive mode), and their purity confirmed by reverse phase HPLC. The solubility of compound 9 was much higher than that of 8 in 20 mmollL Mops I 0.2 mollL KCI (pH 7.2), due to the additional sulfonate groups. Superoxide anions-induced chemiluminescence. To prove the utility of compounds 8 and 9 on the detection of superoxide anions, I performed experiments on the detection of superoxide anions using the hypoxanthine-xanthine oxidase system as a source of superoxide anions. MCLA (1 flmollL) exhibited superoxide anion-induced chemiluminescence peaks at around 460 nm in the luminescence spectrum (Fig. 3-A). In contrast, chemiluminescence spectra of compounds 8 and 9 (1 flmollL) revealed their luminescence maximum only at 785 nm (Fig. 3-B). The absence of chemiluminescence due to the MCLA moiety indicates that the superoxide anion-induced chemiluminescence of compounds 8 and 9 is generated from the indocyanine moiety. These results clearly indicate that the excitation-energy generated from the MCLA moiety is efficiently transferred to the indocyanine moiety. When instead of compounds 8 and 9 a mixture of MCLA (1 flmollL) and indocyanine compound 4 (1 flmol/L) was used in the test, the chemiluminescence spectrum showed "'-max 460 nm (Fig. 3-C), indicating the conjugation of the indocyanine molecule to MCLA functions for CRET. The luminescence intensity of 9 was about 4 times higher than that of 8. Probably, it is due to improvement of the quantum yield of the indocyanine moiety by the additional sulfate groups on the aromatic groupS.6 Compound 10, which consists of N-ethylisoluminol and indocyanine molecules, showed no luminescence under the same condition. The effect of concentration of compound 9 on the intensity of the superoxide anion-induced chemiluminescence using 0.015 unitlmL XOD is shown in Fig. 4-A; at

Synthesis and Characterization of Near-Infrared Chemiluminescent Probes

145

concentrations less than 5 ~mol/L the chemiluminescence intensity increased with increases in probe concentration. The relationship between the concentration of XOD and luminescence intensity with compound 9 (1 ~mol/L) are shown in Fig. 4-B. Because the light sensitivity of the spectrometer used was low, the weak light emission at low concentration ofXOD could not be detected. In this study I have developed a near-infrared chemiluminescenct probe 9 for the detection of superoxide anions. First, the present study proved that CRET with MCLA and indocyanine molecules is useful to generate near-infrared light. Second, the near-infrared chemiluminescent probe 9 is easily synthesized. FinaJly, the suitability of 9 for the detection of superoxide anions was demonstrated. o

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3: R= H 4: R=S03H

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Scheme 1. Synthesis of near-infrared chemiluminescent probes 8 and 9

146

Teranishi K

>,

1400

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Wavelength (om)

Wavelength (om)

Figure 3. Luminescence spectra of MCLA (A), componds 8 and 9 (B), and a mixture of MCLA and compound 4 (C) 2

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100

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Figure 4. Effect of the concentrations of compound 9 (A) and XOD (B) on the intensity of the superoxide-induced luminescence ACKNOWLEDGEMENT I thank Mr. Tsutomu Irie (Atto Corporation, Tokyo, Japan) for spectral measurements. REFERENCES 1. Goto T, Takagi T. Chemiluminescence of a Cypridina luciferin analogue, 2-methyl-6-phenyl-3,7-dihydroimidazo[1 ,2-a]pyrazin-3-one, in the presence of the xanthine-xanthine oxidase system. Bull Chern Soc Jpn 1980;53:833-4. 2. Minakami H, Arai H, Nakano M, Sugioka K, Suzuki S, Sotomatsu A. A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation. Biochem Biophys Res Commun 1988;153:973-8. 3. Teranishi K. Development of imidazopyrazinone red-chemiluminescent probes for detecting superoxide. Luminescence 2007;22:147-56. 4. Lin Y, Weissleder R, Tung C-H. Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chern 2002; 13:605-10. 5. Teranishi K, Komoda A, Hisamatsu M, Yamada T. Synthesis and enhanced chemiluminescence of new cyclomaltooligosaccharide (cyclodextrin)-bound 6-phenylimidazo[I,2-a]pyrazin-3(7H)-one. Carbohydr Res 1998;306: 177-87. 6. Mujumdar R B, Ernst L A, Mujumdar S R, Lewis C J, Waggoner A S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chern 1993;4:105-11.

GENERATION OF HIGH-ENERGY CHEMILUMINOPHORES IN AMBIENT LIGHT

YuB TSAPLEV, RF VASIL'EV, AV TROFIMOV Emanuel Institute 0/ Biochemical Physics, Russian Academy o/Sciences, ul. Kosygina 4, Moscow 119334, Russian Federation E-mail: [email protected]@[email protected]

INTRODUCTION Chemiluminescence is a prominent example of converting chemical energy into light and, thus, may be considered as a reversal of photochemistry. Such a symmetry is illustrated by Scheme I, in which Rg denotes the

Scheme 1

Photochemistry:

Rg + hV ~ Rg*

~

Chemiluminescence:

Pr + hV

Eo-

Pr*

Eo-

Int*

~

Int ~ ...

~

Pr

y,""'...... "round-Slale High-Energy Species

Int*

Eo-

Int Eo-

••• Eo-

Rg

reagent molecules, Pr represents the reaction products and Int refers to intermediate species. Apart from "straight-line" photo- (Rg + hv -+ ... -+ Pr) and chemiexcitation (Rg -+ ... -+ Pr + hv) processes, both photochemical and chemiluminescent reactions may involve energy-accumulation channels, in which relatively persistent molecular "depositories of light" (X) are formed. The said X species exist in the ground state, but possess enhanced internal energy, and the excess may be released in the form of a photon emission upon the relevant external influence on the reaction system (e.g., addition of any triggering reagent or heating). These peculiar "light accumulators" may be generated in different ways, in particular, under irradiation of the reaction mixture. Even simple exposure of the chemical system to the ambient light may convert the reagents into high-energy chemiluminophores. These transform upon subsequent addition of pertinent triggers and are exothermal enough to generate electronically excited products, the emitters of chemiluminescence. We term the latter phenomenon as [ight-f.reated f.hemiluminescence (LCCL/ and below discuss our recent findings in this context along with related issues. MATERIALS AND METHODS The chemuluminescence measurements were performed with a photon-counting 147

148

Tsapiev Yu B et ai.

instrument (Hamamatsu photosensor modules H7467, H7360-02 and H7360-03 supplied with the RS-232C interface). Preparation of the samples has been described before. 2 The quantum-chemical calculations were made using the semiempirical PM3 method. RESULTS AND DISCUSSION The material presented herein encompasses the energy accumulation in organic processes through the formation of high-energy chemiluminophores (X in Scheme I), the latter species are furnished by intermediate dioxetanes and pertinent photo isomers and photodimers. Intermediate dioxetane species. The chemiluminescence upon oxidation of unsaturated organic substrates (most prominently, lipids) provides a relevant chemical model for the generation of bioluminescence in living tissues and, thus, merits particular attention. An important feature revealed by our studies is the occurrence of at least two excited-state generation processes. The first chemiexcitation process constitutes the classical free-radical chemiluminescence channel, while the second one is of molecular origin. Addition of antioxidants to the reaction mixture immediately suppresses the radical part of the overall light emission. Then, the remaining molecular component decreases according to an exponential law (the rate constants are of the order of 10.4 S·I). Estimation of the emitter characteristics (lifetimes, quantum yields and radiation constants) leads to values typical for the phosphorescence of carbonyl compounds. Such behavior shows that the observed molecular chemiluminescence is excited in chemical transformations of oxidation products, which are persistent enough to accumulate in the course of reaction. Possible sources of this molecular contribution to the chemiluminescence of unsaturated substrates are dioxetane intermediates. In nature, these high-energy species may be formed through diverse (e.g., enzymatic or photochemical) mechanisms. 3 However, presently we favor the mechanism, which involves cyclization of alkylperoxy radicals. This may be illustrated by considering the oxidation of 1,1,2-trimethylethylene, H 3CCH=C(CH3)2, as a model alkene substrate. In this case, under a constant reaction-initiation rate, several types of radicals exist in their stationary concentrations. These are carbon-centered radicals, the products of addition of initiating radical r' to the double bond, or of the H-atom abstraction by this radical from methyl groups, for instance: H3CCHrC"(CH3 )2, H 3CC"HCr(CH3)2, H2C'CH=C(CH3)2, H3CCH=C(CH3)C"H 2. The fast addition of O2 transforms C radicals into a number of peroxy-radical species: H3CCHrC(CH3)200', H3CC(00")HCr(CH3)2, H2 C(00")CH=C(CH3)2, H3CCH=C(CH3)CH 200·. As a result, various reactions of these radicals occur. Combination of the ROO' radicals is exothermal, but it gives products that are not luminophoric, i.e., it is not a chemiluminescent process. Disproportionation of ROO' gives the emitters of "classic" free-radical chemiluminescence. However, the chain =C-C-O-O' in radicals H 2C(00,)CH=C(CH3)2 and H3CCH=C(CH3)CHzOO' is flexible enough (the bonds are single) for overlapping the unpaired electron with the Jt MO of the double bond, and

Generation of High-Energy Chemiluminophores in Ambient Light

149

so, for forming a four-membered dioxetane cycle. The latter cycle stores large amounts of chemical energy, that may be easily transformed into the electronic excitation upon decomposition of the dioxetane. Intermediate photoisomeric species. Structural photoisomerisation provides another way for the formation of high-energy chemiluminophoric species, which may be noticeable even under low-intensity irradiation of reactants. Upon subsequent addition of certain triggering agents to such photogenerated chemiluminophores, the electronically excited chemiluminescence emitters are formed. This recently observed light-created chemiluminescence (LCCL) phenomenon may be exemplified by the base-triggered chemiluminescent reaction of salicylaldehyde hydrazone pre-irradiated by visible light (400-500 nm) with the intensity of the order of 1 mW/m 2 . The illumination results in isomerisation of the non-chemiluminescent benzenoid reagent form (Rg) into the quinonoid structure (Rg'), the latter is prone to excited-state generation upon reaction with the base (Scheme 2). Scheme 2

The interaction of both Rg and Rg' isomers with bases is exothermic; however, the energy released in the reaction of Rg with HO' is not sufficient for the electronic excitation of the hydrazone anion, the chemiluminescence emitter. The PM3 computations reveal that the enthalpy of the Rg' formation exceeds that of the Rg species by 130 kllmol, which covers the energy deficiency for the excitation of the light emitter. Thus, the Rg' species constitutes the putative high-energy chemiluminophore, which is readily generated in the ambient light. Intermediate photodimeric species. The following example par excellence for the LCCL manifestation is provided by the base-initiated chemiluminescence of preirradiated 9-anthrone? Addition of bases to the 9-anthrone solutions stored in the dark does not trigger a chemiluminescence process. However, exposure of the reactant to ambient light and subsequent addition of base results in excited-state generation as manifested by the chemiluminescence emission. The products of the 9-anthrone photolysis in dioxane were separated by high-performance thin-layer chromatography.

150

Tsaplev Yu B et al.

The analysis of chromatograms has disclosed that the maximum chemiluminescent activity corresponds to the spot of dianthrol (DA, with retention index Rf = 0.25) rather than to that of peroxide species (Rf '" 0.37), whose intervention into the anthrone photochemiluminescence process was suggested in the literature. Thus, DA, which exists in two isomeric forms (Scheme 3), furnishes the relevant high-energy

Scheme 3

*==~hV

~~ H

hj

H

H

KH

EH DA+HO- __ DA-+HzO DA - + HO- - - (E-)* + E- + H 20 (E-)*-- E-+hv

"Head-ta-Head"

"Head-to-TalP'

r

DA

HO-

hv

chemiluminophore species; its base-triggered decomposition results in the electronically excited anthrol anions, K (the chemiluminescence spectrum matches the fluorescence emission of K). It is noteworthy that the DA species may be formed not only under in vitro conditions, but also in nature (the pertinent example is furnished by DA found in st. John's wort [Hypericum perjoratum), as it has been established in the present chemiluminescence studies. At a first glance, the ability of ambient laboratory light to create chemiluminescence seems astonishing. However, under ordinary illumination (only a few mW/m\ more 13 than 10 photons pass through an area of 1 cm z per second, which is 10 9 times greater than the detection limit of laboratory chemiluminometers. Clearly, this gap can be filled by numerous chemical systems, for which the absorption efficiencies mUltiplied by the photochemical transformation and the chemiluminescence yields are no less 9 than 10- . This implies that the list of known chemiluminescent reactions needs to be "revisited" to disclose the systems, which may be particularly influenced by the ambient illumination while preparing the reaction mixtures.

ACKNOWLEDGEMENTS The work was supported by the Russian Foundation of Basic Research, the Russian Academy of Sciences and the Russian Science Support Foundation.

REFERENCES 1. 2. 3.

Vasil'ev RF, Tsaplev YuB. Light-created chemiluminescence. Russ Chern Rev 2006; 75:989-1002. Tsaplev YuB, Vasil'ev RF. The nature of chemiluminescence of the photolyzed anthrone solutions. Russ J Phys Chern 2006;80:795-8. Adam W, Trofimov AV. Contemporary trends in dioxetane chemistry. In: Rappoport Z, ed. The Chemistry of Peroxides (Patai Series). Chichester: Wiley, 2006: 1171-1209.

ALKALINE METAL ION ENHANCED CHEMILUMINESCENCE OF BICYCLIC DIOXETANES BEARING A 3-HYDROXYNAPHTHALEN-2-YL GROUP N WATANABE, F KAKUNO, N HOSHIYA, HK IJUIN, M MATSUMOTO Dept a/Chemistry, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan

INTRODUCTION A dioxetane substituted with an oxyaryl group forms, after deprotonation or deprotection, an unstable dioxetane bearing an oxidoaryl anion which acts as electron donor for the intramolecular charge-transfer (CT) induced chemiluminescence.'·2 The position of the oxido anion relative to the attachment point of the dioxetane on the aryl group influences significantly the chemiluminescence properties. For instance, dioxetanes 1 substituted with a naphthalen-2-yl group bearing an oxido anion at the 4-, 5-, or 7-position ("odd' pattern) give off light more effectively than dioxetane analogs with an oxido anion at the 3-, 6-, or 8-position ("even" pattern) (Fig. 1).3 The phenomenon recognized as the "odd/even" relationship'" has been observed also for dioxetanes substituted with various oxyaryl groups such as oxyphenyl, oxybenzo[b]furan-2-yl, or oxybenzo[b]thiophen-2-yl group? These facts suggest a simple but important question as to whether chemiluminescence with high efficiency can be realized even from a dioxetane bearing an oxyaryl with "even" pattern by modifying the reaction conditions. In the course of our investigation of CTICL-active dioxetanes, we found a rather simple clue to answer the question.

1

: even odd

Fig. 1. Charge-transfer-induced chemiluminescent decomposition of dioxetanes bearing an oxidonaphthalen-2-yl group

RESULTS AND DISCUSSION Bicyclic dioxetane bearing a 3-hydroxynaphthalen-2-yl group 2 has very recently been found to possess a structure in which the 3-hydroxy group forms intramolecular hydrogen bonding with oxygen atom of the dihydrofuran ring, as illustrated in Fig. 2.5 This finding stimulated us to investigate decomposition of 2 in the presence of a metal 151

152

Watanabe Net at.

ion which should coordinate with both 3-oxidonaphthyl anion and oxygen of the dihydrofuran ring to cause expectedly unique chemiluminescence.

3-M

4-M

Fig. 2. Base-induced chemiluminescent decomposition of a dioxetane 2 bearing a 3-hydroxynaphthalen-2-yl group

When a solution of dioxetane 2 in THF (1.0 x 10-4 M, 1 mL) was added to a solution of tetrabutylammonium fluoride (TBAF) in THF (1.0 x 10-2 M, 2 mL) at 25 DC, 2 decomposed through unstable anionic dioxetane 3 to give keto ester 4 with an accompanying emission of orange light with maximum wavelength Amax CTICL = 552 nm and decomposition rate kCTICL = 1.7 X 10-2 S-I. However, chemiluminescence efficiency cpCTICL was quite poor (2.4 x 10-5) and a typical one for "even" pattern, as expected. On the other hand, when 2 was similarly treated with a large excess of sodium t-butoxide in place of TBAF, 2 decomposed to give intense light with TICL Ama/ = 535 nm, chemiluminescence efficiency cpCTICL = 1.5 X 10-3, and decomposition rate kCTICL = 4.0 X 10-2 S-I. This cpCTICL value was more than 60 times of that in TBAF / THF and was comparable to that for naphthyl-substituted dioxetane with "odd' pattern. 3 Comparing AmaxCTICL and kCTICL for sodium t-butoxide in THF with those in TBAF / THF, we see that AmaxCTICL shifted to blue considerably and kCTICL increased. On treatment with potassium t-butoxide instead of sodium t-butoxide in THF, dioxetane 2 decomposed more rapidly (kCTICL = 0.11 S-I) to emit a flash of light with Amax CTICL = 539 nm. The chemiluminescence spectrum is illustrated in Fig. 3 together with chemiluminescence spectra for TBAF system and for the other alkaline metal t-butoxide (vide infra). cpCTICL decreased to 4.5 x 10-4 though it was yet effective 10 times more than that in the TBAF system. Finally, we carried out the decomposition of 2 with lithium t-butoxide in THF. Lithium ion in general coordinates strongly to oxidoaryl anion in an organic solvent because of the smallest ion radius, thus, we did

Alkaline Metal Ion Enhanced Chemiluminescence of Bicyclic Dioxetanes

153

not expected so much that lithium t-butoxide functioned as a base for CTICL. In fact, dioxetane analog bearing a 3-hydroxyphenyl 4 decomposed sluggishly with lithium t-butoxide even in DMSO. However, the decomposition of 2 was effectively induced by lithium t-butoxide in THF at 45°C to give intense light with Amax CTlCL = 535 nm and kCTICL = 4.9 X 10-3 S-I. Astonishingly, the q,CTICL was enhanced more than 200 times of that in TBAF / THF.

450

500

550 600 wavelength / nm

650

700

Fig. 3. Chemiluminescence of dioxetane 2 in THF Effects of alkaline metal ion on the CTICL of dioxetane 2 in THF observed here are summarized as follows. First, all chemiluminescence spectra in the presence of alkaline metal ion shifted 12-17 nm to blue compared to the case in TBAF. Second, the decomposition rate constant kCTICL increased in the order t-BuOK < t-BuONa < t-BuOLi. This order coincides with the order of ion radius decreasing from K+ to Li+. This suggests strongly that alkaline ion coordinates to oxidonaphthyl ion of 3 and Lt does the most tightly among these three alkaline metal ions. Thus, CTICL of 2 proceeded most likely through anionic dioxetane forming chelate 3-M to give chelated keto ester 4-M with the accompanying emission of light. Third, q,CTICL increased in the order TBAF « t-BuOK « t-BuONa «t-BuOLi. This result suggests that conformation of oxidonaphthyl group as an electron donor relative to dioxetane ring affects likely efficiency of singlet-chemiexciation process. Next, we investigated CTICL of a dioxetane bearing a 7-hydroxynaphthalen-2-yl group 5 induced by alkaline metal t-butoxide in THF. Dioxetane 5 belongs to "odd' pattern and is known to emit light effectively, though it cannot form a chelate with a metal ion. When 5 was treated with TBAF in THF, 5 decomposed to give off light effectively with Amax CTICL = 580 nm, chemiluminescence efficiency q,CTICL = 2.3 X 10-2 , and decomposition rate 3 kCTICL = 1.7 X 10- S-I. On the other hand, although 5 decomposed to give very weak light with Ama/TlCL = 564 nm, neither q,CTICL nor kCTICL could not be estimated.

154

Watanabe Net at.

~

o-& 0-0

HO

t-Bu

0

4

HO 5

Fig. 4. BicycIic dioxetanes bearing a 3-hydroxyphenyl4 or 7-hydroxynaphthalen-2-yl group 5

In conclusion, we have discovered that chelation with alkaline metal ion, especially Li+, enhances markedly chemiluminescence efficiency of CTICL for an "even" pattern" hydroxyaryl-substituted dioxetane, i.e., bicycIic dioxetane bearing a 3-hydroxynaphthalene-2-yl group 1 in THF. ACKNOWLEDGEMENTS The authors (MM and NW) gratefully acknowledge financial assistance provided by Grants-in aid (No. 1550043, and No. 17550050) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

REFERENCES 1. Beck S, Koster H. Applications of dioxetane chemiluminescent probes to molecular biology. Anal Chern 2000;45 :2258-70. 2. Matsumoto M. Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence. J Photochem Photobiol C Photochem Rev 2004;5:27-53. 3. Hoshiya N, Fukuda N, Maeda H, Watanabe N, Matsumoto M. Synthesis and fluoride-induced chemiluminescent decomposition of bicyclic dioxetanes substituted with a 2-hydroxynaphthyl group. Tetrahedron 2006;62:5808-20. 4. Edwards B, Sparks A, Voyta JC, Bronstein I. Unusual luminescent properties of odd- and even-substituted naphthyl-derivatized dioxetanes. J Biolumin Chemilumin 1990;5:1-4. 5. Hoshiya N, Watanabe N, Ijuin HK, Matsumoto M. Effect of intramolecular hydrogen bonding on thermolysis of dioxetane: unusual instability of bicyclic dioxetanes bearing a hydroxynaphthyl group with vicinal substitution pattern. Chern Lett 2007;36:516-7.

PART 4 APPLIED CHEMILUMINESCENCE

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ANALYTICAL CHALLENGES FOR LUMINESCENCE-BASED POINT-OFCARE TESTING DEVICES IN BIOMEDICAL DIAGNOSTICS ARODA,' M GUARDIGLI,' M MIRASOLI,' E MICHELINI,' LS DOLCI,' M MUSIANI 2 JDept of Pharmaceutical Sciences, University of Bologna, Bologna 40126, Italy 2 Dept of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna 40138, Italy, Email: [email protected]

INTRODUCTION Point-of-care tests (POCT) are clinical laboratory tests performed in a self-contained analytical platform that can be operated by non-laboratory healthcare professionals. POCT allow quicker results than centralized laboratory services due to reduced assay time and because the analysis is performed at the site where the sample is drawn or clinical care must be delivered. The recent technology improvements with the possibility to achieve high detectability (down to the micro- and nanomolar levels) for a wide number of analytes allows one to envisage a new generation of diagnostic POCT devices for the detection in biological fluids (blood, serum, saliva) of a panel of biomarkers of a given disease. A POCT device should combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. Microfluidics-based integrated devices relying on biospecific recognition reactions combined with ultrasensitive luminescence detection techniques such as bio-chemiluminescence (BLlCL), electrochemiluminescence (ECL) and photoluminescence, represent one of the most promising options. Indeed, this experimental approach could gain enough sensitivity and selectivity to perform the simultaneous detection of different target analytes present in very low concentrations in complex samples. BIOSPECIFIC RECOGNITION SYSTEMS Different biospecific recognition elements, such as enzymes, antibodies, nucleic acids and receptors, could be used for mUltiplexed assays in a POCT device. Potentially, the device could perform immunoassays (e.g., for hormones, proteins, viruses, bacteria) or nucleic acid hybridization reactions (e.g., for evaluating gene expression and detecting gene mutations, single nucleotide polymorphisms, DNA and RNA sequences). In most cases, the highest detectability is achieved with non-competitive analytical formats, in which the target analyte is captured by a specific probe and revealed by a second labelled probe. Capture probes can be immobilized directly on the surface of the sensor (e.g., on glass, plastic, transparent conductive materials) or, to increase immobilization efficiency, on nanostructured supports (e.g., nanospheres, nanowires, nanofibers). In addition, single or coupled enzyme reactions could be used for the quantification of enzyme activities or enzyme substrates. 157

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RodaA etal.

LUMINESCENCE DETECTION Luminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in ultra-small sample volumes (down to nanoliters) and accurate signal localization and quantification. Detectability can be further increased by passive luminescence signal amplification, i.e., by labeling the probes with nanostructured supports (e.g., nanospheres, molecular dendrimers, biotin-streptavidin scaffolds) bearing a large number of luminescent species. Moreover, various luminescent detection techniques and/or luminescent labels can be combined in a single device to obtain separate quantification of several analytes by spectral- or time-resolved signal acquisition or by independent signal trigger. We have developed luminescent labels with improved characteristics suitable for miniaturized analytical devices and multiplexed assays. For example, we reported a strategy for obtaining BODIPY -based fluorophores for multiplexed assays consisting in the introduction of a common light absorption moiety in the dye molecules. The resulting fluorescent species maintain the emission bands of the parent dyes and can be excited simultaneously at the same wavelength, thus only one excitation source is required.' Time-resolved luminescence is a promising detection technique because it is not affected by sample autofluorescence, but the available labels are often characterized by low quantum yields. We recently described a new chelating ligand that gave water-stable and strongly luminescent lanthanide complexes and demonstrated its suitability for bioanalytical time-resolved luminescence microscopy.' Concerning BL, one of the main limitations of the use of luciferases as labels is their poor stability. To overcome this problem and to facilitate multiplexing, we have obtained new luciferases by cloning the luciferase gene from other species (e.g., Luciola italica), or by producing new mutants characterized by higher thermostability and red-shifted emission spectra.' In addition, it is also desirable to improve the performance of the currently available luminescent labels. We have recently demonstrated that the incorporation of an acylation catalyst in an enhancer/luminol/oxidant CL substrate for horseradish peroxidase resulted in a very significant increase in light output, thus in an improvement of CL assays sensitivity.' In the context of ECL detection, we recently designed a transparent electrochemical cell for ECL imaging (described below) that is also suitable for BLlCL measurements and can thus be employed as a luminescence reading device of general applicability. ANALYTICAL DEVICES The analytical challenge is to achieve multiplexed analysis in the same device, i.e., to perform the biospecific recognition reactions and to reveal them independently by luminescence detection. Multiplexing could be achieved by "position encoding" and "reaction encoding" strategies. In "position encoding" the recognition reactions corresponding to the different analytes take place in different positions within the device and the resulting luminescent signals are detected by a luminescence imaging technique. In "reaction encoding" the various recognition reaction are associated with different luminescence reporting processes and/or labels and the detection rely on the independent trigger of the luminescence processes (e.g., addition of specific BLlCL

,wl,,'u'nl

Challenges Jor Luminescence-Based Point-oj-Care Testing Devices

159

substrates) or on the spectral resolution of light emission. A possible layout of the reading device is shown in Fig. I. The device consists of a on which the various biospecific recognition molecules are immobilized within an array. The analytes are captured by the biospecific rf>C"r.crrIlT,r.n molecules and revealed by a set of bioprobes labelled with luminescent species. The is in contact with an imaging light sensor (CCD, CMOS photosensor array, "avalanche" diodes array) able to localize and quantify the luminescent "Contact" detection, in which the signal is produced on (or very close to) the detection allows it to achieve a much higher optical efficiency than that of conventional camera-based imaging systems. The limitations on the resolution of contact detection can be overcome by a suitable design of the sensor photodiodes can be sized with respect to a given application) and applying suitable cross-talk reduction algorithms. The device will also include a fluidic system for and reagents.

Fluidics system "

Transparent support with biospccific ,wobes immobilized in defined ""cas ........... Imaging light detector (CCD _ or """"y of diodes)

A device for luminescence multiplexed assays based on "position

""",vu ... !;

the device will be suitable for BLlCL measurements. luminescence technique, it will also contain the other elements necessary for generation and measurement, for example excitation sources laser and filters for spectral selection for fluorescence detection or addressable miniaturized electrodes for ECL detection. We have a prototype device to perform ECL imaging detection of biospecific labelled with ECL-active species, such as Ru 2+ complexes. The device is conductive fluorine-doped tin oxide (FTO)-coated which was chemically etched to obtain a three-electrodes electrochemical cell with a silver quasi-reference electrode. Experiments conducted on model systems (micron-sized beads) showed that the generation of the ECL could be switched on/off during the measurement process and that the emitted could be detected with a micrometer-scale resolution by luminescence The characteristics of such device made it suitable also for BLlCL measurements, in which the luminescent signal is triggered by addition of a BLlCL substrate. MICROFLUIDICS A POCT device should be equipped with pumps, valves, sensors,

reservoirs

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Roda A et al.

and all other components required for its operation, comprising an integrated control system for the management of the whole analytical procedure and the acquisition and evaluation of the results. Microfluidics, which is fundamental for POCT development, requires the fabrication of highly integrated devices for performing several different functions on the same substrate chip. In a manner similar to that for microelectronics, the components of a POCT device could be easily produced by microfabrication techniques, which are relatively inexpensive and amenable both to highly elaborate devices and also to mass production. An integrated module for on-line sample pre-analytical treatment and/or clean-up could be also included in the device. With this respect, field-flow fractionation (FFF) techniques, which can separate analytes based on their morphological characteristics (size, shape and superficial properties) can be exploited to develop pre-analytical modules for cells or macromolecules (e.g., proteins, protein complexes or adducts) fractionation, thus providing a selectively enriched fraction for the analysis.

CONCLUSIONS The recent advancements in molecular biology, luminescent labels and detection systems, and micro fabrication techniques would allow the development of POCT devices for multiplexed assays. Biospecific recognition reactions combined with luminescence detection represent the most promising approach due to their high sensitivity, wide range of detectable analytes and possibility of miniaturization. New POCT services would provide numerous advantages and significant patient benefits, but the quality of results could be affected by inadequate training or inappropriate use. All analytical tests, whether performed in the laboratory or not, can run into problems but laboratory staff have more knowledge and experience to recognize and address these situations. Therefore, it is important to get the balance right and maximize the benefits that this exciting technology offers while ensuring that the quality of results and patient safety is not compromised. REFERENCES 1. Weibel N, Charbonniere LJ, Guardigli M, Roda A, Ziessel R. Pyrromethene dialkynyl borane complexes for "cascatelle" energy transfer and protein labeling. Angew Chern Int Ed EngI2005;44:3694-8. 2. Ulrich G, Goze C, Guardigli M, Roda A, Ziessel R. Engineering of highly luminescent lanthanide tags suitable for protein labeling and time-resolved luminescence imaging. J Am Chern Soc 2004;126:4888-96. 3. Branchini BR, Southworth TL, De Angelis J, Michelini E, Roda A. Isolated luciferase gene of L. italica. US Patent 102816-100,2006. 4. Branchini BR, Southworth TL, Khattak NF, Michelini E, Roda A. Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 2005;345:140-8. 5. Marzocchi E, Grilli S, Della Ciana L, Mirasoli M, Prodi L, Roda A. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts. Anal Biochem, accepted for publication.

MOLECULAR IMPRINTED POLYMER· BASED CHEMILUMINESCENCE SENSORS ZHUJUN ZHANG Department of Chemistry, Shaanxi Normal University, Xi' an 710062, China; Email: zzj18 @hotmail.com

INTRODUCTION

Chemiluminescence (CL) is a light emission from a chemical reaction.

CL

analysis has advantages of high sensitivity, wide linear range and simple instrumentation. It has become an attractive analytical tool in biological and chemical analysis. However, CL analysis also has some disadvantages. The main disadvantage is that it has very poor selectivity, which has limited its application. How to solve this problem? One approach is to combine it with a high performance separation method such as high performance liquid chromatography or capillary electrofluorescence. Another approach is to combine it with a specific molecular recognition method such as an enzyme, antibody or receptor, nucleic acid, aptamer, DNA or RNA and molecular imprinted polymer (MIP).l.g Molecular imprinting belongs to the category of host-guest chemistry in super-molecule

chemistry.

It

is

an

interdisciplinary

subject,

involving

macromolecular chemistry, biochemistry, and material chemistry. Molecular imprinting polymerization is a technique of making molecular recognition sites for an analyte molecule in a synthetic polymeric substrate. MIP is synthesized by a polymerization reaction of a mixed solution containing functional monomers, crosslinker and a target molecule in an organic solvent. A complex is formed between the target molecule and the functional monomer through polar interactions. Subsequent polymerization with the cross-linker fixes the positions of the polar groups of the functional monomer. After removing the target molecules, the molecular recognition sites will be made with shape, size and function 161

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Zhang Z et al.

complementary to the analyte, and they can rebind the analyte molecules in preference to other closely related structures. MIP is stable and resistant to a wide range of pH, humidity and temperature, so that a sensor modified with a MIP is easily stored and prepared. It also has a long lifetime. Potential target molecules include carbohydrates, amino acids, nucleic acids, alkaloids, drugs, vitamins, proteins, antibodies and bacteria. The target molecule may be covalently or noncovalentIy linked to a functional monomer. Usually, with an increase in binding sites between target molecule and functional monomer, the specific molecular recognition will be increased, but the reversibility of sensor will be decreased. Under certain conditions, we are only interested in specific molecular recognition. For example, we prepared a MIP as an alternative to an antibody in an immunoassay. In this application, the reversibility of MIP is not important. However in other situations, we have to achieve a balance between the specific recognition and reversibility for sensor design. Molecular imprinted polymer-based CL imaging sensors.

When using MIP

as the recognition element in a CL sensor, the selectivity of the CL method can be greatly improved and the interference of some species commonly present in samples can be eliminated.

So, an MIP-based CL sensor can be used directly to

determine the analyte in real samples. Along with the development of CL analysis, significant progress has been made in techniques to measure CL. The CL signal may be detected by both conventional photomultiplier (PMT)-based luminometers and high resolution imaging detectors. Light emission down to the single-photon level can be localized and quantified by CL imaging techniques. Imaging techniques are advantageously used when the spatial distribution of the luminescence signal represents crucial analytical information. MIP combined with CL imaging assay has the advantages of simplicity, high selectivity, high sensitivity and high throughput. Due to the beneficial health effects of trans-resveratrol in grape wine, many methods have been developed for its detection and quantification. 1 In our laboratory, a MIP-based CL imaging sensor for determination of trans-resveratrol in grape wine has been developed. The MIP of trans-resveratrol was prepared by a precipitation polymerization using trans-resveratrol as the target molecule,

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

acid

(MAA)

as

the

functional

monomer,

163

ethylene

dimethacrylate (EGDMA) as the cross linker. The trans-resveratrol in the MIP can be washed out using methanol containing 10% acetic acid (v/v) to uniform MIP microspheres (average diameter -l.5 Ilm) (Fig. 1). Microtiter with 96 wells were coated with trans-resveratrol-MIP, which were fixed in polyvinyl alcohol as glue. The amount of polymer-bound trans-resveratrol was

using the imidazole (IMZ)-catalyzed peroxyoxalate CL reaction.

The

produced was measured with a high-resolution CCD camera. The

exposure time was optimized to 1 min. The intensity of the spots was determined the

FC software, which combines the pixel intensities.

1.

The SEM image of trans-resveratrol-MIP microspheres.

Table 1 shows the tolerable ratios for some interfering substances in grape wine. The trans- resveratrol , cis-resveratroL trans-piceid and cis- piceid have similar structure and in the absence of trans-resveratrol with MIP, the tolerable ratio is 1. When the sensor was prepared with MIP, the tolerable ratio increase to 100. These results show that these substances in grape wine in the normal concentration range did not interfere with the determination of trans-resveratrol. Other substances existing in wine such as fructose, citric acid and many amino acids have no CL

and did not interfere with the trans-resveratrol analysis.

The chiral molecular recognition of neutral molecules has become an important in the fields of analytical, biochemical and pharmaceutical technologies.

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Zhang Z et al.

Table 1. The tolerable ratios for some interfering substances in grape wme. Intelierencc substo'lllces

Without MIP

100 100 100 50

Trt1/IJ -piceid

Cis-resvemtroI Cis-pkeid

1

Vitamin B I. B2. B6. B 12

0.1

With f<.IIP

Threonine. tryptophan, methionine

5()()

800

Fructose. citric add

450

5uo

The design and development of effective chiral separation and recognition of enantiomers is the key point of the chiral technique. Many technologies have been developed for chiral recognition and separation of amino acids and their derivatives including HPLC, CE and electrochemical sensors. However, they are expensive techniques in terms of time and reagent consumption. Accordingly, there is considerable interest in the development of simple, rapid and economical methods that will afford the analysis of enantiomeric species. The aim of our work was to develop a fast, simple and selective CL imaging assay coupled with MIP for the chiral recognition of fluorescence labeled phenylalanine. The

precipitation

polymerization

method

was

used

for

preparing

MIP

micro spheres with uniform shape. Figure 2 shows the dansyl-L-phenylalanine molecular imprinting process. The average diameter of microspheres was about 0.7 flm. We know that the TCPO-HzOz CL system requires a suitable fluorescent species. This limitation was overcome in this work by using dansyl-based amino acid as the signaling element. The light produced by the CL reaction was measured by using the Alpha Innotech Fluorescence and CL imaging system. The control experiments using capillary the electrophoresis method showed that there was no significant difference between the proposed method and the control method (confidence level 95%).

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

165

Pre-assembly

Removing the template

2.

The method can To obtain

Dns-L-phe molecular imprinting process.

96 independent measurements simultaneously in 30 min.

better understanding of the nature of the interactions between the sites of the MIP and its target molecules, and to determine the to recognize the imprinted molecules, adsorption of other acids on the MIP was investigated with CL imaging. The responses ,.n"'VT"~

of tile

to the imprint molecule. The response for

the

and then dansyl-D-Trp. This may be due to the Try and

the are

are shown in Fig. 3. The imprinted polymer showed

than Phe. The responses of dansyl-L-amino acids were acids. The lower response can also be

of the effect. The

was molecules than those a sterk

results indicated that the application of an

as a chiral discrimination element, combined with CL CCD camera may allow the detection of the analyte enantiomeric composition from a matrix without separation.

Similar designs have also been used for

determination of polycyclic aromatic hydrocarbons (PARs), dipyridamole and (6-MP).

166

Zhang Z et al. 4000 ---.- Dansyl-L-Phe _Dansyl-I...-Oy

3200

.......-Dnasy~

---4-Dansyl-L-Trp

2400

0

-J(-..-..-

>

-<

Dansyl-LTry

---+- Dansyl-D-Oy 1600

--+- Dansyl-D-Ou

-

Dansyl-D-Trp

-I'r- Dansyl-D-Tty

800 0 0

50

100

150

200

250

300

Concentrati>n (1M)

Fig. 3.

The corresponding CL imaging with dansy-L-phe imprinted polymer.

Molecular imprinted polymer-based chemiluminescence micro flow sensor on a chip. The field of J.lTAS has grown rapidly following the introduction of the concept of micro total analysis system (J.l TAS) by Manz et al. in 1990. The ultimate purpose of J.lTAS is the integration of the entire analytical process on a micro-device. The micro-flow sensor chip is a part of J.lTAS. 9- IZ A novel chemiluminescence (CL) micro flow sensor on a chip for the determination of clenbuterol, salbutamol and terbutaline in human or animal urine based on a molecularly imprinted polymer (MIP) as the recognition element has been described. 5,6 Clenbuterol, salbutamol and terbutaline are p-agonists and can accelerate animal growth, and produce more muscle. However, residues of p-agonists can lead to food poisoning. They are also doping agents forbidden by International Olympic Committee. Uniform MIP microspheres (average diameter -0.5J.lm) were prepared by a precipitation polymerization using clenbuterol, salbutamol or terbutaline as the target molecule respectively, MAA as a monomer, EGDMA as a cross-linker, AIBN as an initiator and acetonitrile as porogen. The maximum binding capacity ofMIP was 1-1.5 mg/g of target molecule. We found that clenbuterol, salbutamol and terbutaline can enhance the CL reaction of luminol with ferricyanide.

The

micro flow sensor chip (Fig. 4) was fabricated from two 50x40x5mm transparent poly (methylmethacrylate)(PMMA) slices. The microchannels on the chip, etched by a COz laser, were 200 J.lm wide and 150 J.lm deep. The micro flow sensor cell

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

167

filled with 2 mg MIP for on-line selectively adsorbing clenbuterol was 10 mm long, 1 mm wide and 0.5 mm deep. A top plate was placed on the base plate and bonded at a pressure of 1.5 MPa at 80°C for 20 min. The injection pump with accurate time control was used for monitoring all reagent solutions including the sample. The precision of the timer is 0.01 sec. The on-line adsorbed clenbuterol by the MIP can enhance the CL intensity of the reaction of luminol with ferricyanide. The enhanced CL intensity is linear with clenbuterol concentration from 2.0 to 100 ng/mL The chip with a detection limit of 1.0 ng/mL (30'). The micro flow sensor on a chip provides for good reproducibility with the relative standard deviation of 3.6% (n=7) for 10 ng/mL clenbuterol. We have also combined microdialysis and CL micro flow sensor. chip for in vivo and real-time monitoring the metabolism of tramadol in rabbit blood (Fig. 5) and real-time monitoring the metabolism of Pb, Cu, Hg and Cd in rabbit blood. Similar designs have also been used for determination of isoniazid, dipyridamole, chlorpromazine and gallic acid etc.

~

.-:~U::::H::L:::::: :::::~:-:-

! ! ']:\ '-.,,',' 1 -

Luminol "

"" __ ------ /,J;;!,:.

Stainle~ screw ~

/:/

"

"

I

~',-

--:.-Topp!ate

~. Sa !at ---\:-~ sep e

:.t"

Fig. 4.

Fig. 5.

Structure of micro flowsensor on a chip.

Schematic of the microdialysis/CL flow sensor chip system for in vivo and real time monitoring the metabolism oftramadol in rabbit blood.

168

Zhang Z et ai.

Although MIP has many advantages, a number of problems still exist. First, the main disadvantage is the difficulty in removing all of the target analyte molecule, even after extensive washing. In fact, the better selectivity MIP provides, the more difficult it is to remove the target by washing. This leads to time-consuming washing, less reversibility and inaccurate results. However, an ideal sensor should have the characteristics of both high selectivity and reversible binding of the analyte. In the present sensor chip, because the analyte can be destroyed through the CL reaction, the reacted analyte can be easily washed off from the MIP by using water as the eluent. The cavities can be released and retain their memory for the target analyte. Therefore, the proposed micro flow sensor on a chip is reversible and reusable. Second, the use of organic reagents and buffer solutions as the eluent, which can affect the CL reaction was avoided. The analysis procedure is hereby simplified and the sensitivity of the CL reaction is improved. Third, the MIP can on-line enrich the analyte for the CL determination. The enrichment effect of the MIP can also improve the sensitivity of the CL reaction. At last, all the reagents are controlled by the injection pump with an accurate timer. Therefore, the micro flow sensor on a chip provides for very reproducible measurements. Molecular imprinted polymer recognition and on line electrogenerated chemiluminescence detection.

Most CL results from a direct oxidation reaction

or an oxidation reaction with energy transfer. Commonly used oxidants include hydroperoxide, oxygen, potassium permanganate, ferricyanide, tetravalent cerium ion, lead dioxide and oxygen free radical such as superoxide anion (-0 2 -), hydroxy radical (-OH) and nitric oxide (NO). Some transition metals exist in their highest oxidation state such as copper in trivalent state as [Cu(HI0 6ht and Cu(H2 Te06)2t, nickel in tetravalent state exists as [Ni(H2 Te06hf, silver in trivalent state exists as the form of [Ag(HI06 ht and [Ag(H2Te06h]5-, iron in the hexavalent state exists as K2Fe04. Hitherto, a considerable amount of research has been reported on the reaction rate of transition metals in their highest oxidation state or in the form of an active reaction center. It has been proved that these transition metal cations in the highest oxidation state can be stabilized by chelation with suitable polydentate ligands in

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

169

aqueous solution. We

that these polydentate chelates of transition metals in the

oxidation state showed a supernormal catalysis effect on the CL reaction of peroxide. As shown in Fig. 6, at the lower concentration of mollL) and hydrogen peroxide «10-8 moIlL). The

luminol

trivalent copper complex exceeded other catalysts such as which are regarded as the strongest of trivalent copper complex is also

reaction.

of the CL ur,rm'Cfpr

to prepare the nanoparticles of a

than HRP. of trivalent

as a new CL label for immunoassay.

Cr(1II1

Cu(ll) 4 Co(ll)

of luminol concentration under the catalytic activity of 5XlO-9 mollL,

, HRP and

HRP and DPC 10-4 moliL. Transition metals in reactions

oxidation state also showed

redox

most are difficult to use directly because they are not stable. In

order to overcome this drawback, online electrogenerated unstable from a stable oxidation state of the same element in flow CL in our laboratory. 13-17

were

For example, a molecularly

and on-line electrogenerated complex of trivalent copper and luminol chemiluminescence system for determination of glucocorticoid residues in

liver was proposed. 13 Fig. 7 shows the design of the

The solutions were delivered by two peristaltic pumps. A PTFE tube (2 mm id

Zhang Z et al.

170

cm length) packed with MIPs was used as MISPE micro column and placed on an eight-way injection valve. The sample solution passes through the column of MIP for on-line selective pre-concentration of glucocorticoid, then changed to the b line. Another example is determination of tetracycline residues in fish using MIP recognition and on line electrogenerated Ag (II) direct oxidation CL. The MIP particles were packed into a PIFE tube, which was connected into the sample loop of an eight-way injection valve to serve as the MIP solid phase extraction column. The sample solution passes through the column of MIP for on line selective pre-concentration for on-line extraction of the absorbed tetracycline. Then an intermediate differential pulsed elution (DPE) step used 3% acetic acid to remove oxytetracycline and other interfering substances. Finally, a mixed solution of acetonitrile and nitric acid ( 0.01 mollL) was used as a pulsed elution (PE) for extraction the adsorbed tetracycline, which could be detected by direction CL reaction of tetracycline and bivalent silver, which was on-line electrogenerated by constant current oxidizing silver nitrate in 5.0 mollL nitric acid medium in FEC. The bivalent silver concentration could be readily adjusted on line over a wide concentration rang with changing the electrolytic current. pump

sampl e

_++_---,

el uent-++---{(~}-----....,

lWlinol electrolyte

--==tt;:J------t~

-

Wa.te

Counter electrode, samJllr

I"lu('nt

w;tstr

to now {eU

Frit glass "

~

:vorkingelectrode

I/

Pt wire

Electrolyte~

Out

Structure of FEC

Structure of Valve Fig. 7.

Design of MIP recognition and on line electrogenerated reagent CL detection.

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

171

ACKNOWLEDGEMENTS We gratefully acknowledge National Natural Science Foundation of China (Grant No. 20175039 and 30470886) and Science and Technique Ministry of China (Grant No. 2002AA2Z2031 and 2003BA31 OA05) for financial support

REFERENCES 1.

Wang L, Zhang Z. Molecular imprinted polymer-based chemiluminescence imaging sensor for the detection of trans-resveratrol.

Anal Chim Acta

2007;592: 115-20. 2.

Hou L, Zhang Z, Luo L. Chemiluminescent imaging analysis of interferon alpha in serum samples.

3.

Wang L, Zhang Z, chemiluminescence

Anal Bioanal Chern 2007; 387:925-31. L.

Huang imaging

Molecular for

imprinted

the

chiral

polymer based recognition

of

dansyl-phenylalanine. Anal Bioanal Chern 2008;390: 1431-6. 4.

Wang L, Zhang Z. Chemiluminescence imaging assay dipyridamole based on molecular imprinted polymer as recognition material. Sens Actuators B, on line - DOl: 10.1016/j.snb.2001.01.051.

5.

Zhou H, Zhang Z, He D, Hu Y. Flow chemiluminescence sensor for determination of clenbuterol based on molecularly imprinted polymer. Anal Chim Acta 2004;523:237-42.

6.

Zhou H, Zhang Z, He D, Xiong Y. Flow through chemiluminescence sensor using molecularly imprinted polymer as recognition elements for detection of salbutamol. Sens Actuators B 2005;107:798-804.

7.

Xiong Y, Zhou H, Zhang Z. Molecularly imprinted on-line solid-phase extraction

combined

with

flow-injection

chemiluminescence

for

the

determination of tetracycline. Analyst 2006; 131 :829. 8.

Luo L, Zhang

Z,

Ma L.

Chemiluminescent

imaging detection

staphylococcal enterotoxin C, in milk and water samples. 2006;97:355-60.

of

Food Chern

172

9.

Zhang Z et al.

Liu W, Zhang Z, Yang L. Chemiluminescence microfluidic chip fabricated in PMMA for determination of benzoyl peroxide in flour. Food Chern 2006;95:693-8.

10. He D, Zhang Z, Zhou H, Huang Y. Micro flow sensor on a chip for the determination ofterbutaline in human serum based on chemiluminescence and a molecularly imprinted polymer. Talanta 2006;69:1215-20. 11. Xiong Y, Zhou H, Zhang Z, He D, He C. Determination of hydralazine with flow

injection chemiluminescence sensor using molecularly imprinted

polymer as recognition element.

J Pharm Biomed Anal 2006;41 :694-700.

12. He C, Zhang Z, He D, Xiong Y. Chemiluminescence determination of metformin based on hydroxyl radical reaction and molecularly imprinted polymer on-line enrichment. Anal Bioanal Chern 2006;385:128-33. 13. Zhang Y, Zhang Z, Song Y, Wei Y. Detection of glucocorticoid residues in pig

liver

by

electrogenerated

high-performance

liquid

[Cu(HI06)z]5--luminol

chromatography

with

on-line

chemiluminescence detection.

J

Chromatogr A 2007; 114:260-8. 14. Zhang Y, Zhang Z, Song Y, Wei Y. Detection of indomethacin by high-performance liquid chromatography with in situ electrogenerated Mn(III) chemiluminescence detection. Anal Chim Acta 2007;582:229-34. 15. Hu Y, Zhang Z, Yang C. The determination of hydrogen peroxide generated from

cigarette

smoke

with

an

ultrasensitive

and

highly

selective

chemiluminescence method. Anal Chim Acta 2007;601 :95-1 00. 16

Hu Y, Zhang Z, Yang C. A Sensitive determination

of

Hz0 2

in

exhaled

chemiluminescence method for the breath

condensate.

Anal

Sci

2008;24:201-5. 17. Hu Y, Zhang Z, Yang C. Measurement of hydroxyl radical production in ultrasonic aqueous solution by a novel chemiluminescence method. Ultrason Sonochem 2008;15:665-72.

FLOW INJECTION ANALYSIS CHEMILUMINESCENCE DETERMINATION OF HYDROXYLAMINE HYDROCHLORIDE MOHAMMAD REZA BAEZZA T, MARY AM IZADPANAH Department o/Chemistry, Fars Bioconversion and Environmental Research Institute, Shiraz, Iran.

INTRODUCTION Chemiluminescence (CL) arises when a chemical reaction produces an electronically state which emits light as it returns to its ground state. I Light measurement is a relative indicator of the amount of luminescent material present in the sample of interest. The use of CL in analysis for organic and inorganic species at trace levels has received attention mainly because of the simplicity of the instrumentation, the low detection limit and the wide dynamic range. CL reactions based on reagents such as luminol, lucigenin, lophin and some oxalate esters 2 are most common. 2,3 Hydroxylamine hydrochloride is mainly used as reducer and developer, during organic synthesis, it can be employed to make oxime inorganic synthesis and material for the compound of anticancer medicine (hydroxyurea), sulphonamide (sulfamethoxazole) and pesticide (medorwy).

MATERIALS AND METHODS Reagents. Analytical reagent-grade chemical and triple-distilled water were used. Hydroxylamine hydrochloride stock solution (l.00x1O-2M) was prepared by dissolving O.l737g hydroxylamine hydrochloride (Merck) in water and diluting to 250 mL with water. Na202 stock solution (2.00x 10-IM) was prepared daily by dissolving 3.898g ofNa202 (Merck) in water and diluting to 250 mL with water. Apparatus. The injection manifold used was used as in previous work.' The carrier stream mixing and reacting with oxidant stream in a homemade flow cell (40flL) in front of a photomultiplier tube (PMT). The signal from the Filter-Fluorimeter (Perkin-Elmer RI OOA) was sent to a computer.

RESULTS AND DISCUSSION In this CL reaction hydroxylamine hydrochloride is oxidized by sodium peroxide in acidic medium. This method development includes optimization of reagent concentration and flow conditions. Effect of hydrochloric acid concentration. The effect of hydrochloric acid concentration was investigated over the range 0.02-0.14 M. Sample solutions contained hydroxylamine hydrochloride (3.00 x 10- 6 M) added in different concentration of hydrochloric acid were reacted with Na202 (5.00 x 10-2 M); each stream pumped at 2 mUm in. The optimal concentration of hydrochloride acid for detection of hydroxylamine hydrochloride was 1.20x 1O-I M. 173

174

Baezzat MR & lzadpanah M

Effect of NazOz concentration. The effect of Na202 concentration was investigated over the range 0.005-0.100 M. Sample solutions contained hydroxylamine hydrochloride (3.00 X lO- 6M) and hydrochloric acid (1.200 x lO- 1M) reacted with different concentration Na202; each stream pumped at 2 mLimin. Increasing the concentration of Na202 resulted in an increase in the chemiluminescence observed up to 5.000x 1O-2M and decrease in light intensity at high concentrations. Effect of flow rate. The effect of flow rate on the chemiluminescence emission intensity was studied over the range 0.50-3.00 mLimin in each stream. An increase in the emission intensity was obtained with increase in flow rate in range 0.50-2.00 mLimin. Thus the chosen optimal flow rate was 2.00 mLimin in each channel. Analytical performance. Calibration graphs were obtained for each of the CL system described above in Fig. 1. Under the optimum conditions described above, hydroxylamine concentration was linear over the range 3.00x 10- 7_ 5.00 x l0- 6 M Table 1. Precision and accuracy of the method (conditions: 1.20x lO-IM hydrochloric acid, 5.00x 10-2M Na202 and 2.00 mLimin flow rate). Hydroxylamine present (M) 5.00xlO-'

Hydroxylamine found (M) 5.00xl0-'

RSD (%) (n=8)

7.00 x lO-'

6.96xl0-'

1.27

1.30

450 400 ~

350

~ 300

.S 250 ..J

~

~

;Ji

200 150 100

50

[Hydroxyl aminej x lO'6 (M)

Fig. 1. Analytical calibration graph for determination of hydroxylamine I (conditions:1.20 x lO- M hydrochloride acid, 5.00 x lO- 2M Na202 and 2.00 mLimin flow rate).

Flow Injection Chemiluminescence Determination of Hydroxylamine Hydrochloride

175

hydroxylamine hydrochloride (r = 0.9979, n=IO). The 3(} limit of detection 5 was 8 1.60x I 0- M hydroxylamine hydrochloride. The accuracy and precision for the analysis of eight replicates of a series of samples containing various concentrations of hydroxylamine are shown in Table I. Effect of various ions. In order to investigate the selectivity of this reaction, various cations and anions were tested. The results are given in Table 2, and indicate that the method provides good selectivity. Table 2. Effect of foreign species on the determination of 3.00x 10-6 M hydroxylamine (conditions: 3.00x 10-6M hydroxylamine, 1.20x 1O-1M hydrochloride 2 acid, 5.00x 1O- M Na202 and 2.00 mLimin flow rate). Species Na+, K+, Ca2+, Zn 2+, Pb 2+, y5+, Mn2+,Ba 2+,Bi3+,Fe 3 +, Mg2+, M0 6+, , P0 4 -3 - ,N0 3-,N0 2-, solo, Cr 20?,S2Sn L +, Cu H HgH, Ni L + Ag+,Cd 1 + C0 2+

Tolerance limit ratio a (moll mol) b 500

SOc 10C 10d 3°

5% relative error. Largest amount tested. d Caused increase in emission intensity

a

cCaused decrease in emission intensity.

b

Table 3. Determination of hydroxylamine added to water samples (conditions: 1.20x1O- 1M hydrochloric acid, 5.00x1O-2M Na202 and 2.00 mLimin flow rate). hydroxylamine concentration (M) Sample Drinking water

Recovery (%)

Found 6.93xI0-

Added 7.00xI02.00x1O-

6

4.00xl0-

6

2.03 x I0-

99.00 o

101.50

6

101.75

4.07x 10-

176

Baezzat MR & /z£ dpanah M

Application to water samples. Hydroxylamine was determined after addition to water samples. Table 3 shows the results, the recoveries being close to 100%, indicating that there is no serious interference in such water samples. CONCLUSIONS The proposed method, based on the CL reaction between hydroxylamine and Na202, provides a simple and sensitive approach for the determination of hydroxylamine. No sample pre-treatment is necessary and the procedure is very rapid using the FIA technique. The dynamic range of the method is 3.00xlO-7 - 5.00xlO-6 M hydroxylamine with detection limit of 1.60x 10- 8 M hydroxylamine. REFERENCES 1. Isacasson U, Wettermark G. Chemiluminescence in analytical chemistry. Anal Chim Acta 1974;68:339-62. 2. Isacasson U, Wettermark G. Chemiluminescence in analytical chemistry. Anal Chim Acta 1976;83:277-301. 3. Knight AW, Greenway GM. Relationship between structural attributes and observed ECL activity of tertiary amines as potential analytes for the tris(2,2bipyridine)ruthenium(Il) ECL reaction. Analyst 1996; 121: 101-6. 4. Safavi A, Baezzat MR. Flow injection chemiluminescence determination of pyrogallol. Anal Chim Acta 1998;368: 113-6 . 5. Miller JC, Miller IN. Statistics for analytical chemistry. Chichester:Ellis Horwood, 1986.

STUDY ON GOLD-SENSITISED CHEMILUMINESCENCE FOR THE DETERMINATION OF NORFLOXACIN

JUN-FANG BAO, ZHENG-HAI JIANG, XI-JUAN YU* College a/Chemistry and Molecular Engineering, Qingdao University a/Science and Technology, Qingdao 266042, China Email: [email protected]

INTRODUCTION Gold nanoparticles are one of the most widely used nanomaterials in recent decades. Gold NP catalysis for gas-phase' or liquid-phase 2-4 reactions is now an expanding area. Herein, we found that the CL intensity of Ce(IV)-Na2S03 catalyzed by gold NPs was strongly increased in the presence of norfloxacin (NFLX). This method has the advantages of simple equipment, high sensitivity detection, low background signals and low cost for the chemical reagents. EXPERIMENT AL Chemicals and solutions. NFLX was obtained from the National Institute for the Control of Pharmaceutical and Biological Products. All the reagents were of analytical grade, and solutions were prepared with de-ionized, distilled water obtained from SZ-93 automatic double-distilled water (Shanghai, China). Synthesis of gold colloids. HAuCl 4 AH 2 0 (47.8 % w/w) was obtained from Tianjin Yingda Sparseness & Noble Reagent Chemical Factory (Tianjin, China). The colloidal solutions of 5 ± 2 nm gold NPs were synthesized by the borohydride reduction method,S colloidal solutions of 30 ± 2 nm gold NPs were synthesized by the citrate reduction method." while the other colloidal solutions of gold NPs synthesized' were 3 ± 1, 12 ± 3, 17 ± 3 nm in diameter, respectively. The size and shape of the synthesized gold NPs were characterized by JEM-2000EX (Japan). CL measurements. The CL was conducted on a Remex IFFM-E client system (Xi'an Remex Analysis Instrument Co., Ltd.). De-ionized, distilled water was used as a carrier for Na,SO) to mix with Ce(IV) with the colloidal solution of gold NPs and NFLX of the optimized concentrations. The CL signals were monitored by a photomultiplier tube (PMT) adjacent to the flow CL cell. Spectral measurements. Fluorescence (FL) spectra and UV-Visible absorption spectra were measured on a model Hitachi-F4500 spectrofluorometer (Hitachi, Japan) and a model Cary 50 UV -Visible Spectrophotometer (Varian, USA), respectively. 177

178

Bao

al.

Colorimetric detection of NFL X in the presence (a) and absence nanoparticIes.

RESULTS AND DISCUSSION typical colorimetric detection of After mixing with 100 47.6 JlM gold NPs the color from red to purple. The color change of gold NPs by the attraction and attachment color change was visible, suggesting that the gold NPs are sensitive to presence ofNFLX. enhancement of NFLX CL. The effects of gold colloids of different on the Ce(IV)-Na,SO,-NFLX-NPs system were The was enhanced by the gold colloids, and the strongest CL colloids. mechanism. Ce(lV) could oxidize Na2S03 and produce energy the NPs that induced the transition of oxidized . When the energy state of the excited oxidized SO, * returned to the state of ground, it could transfer energy to state of NFL X and form NFLX*. The NFLX* then returned to state, with a CL emission. The CL might be attributed to the I).

Study on Gold-Sensitised Chemiluminescence for the Determination of Norfloxacin

179

Ce(lV)+ H80 J' - - -. ~ . H80 3 +Ce(lll)

G Au

2H80 3 '

..

8 Z0 6Z'+2H+

8 zol'

...

80/'+802 *

NFLX

---... ~

----~. ~ .

8 Au

8 _. Au

..

-NFLX

~:_

SO"

'-- NFLX NFLX * ~ + 80z-----...

"'" NFLX*

.NFLX

80,*

•

•

"::0"~

80z*'"

8

· ·-·

"'NFLX NFLX

Au"

+

h"

'-- NFLX

Scheme 1. Scheme of possible reaction mechanism.

Sample analytical detection. The proposed method was applied to detect NFLX spiked into urine. For values that were over the upper limit of the linear range, a dilution was required for analysis. In this work, the urine samples were diluted 10fold with water before analysis. Concentration of 27.6 Ilg/mL NFLX was achieved after the administration of a 400 mg dose drug by mouth for 1 h. In addition, a recovery test was performed by adding NFLX to the diluted urine samples. The recovery from urine, calculated by comparing the found value to the added value, is shown in Table 1. Many metabolites in human urine such as uric acid might interfere with the determination of NFLX. So the proposed method could assay the real sample by combining it with an highly effective separation technology such as HPLC or CEo

Sample 1 2 3

T a bl e 1 D etermmatlOn resu ts 0 f recovery 0 f UrIne n= 5) Recovery (%) Added (p.1g) Found (p.1g) R.S.D. (%) 99.0 30.0 29.7 1.5 40.0 100.7 0.7 40.3 49.1 98.2 50.0 1.1

CONCLUSIONS When NFLX was mixed with gold NPs, the CL intensity of Ce(IV)-Na2S03 increased dramatically. Under the optimum conditions, the CL intensity was proportional to the concentration of NFLX. So a method for NFLX detection based on the catalysis by gold NPs was developed and we also explored the possible CL mechanism of the system. The method exhibited good results in determination of NFLX in human urine.

180

Bao J-F et at.

ACKNOWLEDG EMENTS This research was supported by the National Natural Science Foundation of China (No. 20505004), and Doctoral Found of QUST (No. 0022235). REFERENCES 1. Wallace WT, Whetten RL. Coadsorption of CO and O 2 on selected gold clusters: Evidence for efficient room-temperature CO 2 generation. 1 Am Chem Soc 2002;124:7499-505. 2. Cui H, Zhang ZF, Shi MI. Chemiluminescent reactions induced by gold nanoparticles.1 Phys Chem B, 2005;109:3099-113. 3. Zhang ZF, Cui Hand Shi MJ. Chemiluminescence accompanied by the reaction of gold nanoparticles with potassium permanganate. Phys Chem Chem Phys 2006;8:1017-21. 4. Cui H, Zhang ZF, Shi MJ, Xu Y, Wu YL. Light Emission of gold nanoparticles induced by the reaction of bis(2,4,6-trichlorophenyl) oxalate and hydrogen peroxide. Anal Chem 2005;77:6402-6. 5. Wang L, Yang P, Li YX, Chen HQ, Li MG, Luo FB. A flow injection chemiluminescence method for the determination of fluoroquinolone derivative using the reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticles. Talanta 2007;72: 1066-72. 6. Maxwell DJ, Taylor JR, Nie S. Self-assembled nanoparticle probes for recognition and detection of biomolecules. 1 Am Chem Soc 2002;124:960612. 7. Wu ZS, Jianga JH, Fua L, Shen GL, Yua RQ. Preparation of oligonucleotidefunctionalized gold nanoparticles. Anal Biochem 2006;353:22-9.

CONJUGATES OF (ACRIDINIUM)x-BSA ANTI-HCV CORE TO ENHANCE THE DETECTION OF HCV CORE ANTIGEN CD CHANG, KY CHANG, L JIANG, VA SABLILLA, DO SHAH Infectious Disease Diagnostics R&D, Abbott Laboratories, Abbott Park, IL 60064 USA E-mail: [email protected]

INTRODUCTION Since its discovery in 1989, it has been demonstrated that the hepatitis C virus (HCV) is a major cause of chronic hepatitis throughout the world. HCV core Ag can be detected in the preseroconversion window period and HCV core antigen levels correlate well with HCV RNA levels. l Direct detection of HCV core antigen (A g) have been developed and these assays was showed useful in monitoring HCV therapy and early detection of patients with HCV infections. It has been suggested that the HCV Ag testing could be successfully used in situations or countries where nucleic acid testing (NAT) unavailable or unaffordable for the management of HCV? The HCV core Ag occurs at a very low level in pg or fmole per ml of specimen, especially during early seroconversion, that was barely detectable using directly labeled antibody (Ab) for the detection. 3 Sulfonamide acridinium derivatives have been utilized as signal labels for sensitive immunoassays because of their stability and high chemiluminescence yield. Two acridinium derivatives, N-sulfopropyl N-tosylamide acridinium [Acr or Acr(spcp)] and N,N -disulfopropy I N -p-(3 -carboxypropy l)pheny lsulfonamide acridin ium [Acr(spsp)], were used in our conjugates. 4 Their structures and chemiluminescence reaction mechanism are illustrated in Fig 1.

Acr: R

=

Ar~

carboxypropyJ

Phenyl

Acr(spsp): R ~ Sulfopropyl Ar ~ p.(3·Carboxypropyl)phenyl

. ,

I

["yN~

~ o

Fig. 1. Structures of two sulfonamide acridinium derivatives and the chemiluminescent reaction mechanism. 181

182

Chang CD et al.

Signal response of conjugate usually goes up with higher loading of labels. Due to alternation or steric hindrance of binding sites in random labeling of antibody, the label/Ab molar ratio of directly labeled antibody, generally, has an optimal limit about 5 or less. Detrimental effects to assay sensitivity and specificity were commonly observed in immunoassays using conjugates with a high molar ratio of label to Ab. Herein, we reported a new kind of conjugates having over 10 copies of acridinium label per Ab to achieve the sensitivity by many folds in the detection of HCY core Ag. MATERIALS AND METHODS Conjugates of Acridinium and MAb Anti-HCV core. Acr-cll-lO IgG (Control Conjugate). Monoclonal antibody (MAb) cll-IO anti-HCY core IgG was directly labeled with N-hydroxysuccinimide ester of N-sulfopropyl N-tosylamide acridinium (Fig 1) in a phosphate buffer. After conjugation, the conjugate was separated by secHPLC into fractions with Acr/IgG molar ratios ranging from 3 to 5 estimated by A370 and A 2S0 . Acr-BSA and activation with SMCC. Bovine albumin (BSA) was labeled with active ester of N-sulfopropyl N-tosylamide acridinium in a phosphate buffer. AcrBSA was separated from free acridinium through a disposable G-25 column. Multiple lots of Acr-BSA were made and their Acr/BSA ratios ranging from 10 to 25 were estimated by A370 and A 2S0 . Acr-BSA was activated with LC-SMCC active ester (Pierce Biotech, Rockford, IL, USA) in a phosphate buffer. The LC-SMCC linked Acr-BSA was separated from free SMCC with a G-25 column. Acr-BSA-cll-IO IgG. cll-IO IgG was reduced with dithiothreitol (OTT) briefly and separated from free OTT through a G-25 column. The reduced IgG was mixed immediately with fresh LC-SMCC linked Acr-BSA. After overnight reaction, the Acr-BSA-cll-IO IgG was isolated with sec-HPLC. Acr-BSA-cll-IO Fab'. cll-IO (Fab')2 was prepared from cll-IO IgG by pepsin digestion and sec-HPLC separation. Acr-BSA-cll-IO Fab' was made by following the procedure described above for Acr-BSA-cll-IO IgG. Acr(spsp)-BSA-cI 1-10 IgG. Three lots of Acr(spsp)-BSA was prepared similarly as Acr-BSA above and having Acr(spsp )/BSA ratios ranging from 15 to 28. Each of these Acr(spsp )-BSA was conjugated with c 11-10 IgG as above to yield the conjugate. Assay Reagents, Controls, and Samples in the PRISM HCV core Ag Assay. Solid-phase. Microparticles coated with MAb cI 1-7 or cll-14 anti-HCY core IgG. Specimen Diluent Buffer (SOB): SOB contained surfactants, blockers, and buffer salts to enhance specific binding and minimize nonspecific binding. Conjugate. Acridinium conjugates described above diluted to 50 to 100 ng/mL. Assay Controls. Negative Control is re-calcified negative human plasma. HCY core Ag Positive Control (AgPC) was diluted from a unit of HCY RNA positive I HCY Ab negative human plasma.

Conjugates of (Acridinium)x-BSA Anti-HCV Core

described

The HCV core assay was run on the Abbott 3,5 steps and reactions are illustrated in

183

as

HCVcore

+

SDB

Acridinylated Anti~c()re

Coupled

ABBOTT

HCV core Antigen

Format

assay starts with 50 fAl of sample and incubates with 50 anti-HCV core coated microparticles in the sample well of a After the first incubation, the tray is moved to the transfer station the reaction mixture is flushed into the sandwich reaction well buffer and excessive fluid is absorbed by a blotter underneath. In the acridinium anti-HCV conjugate is dispensed to the reaction well station. After the incubation, unbound of an alkaline hydrogen solution is then the blotter. 50 station to trigger chemiluminescence from acridinium in the reaction well. The intensity of chemiluminescent is to the amount ofHCV core Ag in the sample.

RESULTS AND DISCUSSION The of directly labeled cll-l 0 IgG conjugate was determined to be more copies of acridinium label per Ab were achieved BSA as a macromolecular carrier for indirect attachment of many the conjugation of Acr-BSA and DTT reduced cll-IO Ab with LC-SMCC away from the binding site at N-terminal domain of Fab' would preserve the

184

Chang CD et al.

Table 1. Comparison of conjugates for the HCV core Ag detection Coniw>ate Acr( spcp )-c 11-10 IgG Acr( spcp)-BSA -c 11-10 IgG Acr(spsp)-BSA-cll-IO IgG Acr(spcp)-BSA-cll-IO Fab'

Acr/Ab 3-5 10- 12 20 - 30 20 - 26

Range of PIN ratios 5-8 20 - 30 40 - 50 40 - 52

Solubility was a limiting factor for Acr-BSA-c1I-IO IgG in the conjugation of cll10 IgG with Acr-BSA having Acr/BSA ratios over 14. However, more than 20 copies of acridinium label per Ab were achieved in the preparation of Acr(spsp)BSA-cll-IO IgG and Acr(spcp)-BSA-c11-10 Fab' (Table I). These results are attributed to better solubility of either Acr(spsp) over Acr(spcp) or Fab' over I~G. Each conjugate has been evaluated in multiple lots many times in the PRISM HCV core Ag assay. The test results were summarized in Table I. Conjugates of AcrBSA-c11-10 showed significantly better sensitivity, at least 4 to 6-fold increases in PIN ratios, compared to the control conjugate of Acr-cll-l0 IgG in the assay. Apparently, the sensitivity increase correlates with the Acr/Ab ratio. Higher loading of acridinium labels to Ab was achieved through an indirect labeling strategy using BSA as a carrier.

REFERENCES 1.

2.

3.

4.

5.

Lorenzo J, Castro A, Aguilera A, et al. Total HCV core antigen assay. A new marker of HCV viremia and its application during treatment of chronic hepatitis C. JViroIMethods2004;120:173-7. Seme K, Poljak M, Babic DZ, Mocilnik T, Vince A. The role of core antigen detection in management of hepatitis C: a critical review. J Clin Virol 2005 ;32 :92-101. Muerhoff SA, Jiang L, Shah DO, et al.. Detection of hepatitis C virus core antigen in human serum and plasma using an automated chemiluminescent immunoassay. Transfusion 2002;42:346-56. Adamczyk M, Chen YY, Mattingly PG, Pan Y, Rege S. Neopentyl 3trifly loxypropane sulfonate. A reactive sulfopropylation reagent for the preparation ofchemiluminescent labels. J Org Chern 1998;63:5636-9. Shah DO, Stewart J. Automated panel analyzers PRISM.® In: The Immunoassay nd Handbook - 2 Edition, Wild D, Ed. London:Nature Publishing, 2001 ;297-303.

CHEMILUMINESCENCE DETERMINATION OF RUTIN BASED ON A MICELLE-SENSITIZING N-BROMOSUCCINIMIDE-H 20 2 REACTION JX DU,' L HAO,'" JR LU' 'Key Laboratory ofAnalytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xi'an 710062, China: 2Department of Chemistry, Shaanxi University of Technology, Hanzhong 723001, China Email: [email protected]

INTRODUCTION Rutin is an active component of many Chinese medicinal herbs, It has been known to have some therapeutic and pharmacological activities and usually used as an oral capillary protective drug for the prevention and cure of cardiac blood vessel disease,' It also has potential pharmacological activities in anticarcinogesis,' Therefore, the determination of rutin is of importance for the treatment of the diseases, the study of plant physiology, and the improvement of the plant quality, Various techniques have been reported for the determination of rutin, including spectrophotometry, fluorimetry, electrochemical method, chromatography and capillary electrophoresis, Chemiluminescence (CL) has also been exploited for the determination of rutin, which were based on luminol reaction,],4 K3Fe(CN)3 reactionS and KMn04 reaction, 6 Rutin is found to react with N-bromosuccinimide (NBS) and H 2 0 2 to produce weak CL in alkaline medium, The CL signal is enhanced significantly in the presence of cetyltrimethylammonium bromide (CTMAB) surfactant micelles, The experimental conditions affecting the CL reaction were optimized and a new FI-CL method for the determination of rutin was developed, The proposed method was applied to the determination of rutin in traditional Chinese medicinal herbs with satisfactory results. MATERIALS AND METHODS Apparatus. Fig, I depicts the schematic diagram of CL flow system used, which consists of a peristaltic pump, a six-way valve and a CRI05 photomultiplier tube (Beijing Hamamatsu Photo Techniques Inc.). PTFE tubing (0.8 mm id) was used to connect all components in the flow system, CL data acquisition and treatment were performed using IFFL-D data processing system (Xi'an Remex Eletronic Science-Tech Co, Ltd.). CL spectra were obtained by means of a series of cut-off filters (Institute Biophysics Chinese Academy of Science). Fluorescence spectra were taken on a CRT-970 fluorescence spectrophotometer (Shanghai 3rd Analytical Instrumental Plant). Absorbance spectra were measured with a TU-190 1 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.). 185

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Chemicals. All chemicals used were of analytical grade; doubly distilled water was used throughout. Rutin was obtained from the National Institute for the Control of Pharmaceutical and Biological Products of China. Other chemicals were the products of Xi'an Chemical Plant. Rutin stock solution (500 mg/L) was prepared in methanol. Working solutions were freshly prepared by the dilution of the stock solution with water. NBS solution (0.01 mollL) and H 2 0 2 solution (0.1 mollL) was prepared in water. CTMAB solution (0.2 %) was prepared in pH of 12.5 NaOH solution. Procedure. Flow lines were connected with rutin standard/sample solution, NBS solution, CTMAB solution and H 2 0 2 solution, respectively. The CL signal was measured by injection 50 ilL of rutin solution into the merged stream of NBS solution with CTMAB solution by means of the six-way valve, which then mixed with H 2 0 2 stream to produce CL signal. The concentration of rutin was quantified by the CL intensity (peak height). Rutin

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RESUL TS AND DISCUSSION Optimization of experimental conditions. Preliminary experiments showed that the weak CL signal was generated from the reaction of rutin, NBS and hydrogen peroxide in alkaline solution. To increase the sensitivity, the following surfactants were tested: CTMAB, cetylpyridinium bromide, sodium dodecyl sulphate, Tween 80 and polyethylene glycol 400. It was found that CTMAB was the most effective surfactant, giving a maximum enhancement on the CL signal. The CL signal increased with increasing CTMAB concentration up to 0.2 %. Above this concentration, the CL signal changed slightly. The 0.2 % CTMAB corresponds to 5.5 mmollL, which is above the critical micelle concentration of CTMAB (0.92 mmollL). The CL signal was strongly dependent on the pH of reaction medium. No CL signal was recorded when the pH of reaction medium was below 12. A maximum CL signal was achieved at pH 12.5. The effect of NBS concentration on the CL reaction was examined in the range of 0~0.05 mollL. No CL signal was detected in the absence of NBS. The maximum CL signal was achieved when using 0.01 mollL NBS. The CL signal increased with increasing H 2 0 2 concentration up to 0.1 mollL. Above 0.1 mollL the CL signal remained almost constant.

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The CL signal continued to increase with increase in flow rate in the range of 0.7-3.5 mLimin. Finally, 2.S mLimin was employed by the considering the sensitivity, reagent consumption and reproducibility. Linear range, detection limit and precision. Under the selected conditions, the calibration graph of CL signal (I, au) versus rutin concentration (C, mg/L) was linear in the range of 0.1-10.0 mg/L. The regression equation is J= IS.54 C + 7.59 and the correlation coefficient is 0.9959 (n= 10). The detection limit is 0.07 mg/L rutin and the relative standard deviation for 1.0 mg/L rutin solution is 2.2% (n= 11). Interference. The effect of foreign substances was investigated by using 6.0 mg/L rutin standard solution. The tolerable limit was taken as the amount that caused an error of ±5% in peak height. No interference was found when including up to 1000-fold glucose, lactose, starch, 100-fold sot, cr, PO/", N0 3', 50-fold Zn 2+, IO-fold AI3+, ci+, Mg2+, SO/·, cysteine, 0.5-fold quercetin, O.l-fold Pb 2+, and O.OI-fold Cu2+, C0 2+, Fe 2+, Mn2+. Transition metal ions did not interfere with the determination of rutin in sample since the sample solution was a methanolic extract. Determination of rutin in Chinese medicinal herbs. The Chinese medicinal herbs samples were treated similar to the procedure described in the Chinese pharmacopoeia' and determined by the proposed method. The results are summarized in Table 1. Recovery studies were also carried out on the samples to which a known amount of rutin had been added. As can be seen from Table 1, the recoveries of added rutin were quantitative. Table 1. Results for the determination of rutin Samples Added Found (mg/L) (mg/L) Flower-buds of 0 0.97 Sophora 1.62 0.6 japonica 2.01 1.0 3.19 Flowers of 0 Sophora 3.S0 0.6 japonica 4.20 1.0 0.44 Forsythia 0 1.0S 0.6 14.66 1.0 1.24 0 Hawkthorn 1.79 0.6 2.21 1.0

in Chinese medicinal herbs Recovery Content±SD (%) (mg/g) (N=5) 236.3±5.4 IOS.I 103.9 7S.6±0.5 101.7 9S.0 10.6±0.5 106.7 101.l 25.7±0.5 91.7 97.0

Possible CL reaction mechanism. No obvious difference has been observed between the UV spectrum taken from rutin alone and that from rutin-CTMAB mixture, which indicated no reaction occurred between rutin and CTMAB. In the UV spectrum of the reaction mixture, the absorption peak of rutin at 350 nm

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disappeared and a new absorption band with an absorption peak at 400 nm emerged, which indicated that rutin participated in the CL reaction. No fluorescence spectrum was recorded for rutin solution alone probably because of its low fluorescence quantum efficiency. The fluorescence spectrum recorded for rutin-CTMAB mixture had one strong fluorescence peak (t...ex=425.6 nm, t...em=537.9 nm), which indicated the presence of CTMAB surfactant micelles could enhance the fluorescence quantum efficiency of rutin. The fluorescence spectrum of the reaction mixture also had one strong fluorescence peak (t...ex=461.3 nm, t...em=539.0 nm), which suggested that the reaction product of rutin was a strongly fluorescent compound. The CL spectra of the reaction with and without rutin was examined in the range of 400-700 nm by means of a series of cut-off filters. When rutin was absent, no CL spectrum was obtained from the weak CL from NBS-H 20 2 . The more intense CL spectrum in the presence of rutin showed one peak band, 490-640 nm, which coincided with the fluorescence spectrum of the reaction mixture. Therefore, it was proposed that the emitter in the present reaction is the excited state of the reaction product of rutin. REFERENCES I. Society of Pharmacopoeia of Hygiene Department. Chinese Pharmacopoeia (Part I): Chemical Industry Press, Beijing, 2000. 2. Mahmoud NN, Carothers AM, Grunberger D, et al. Plant phenolics decrease intestinal tumors in an animal model of familial adenomatous polyposis. Carcinogenesis 2000;5 :921-7. 3. Han SQ. Capillary electrophoresis with chemiluminescence detection of rutin and chlorogenic acid based on its enhancing effect for the luminol-ferricyanide system. Anal Sci 2005;21:1371-4. 4. He CX, Cui H, Zhao XV, Zhao HZ, Zhao GW. Determination of rutin by flow-injection inhibited chemiluminescence detection. Anal Lett 1999;32:2751-9. 5. Li BX, Liu W, Zhang ZJ. Ferricyanide chemiluminescence system for the determination of rutin. Chin J Anal Chem 2001;29: 428-30. 6. Costin JW, Barnett NW, Lewis SW, McGillivery OJ. Monitoring the total phenolic/antioxidant levels in wine using flow injection analysis with acidic potassium permanganate chemiluminescence detection. Anal Chim Acta 2003;499:47-56.

LUMINOL-DEPENDENT CHEMILUMINESCENCE INCREASES WITH FORMA TION OF PHENOTHIAZINE CATION RADICALS BY HORSERADISH PEROXIDASE VA HADJIMITOV A, 1 T TRA YKOV, 1 R BAKALOVA 2 1Dept of Physics and Biophysics, Medical University-Sofia 1421, Bulgaria, 2 Dept of Biophysics, Molecular Imaging Center, NIRS, Chiba 263-8555, Japan, Email:[email protected]

INTRODUCTION Horseradish peroxidase (HRP) is an archetypal heme peroxidase. It is a nonspecific enzyme used for studying the effect of various substances on HRP-catalyzed electronl z transfer reactions. • For the enzyme activity determinations, many substrates and types of methods are used. One of these methods is luminol-dependent chemiluminescence (CL). The combination of the enzyme HRP/hydrogen peroxide (HzO z) system and a chemiluminescent method of detection allows for information to be obtained both about the result of the process and its course. The results presented in this article are the continuation of our studies of side effects 3 ofphenothiazines. These neuroleptics are known for their numerous desired and also undesired side effects. Our investigations were carried out mainly using luminoldependent CL in reactive oxygen species (ROS)-containing systems, both in the presence and in the absence of cells. 4.5 The purpose of the present work is to study the effects of five phenothiazines: chlorpromazine (CPZ), promethazine (PMTZ), thioridazine (TRDZ), trifluoperazine (TFPZ) and levomepromazine (LVPZ) upon a HRPlHzOz/luminol system. There is data indicating that for some phenothiazines, i.e. CPZ, PMTZ, TRDZ, TFPZ, that cation radicals of these drugs are generated in the presence of HRPI H Z0 2 • These radicals are important, as they can cause inactivation of enzymes, and have other freeradical effects. ,7 There is no information about the effects of these phenothiazines on o HRPlH 20 z/luminol, and about the possibility of LVPZ + generation in a HRPIH 2 0 2 system. MATERIALS AND METHODS Luminol-dependent CL we detected with a CL LKB 1251 luminometer (Bioorbit, Turku, Finland) connected to a PC via serial interface, and MultiUse program veL 1.08 (BioOrbit) was used to collect data, HRP-H 2 02 assay. This method is based on HRPIHz0 2 /1uminoi reaction system. Each sample contained the following substances in 50 mmollL phosphate buffer, pH 7,0: 100 IlmollL luminol, 0,5 lUlL HRP enzyme and tested drugs at different concentrations from 0.05 to 50 IlmollL, or a buffer as a control. The reaction was started by introducing 50 ilL of HzO z solution (1 mmoIlL). Chemiluminescence was measured for 5 m in at 25°C. We calculated the chemiluminescence scavenging index - CL-SI (%), as a ratio of 189

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CL in the presence and in the absence of the same drug at each concentration.

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RESULTS The results are shown in Fig. 1 and Fig. 2. All phenothiazines, included in this investigation, enhanced the light emission induced by the HRP catalysed oxidation of luminol by hydrogen peroxide. The concentrations of the phenothiazines used was biologically relevant. CPZ, PMTZ and TFPZ enhanced CL 2.5 times at a concentration 50 ftmollL (Fig. 1). At a concentration 0.05 ftmol/L the enhancement effect was very small but the trend for an increase in CL was the same for all substances tested. For LVPZ and TRDZ the CL increase was much greater (Fig. 2). At a LVPZ and TRDZ concentration of 5 ftmollL, the CL increase was almost the same as in the other drugs at a concentration of 50 ftmollL. This shows that these two drugs are ten times more effective than the other drugs tested. ~

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We have now shown that in the HRP/ H 2 0 2/ luminol system with LVPZ that the LVPZ·+ cation radical is also generated, and that LVPZ is very effective in increasing CL. Since the formation of cation radicals occurs simultaneously with the CL increase, we presume that the two processes are related. Using chlorprothixene instead of chlorpromazine in the system did not cause a n increase in chemiluminescence (Fig. 3). Chlorprothixene has structure very similar to that of chlorpromazine, but does not allow the formation ofa radical as in the case of chlorpromazine. Fig. 3 shows the chemical structures of these two drugs. CPX causes 25% decrease in CL, the shape of the curve being similar to that of the control. In the case of CPZ, a delay is observed at the beginning of the process, followed by a rapid increase in CL. After the start of the reaction, caused by introducing H20 2 , there was a 5 s delay before CL was detected in the samples containing the control and CPX, while for CPZ a lOs delay was observed. It seems that this delay is a result of the CPZ effect on a certain stage of the enzyme reaction. We conclude that the chemiluminescence increase is related to the formation of phenothiazine radicals, as on one hand there is a relationship between the increase in chemiluminescence and the type of cation radicals, and on the other hand a small difference in structure inhibits the process. REFERENCES 1. Ryan 0, Smyth MR, Fagain CO. Horseradish peroxidase: the analyst's friend. Essays Biochem 1994;28:129-46. 2. Azevedo AM, Martins VC, Prazeres DM, Vojinovic V, Cabral 1M, Fonseca LP. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol Annu Rev 2003;9: 199-247. 3. Traykov T, Hadjimitova V, GoJiysky P, Ribarov St. Effect of phenothiazines on activated macrophage-induced luminol-dependent chemiluminescence. Gen Physio! Biophis 1997;16:3-14. 4. Hadjimitova V, Traykov T, Mileva M, Ribarov St. Effect of some psychotropic drugs on luminol-dependent chemiluminescense induced by 02-,OH, HOCL. Z Naturforsch 2002;57C;1066-71. 5. Hadjimitova V, Bakalova R, Traykov T, Ohba H, Ribarov St. Effect of phenothiazines on protein-kinase C - and calmodulin-dependent activation of peritoneal macrophages. Cell Bioi ToxicoI2003;19:3-12. 6. Gutierrez-Correa J, Krauth-Siegel RL, Stoppani AO. Phenothiazine radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase: enzyme protection by radical scavengers. Free Radic Res 2003;37:281-91. 7. Muraoka S, Miura T. Inactivation of cholinesterase induced by chlorpromazine cation radicals. Pharmacol Toxicol 2003;92: 100-4.

VARIETY OF CHEMILUMINESCENT METHODS FOR ANTIOXIDANT ACTIVITY: INVESTIGATION OF CRATAEGUS OXYCANTHA EXTRACT V A HADJIMITOV A, J T TRA YKOV, J R BAKALOV A 2 1Dept of Physics and Biophysics, Medical University-Sofia 1421, Bulgaria, 2 Dept of Biophysics, Molecular Imaging Center, NIRS, Chiba 263-8555, Japan Email:[email protected]

INTRODUCTION Free radical (FR) processes have been of condiderable interest for medical science in the last few decades. Chemiluminescent methods have been found to be suitable for the detection of reactive oxygen species (ROS) and reactive nitrogen species.' These methods are sensitive, fast, and make it possible to follow the development of the process. Many methods have been developed for FR registration in vivo and in vitro.2 They can be combined with model systems for generating ROS, in order to be used for testing the ability of a drug to influence FR processes. In the present work, a combination of three chemiluminescence (CL) methods is used, based on luminol-dependent CL for registering antioxidant properties of ethanol extract of Crataegus oxycantha leaves and flowers. Sources of the most reactive ROS - hypochlorite and hydroxyl radical were used, as well as a HRPIH z02 system. There is information that this plant favourably influences cardiovascular diseases and other conditions related to free-radical pathology.3.5 Regarding some other Crataegus species, information has been obtained that they show antioxidant activity in some model systems, which is one of the possible 6 explanations of the curative effect of these herbs. MATERIALS AND METHODS Luminol-dependent CL was detected using a LKB 1251 luminometer (Bioorbit, Turku, Finland). It was connected to a PC via serial interface, and MultiUse program ver. 1.08 (Bioorbit) was used to collect data. Preparation of the ethanol extract. Fresh flowers and leaves from Bulgarian Crataegus oxycantha (Rosaceae) were extracted with 95 % ethanol and were distilled in a vacuum rotating evaporator. The viscous residues yielded were dissolved in 50 mmollL potassium phosphate buffer. Iron dependent hydroxyl radical formation (assay I). The sample contained the following substances in ImL 50 mmol/L potassium phosphate buffer, pH 7.4: 50 3 !-lmollL luminol, 100 !-lmollL Fe +, 100 !-lmol/L EDTA, 100 !-lmol/L ascorbate, 500 !-lmollL H 2 0 2 and either the tested extract at the concentrations shown in Fig. I or a buffer as a control. The CL was measured using the "flash assay" option of the MultiUse program, every 50 milliseconds. NaOCI - generated hypochlorite (assay II). The sample contained the following substances in I mL 50 mmollL potassium phosphate buffur pH 7.4: 50 !-lmollL luminol, 60 !-lmollL NaOCI and the extract at the concentrations shown in Fig. I, 193

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or a buffer as a control. The CL was registered after addition of NaOCI USing the "flash assay" option of the MultiUse program, every 50 milliseconds. HRP-HzOz (assay III). This method is based on HRPiH 20 2/1uminol system. Each sample contained the following substances in 50 mmollL phosphate buffer pH 7.0: 50 IlmollL luminol, 0.5 lUlL HRP enzyme and extract at the concentrations shown in Fig. 2, or a buffer as a control. The reaction was started by introducing of 50 ilL ofH20 2 solution (1 mmoIlL). CL was measured for 5 min at 25°C. We calculated a chemiluminescent scavenging index - CL-SI (%), as a ratio of CL in the presence and in the absence of the extract at each concentration. Calculation of C-50. The value concentration that provide CL-SI = 50 % was termed C-50. C-50 was calculated by the data to the "sigmoid" model: CL-SI =100/[1 + lOB (lgC -Ig (C-SO))], 7 where B is the coefficient (slope) and C is the substance concentration.

RESULTS In order to measure the antioxidant properties of Crataegus oxycantha extract, we used luminol-dependent CL. Luminol is a suitable enhancer, because it reacts with all ROS. This allows an estimation of the effect of the extract on the system for ROS generation, as well as a comparing the effects observed in the model systems. Fig. 1 shows the results obtained with regard to OCI- and ·OH. These are the two most reactive ROS, which are biologically relevant, since they are generated during the development of a local oxidative stress in vivo. We found that the extract shows a strong scavanger effect in both systems. For CL registration we used "flash" assays, because as a result of the high reactivity of these radicals their life is very short, and the detection process lasts a few seconds. The CL curves need the same time interval to reach a maximum after the start of the reaction. Fig. I shows that the scavenger effect of the Crataegus oxycantha extract is stronger with respect to hypochlorite than with respect to hydroxyl radicals. When the extract concentration decreases, the scavenger effect decreases as well, the rate of decrease of the two ROS being different. To compare the effect of the extract in the whole concentration interval, we used the value of C-50 (see Materials and Methods). We obtained the following values: under 30 mg/L for the hypochlorite, and 120 mg/L for the hydroxyl radical. These concentrations are very small, which illustrates the great scavenging effectiveness of these ROS.

Variety of Chemiluminescent Methods for Antioxidant Activity

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Fig. 2 shows the results obtained in the HRPIH z0 2/1uminol system. The Crataegus oxycantha extract exhibited a very strong inhibiting effect upon the CL response. The C-50 value was outside the concentration interval investigated, and is below 30 mg/L. CL curves showed a strong inhibition of the enzyme reaction. In order to exclude the possibil ity that the effects observed were due to a quenching effect, we also measured the radicals in a photometric system, i.e. a system different from the CL one. In the photometric experiments, we did not observe a similar effect. Using the combination of these three CL methods we found that the extract from Crataegus oxycantha leaves and flowers has strong antioxidant properties with regard to some of the most active ROS. These properties of Crataegus oxycantha explain part of its favorable effects in the treatment and prophylaxis of diseases related to free radical pathology, such as the cardiovascular disease.

REFERENCES 1. 2. 3.

4. 5.

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Kricka LJ. Chemiluminescent and bioluminescent techniques. Clin Chern. 1991 ;37:1472-81. Allen RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 1986;133:449-93. Jayalakshmi R, Niranjali Devaraj S. Cardioprotective effect of tincture of Crataegus on isoproterenol-induced myocardial infarction in rats. J Pharm Pharmacol. 2004;56:921-6. Zhao B. Natural antioxidants for neurodegenerative diseases. Mol Neurobiol 2005; 31 :283-93. Zhang DL, Zhang YT, Yin JJ, Zhao BL. Oral administration of Crataegus flavonoids protects against ischemialreperfusion brain damage in gerbils. J Neurochem 2004;90:211-9. Chatterjee SS, Koch E, Jaggy H, Krzeminski T. In vitro and in vivo studies on the cardioprotective action of oligomeric procyanidins in a Crataegus extract of leaves and blooms Arzneimittelforschung. 1997;47:821-5. Traykov T, Hadjimitova V, Goliysky P, Ribarov St. Effect of phenothiazines on activated macrophage-induced luminol-dependent chemiluminescence. Gen Physiol Biophis 1997;16:3-14.

SIMULTANEOUS MULTIPLEX BIO- AND CHEMILUMINESCENT ENZYME IMMUNOASSAY FOR PCR PRODUCTS DERIVED FROM GENETICALLY MODIFIED PAPAYA KATSUTOSHI ITO,! YOKO TANAKA,! MASAKO MAEDA,! KEIKO GOMI/ SATOSHI INOUYE,3 HIROSHI AKIYAMA,4 HIDETOSHI ARAKA WA! I School 0/ Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan, 2 Kikkoman Co., 3 Chisso Co., Yokohama Research Center, 5-1 Okawa, Kanazawa-ku, 4National Institute o/Health Sciences, Japan INTRODUCTION We have developed simultaneous luminometric assays using aequorin (Aq), firefly luciferase (Luc) and horseradish peroxidase (HRP). We already established simultaneous bioluminescent assays with Aq and Luc.! In th is study, the measurement of HRP with luminol and H2 0 2 was followed by Aq and Luc assays. The proposed assay was simultaneous luminometric measurement of three enzymes in a single well. Furthermore, the proposed assay was applied to detect PCR products derived from genetically modified (GM) Papaya genes. Two GM Papaya-specific genes and the intrinsic papain gene were amplified by multiplex PCR. These PCR products were analyzed by bio- and chemiluminescent enzyme immunoassay (BCLEIA). The simultaneous BCLEIA could be distinguishable between GM Papaya and non-GM Papaya. MATERIALS AND METHODS Reagents. Recombinant aequorin (Aq) with a free cys residue was from Chisso Corporation (Yokohama, Japan). Thermostable biotinylated firefly luciferase (b-Luc) was obtained from Kikkoman Corporation (Chiba, Japan). Aq labeled anti-Dig Fab fragment and b-Luc/streptavidin complex were produced by previously reported.! HRP-labeled anti-DNP was purchased from LSL Co. (Tokyo). GM Papaya was included gene sequences of ~-glucuronidase (GUS), cauliflower mosaic virus35S promoter (CaMV35S) and nopaline synthase terminater (NOS). GM and non-GM Papaya were detected by PCR product of papain gene. For inspection of GM Papaya, detection of PCR products were regions between sequences of GUS and CaMV35S and between NOS and CaMV35S (Fig. 1). We used forward primers labeled with fluorescein (FITC). Reverse primers were labeled with digoxigenin (Dig), biotin and 2,3-dinitrophenol (DNP), respectively (Fig. 1). Primers were obtained from Sigma-Aldrich Japan Co. Simultaneous luminescent measurement of aequorin, biotinylated firefly luciferase and HRP. Aq, b-Luc and HRP solutions (5 ilL of each) diluted with 0.05 mollL HEPES-KOH buffer (pH 7.0) containing 5 mmollL EGTA and 10% Block Ace (Dainippon SumitomoPharma Co., Ltd., Osaka, Japan) were introduced 197

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to a 96-well white microtiter plate (Nunc, Denmark). CaCl z solution (50 ilL, 50 mmollL) in HEPES-KOH buffer (0.05 mollL, pH 7.0) was added; subsequently, the luminescence intensity of Aq was measured immediately for 1 s with a Centro LB960 luminescent reader (Berthold Technologies, Germany). Next, a 50 ilL aliquot of luminescent reagent for b-Luc (consisting of 40 mmollL ATP, 1.4 mmollL D-Iuciferin and 0.3 mollL MgS04 in 0.05 mollL HEPES-KOH buffer, pH7.0) was added to the same wells of the microtiter plate. Luminescent intensity of b-Luc was measured for 1 s following a delay of I s. The following step, 50 ilL of luminescent reagent of HRP which was 0.3 mmollL Tris-HCI buffer (pH 9.0) containing 3 mmollL luminol, 0.5 mmollL hydrogen peroxide, 1 mmollL p-iodophenol and 0.25% benzalkonium chloride was added to same well. Luminescent intensity of HRP was measured for 1 s after 7 min incubation at room temperature. Benzalkonium chloride and p-iodophenol were added to the luminescent reagent for HRP for inactivation of b-Luc and enhancement of luminescence of HRP, respectively. Simultaneous sandwich BCLEIA for PCR products. The principle of the proposed simultaneous BCLIA is shown in Fig. 1. DNA extraction of freeze dried non-GM Papaya and GM Papaya was performed with DNeasyTM Plant Mini Kit (QUlAGEN Sciences). The extracted gene concentration was adjusted 10 nglllL. Multiplex PCR of gene samples from non-GM Papaya, GM Papaya and positive control plasmid (NIPPON GENE CO., LTD., Tokyo) were conducted. Briefly, the reaction mixture (25 ilL) consisted of DNA (2.5 ilL; 10 ngIIlL), lOx buffer (2.5 IlL),dNTP mixture (4 ilL) and each primer (I ilL; 5 IlmoI/L), Pfu Turbo DNA polymerase (I ilL; 2.5 U) andHzO. Moreover, thermal cycling was performed at 95°C for 10 min as initial denaturation, followed by 30 cycles of denaturation at 95 °c for 0.5 min, annealing at 60°C for 0.5 min and extension at 72 °c for 0.5 min. Finally, incomplete PCR products were extended for 7 min at 72 DC. The PCR product solution was 8-fold diluted with 0.1 mollL Tris-HCI buffer (pH 7.0) containing 25% Block Ace, 0.05% Tween 20, 0.9% NaCI and 0.05% NaN 3 . The wells of the microtiter plate were coated with rabbit anti-fluorescein IgG which were 5 Ilg/mL in 50 mmol/L sodium bicarbonate buffer (pH 9.5). The solution was added 100 ilL to each well. The wells were then post-coated by adding 100 ilL of a I % water-soluble gelatin solution containing 0.05% NaN 3 • The plates were stored at 4°C prior to use. After washing the plate, 100 ilL of a PCR product solution were added to each well. The plate was incubated for 1 h at room temperature. After washing, ISO ilL ofb-Luc/streptavidin complex (lx10- 9 moIlL), Aq-Iabeled anti-Dig Fab fragment (495 ng/mL) and HRP-Iabeled anti-DNP (500-fold dilution) mixed solution was added and allowed to stand for 1 h at room temperature. The microtiter plate was re-washed and Aq, b-Luc and HRP activities were assayed by the simultaneous bio- and chemiluminescent method described above.

Simultaneous Multiplex Bio- and Chemiluminescent Enzyme Immunoassay

199

of the proposed simultaneous bioluminescent

RESULTS AND DISCUSSION simultaneous luminescent assay of Aq, b-Luc and the (blank + 3SD), which is similar to the result of the The intra-assay coefficients of variation each standard point of Aq were from 2.2 to 4.2%. The detection limit was 6.5 x 10- 19 mol/assay. The CVs of 8 with each standard point for b-Luc were from 2.6 to 4.9%. We added benzalkonium chloride to luminescent reagent of HRP for inactivation of because luminescence of b-Luc interfered with the measurement of HRP. The luminescence of b-Luc decayed after 6.5 min. The detection limit of 17 was 1.9 10- mol/assay. The intra-assay CVs of 8 with for HRP were from 2.4 to 7.9%. The CV values were obtained presence of all three enzymes and were similar to individual assay. The measurements of the three enzymes were completed in 13 minutes with a assay medium room temperature.

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Table 1. Determination of Non-GM Papaya, GM Papaya genes and Positive Control

Plasmid Non-GM Papaya

GMPapaya

(mean ± SD, n=12)

(mean ± SD, n=8)

Positive Control Plasmid (mean ± SD, n=8)

Detection primer (Aequorin)

2.6 ± 1.4

330.0 ± 114.1 *

335.7 ± 381.0*

Confirmation primer (b-Luc)

6.2 ±2.9

9.0±9.0

232.6 ± 195.4*

17.5 ± 7.9

13.0 ± 4.3

20.9 ± 5.23

Papaya intrinsic primer (HRP)

SIN ratio * Statistical significance was determined by Student's t test (p<0.005, vs. non-GM Papaya gene).

We determined multiplex PCR products of extraction from non-GM and GM Papaya, positive control plasmid utilizing the proposed BCLEIA. Signal-to-noise (SIN) ratio of PCR products of detection primer and confirmation primer obtained from positive control plasmid were 130-fold and 38-fold higher than compared with PCR products of non-GM Papaya, respectively. The SIN ratio of PCR products of papaya intrinsic primer from positive control plasmid and GM Papaya were same level as that of non-GM Papaya. Although the SIN ratio of PCR product of detection primer of GM papaya was significantly higher than that of non-GM Papaya, the SIN ratio of PCR product of confirmation primer of GM Papaya was slightly higher than that of non-GM Papaya. We analyzed the products of multiplex peR of GM Papaya by agarose gel electrophoresis. The PCR product of confirmation primer of GM papaya was not detected, and we believe that the sequence of the confirmation primer was not suitable for GM Papaya in the proposed multiplex PCR condition. ACKNOWLEDGEMENTS This research was partially supported by "High-Tech Research Center" Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2007-2009. REFERENCES 1. Ito K, Nishimura W, Maeda M, et al. Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase. Anal Chim Acta 2007 ;588:245-51.

EFFECT OF SUGARS ON ALUMINUM-INDUCED OXIDATIVE BURST AND CELL DEATH IN SUSPENSIONS OF TOMATO CELLS TKADONO, TKAWANO, TYUASA, MIWAYA-INOUE Faculty ofAgricAtlture, Kyushu University, Japan Graduate School of EnvironmentJI Engineering, The University of Kitakyushu, Japan Email:[email protected].)p

INTRODUCTION Aluminum ions (AI3+) have toxic effect on plants, and number of studies documented the toxic impact of A 13+ on roots, I hypocotyls,2 and germinating pollen. 3,4 It has been proposed that early effects of Ae+ toxicity at the root apex, such as those on cell division, cell extension or nutrient transport, involve the direct intervention of Al on 5 cell function. Model mechanisms of Ae+ toxicity has been proposed that Al stimulates the NADPH oxidase and induces the generation of superoxide (0,"') that triggers the influx of calcium ion (Ca2+). The resultant reactive oxygen species (ROS) 2 and cytosolic free Ca + concentration elevation may lead to development of 6 phytotoxicity. On the other hand, the protective effect of sugar such as trehalose under oxidative conditions was suggested in yeast. 7 ,s Trehalose, a nonreducing disaccharide in which two glucose molecule are connected in an a-I ,I-glycosidic linkage, is considered to be an important osmoprotectant that has unique abilities that protect biomolecules from environmental stresses. 9 ,10 In this study, we report the effect of sugars (fructose, glucose, sucrose and trehalose) on Ae+-induced cell death and 0," generation in tomato cell suspension culture using cell death detection dye, Evans blue and 020 -specific chemiluminescent probe, Cypridina luciferin analogue .. Then, the possible roles for trehalose in reduction of AI3+ toxic effects in plants are discussed. MATERIALS AND METHODS CLA (2-methyl-6-phenyl-3,7-dihydroimidazo[1 ,2-a]pyrazine-3-one) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). All other reagents were from Nacalai Tesque (Kyoto, Japan)Tomato (Solanum lycopersicum L. cv. Micro-Tom) suspension-cultured cells were prepared and propagated by the same methods of tobacco suspension-cultured cells." Briefly, the culture was maintained in Murashige-Skoog (MS) liquid medium (pH 5.8) containing 3% (w/v) sucrose and 0.2 f.,lg/mL of2,4-dichlorophenoxyacetic acid at 25°C with shaking on a gyratory shaker in darkness and subcultured once a week with a 6% (v/v) inoculum. The cells were harvested 4 days after subculturing, washed with fresh MS medium without sucrose, and resuspended in fresh MS medium supplemented with 100 mM sugar (fructose, glucose, sucrose and trehalose) as substitute for sucrose. After 12 h incubation at 25°C with shaking on a gyratory shaker in darkness, cells were used for the determination of cell death and induction of 0," generation induced by A1 3+ 201

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-induced cell death in the cell suspension culture was determined by the dead cells with Evans blue (0.1 %, w/v) by mixing and incubating the cells and the for 30 min. Then stained cells were observed under microscope For statistic analysis, 3 different fields of cells under the 50 cells to be counted) were acquired and stained cells were counted. After addition of 10 flM CLA, AICh solution (various concentrationc) was added to cell in plastic tubes placed in a luminometer (AB-2200, and oxidative burst was monitored by CLA-chemiluminescence, and "V,WAOC"r! as relative chemiluminescence units per fresh cell weight.

RESULTS AND DISCUSSION studied the effect of sugars on AI 3+-induced cell death in tomato cultured cells derived from dwarf tomato cultivar, Micro-Tom. exposing to could observe the induction of death determined with Evans blue on the exposure time (Fig. 1). Addition of fructose, glucose and sucrose inhibited the cell death induced by AI3+. In the presence of trpl",I,')Q -indueed cell death was drastically suppressed by 60% and 50%, in the exposure of I hand h, These results suggest that trehalose has -induced cell death than other sugars.

Effect of sugars on Ae+ -induced cell death in tomato cell culture ,increases in the the effect of sugars on production of 0,.- by chemiluminescence of CLA specifically reflecting the of O 2 were measured in tomato cell suspension culture that was treated with after incubation with each of the sugars (Fig. 2). Ae+ induced 0," step cell death induced by AI3+.6 Addition of AICb to tomato cell culture resulted in transient production of 0," that reaches to the maximal level after treatment. Then, AI3+-induced generation of 0,'· was shown manner (data not shown). These AI3+ manner in tomato cells were same in BY -2 tobacco cells. Treatment of sugars no effect on the timing of 02- generation after addition of Ae+ but reduced the

Effect of Sugars on Aluminum-Induced Oxidative Burst

203

maximal CLA-chemiluminescence peak represent 0," generation. Addition of glucose and sucrose slightly inhibited the 0," generation induced by AI3+. Exogenous addition of trehalose reduced 0," generation when exposed to A13+. the effect of trehalose on the inhibition of O 2'' generation was lesser than that on the -induced cell death. 1 mM

2. Effect of sugars on Ae+-induced 0," generation in tomato cell culture ROS are also known to cause damage to cell membranes and in some situation, to a extent than damage to proteins. In yeast, trehalose appeared to an role in protecting plasma membrane from oxidative damage. 7 In vitro trehalose interacts specifically with cis-double bond of unsaturated fatty bonding. 12 According to these reports, the ability of trehalose to ofROS to plasma membrane and cellular components indicated the reduce the antioxidant function of trehalose. The increased production of ROS is a key event in toxicity in 6,13 Treatment with antioxidants such as ascorbic acid effectively -induced cell blocked AICh-induced cell death,14 In this study, trehalose reduced death and Trehalose has higher ability of interaction with cell membrane than other sugars.12 Exogenous trehalose coating of plasma membrane may cause inhibition of contact of Ae+ with NADPH oxidase in plasma membrane and suppression of of NADPH oxidase, thus AI 3 +-induced 0," generation is reduced. In addition, exogenous addition of trehalose protects plasma membrane to This study suggested that trehalose acts a protectant from the environmental stresses-induced oxidative burst.

0,.-

ACKNOWLEDGEMENT This research was partially supported by the Japan Society for the Promotion of Grant-in-Aid for lSPS Fellows.

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REFERENCES I. 2.

3.

4. 5.

6.

7.

8.

9. 10. II.

12.

13. 14.

Ma JF. Role of organic acids in detoxification of aluminum in higher plants. Plant Cell Physio12000;41 :383-90. Ma JF, Yamamoto R, Nevin OJ, Matsumoto H, Brown PH. Al binding in the epidermis cell wall inhibits cell elongation of okra hypocotyl, Plant Cell Physiol 1999;40:549-56. Li GM, Qing SF, Zheng QY, Hua LZ, Fu SZ, Da YS. Does aluminum inhibit pollen germination VIa extracellular calmodulin. Plant Cell Physiol 2000;41:372-6. Konishi S, Miyamoto S. Alleviation of aluminum stress and stimulation of tea pollen tube growth by fluorine. Plant Cell Physiol 1983;24:857-62. Lazof DB, Goldsmith JG, Rufty TW, Linton RW. Rapid uptake of aluminum into cells of intact soybean root tips. A microanalytical study using secondary ion mass spectrometry. Plant PhysioI1994;106:1107-14. Kawano T, Kadono T, Furuichi T, Muto S, Lapeyrie F. Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulation in tobacco BY -2 cells. Biochem Biophy Res Commun 2003;308:35-42. Herdeiro RS, Pereira MD, Panek AD, Eleutherio ECA. Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress. Biochim Biophys Acta 2006; 1760:340-6. Flattery-O'Brien J, Collinson LP, Dawes IW. Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide. 1 Gen Microbiol 1993;139:501-7. Nuccio ML, Rodees D, McNeil S, Hanson AD. Metabolic engineering of plants for osmotic stress resistance. Curr Opin Plant Bioi 1999;2:128-34. Penna S. Building stress tolerance through over-producing trehalose in transgenic plants. Trends Plant Sci 2003 ;8:355-7 Kadono T, Yamaguchi Y, Furuichi T, Hirono M, Garrec IP, Kawano T. Ozone-induced cell death mediated with oxidative and calcium signaling pathways in tobacco Bel-W3 and Bel-B cell suspension cultures. Plant Signal Behavior 2006;1 :312-22. Oku K, Watanabe H, Kubota M, et al. NMR and quantum chemical study on the OH ... Jt and CH ... O interactions between trehalose and unsaturated fatty acids: implication for the mechanism of antioxidant function of trehalose. J Am Chern Soc 2003;125:12739-48. Boscolo PRS, Menossi M, Jorge RA. Aluminum-induced oxidative stress in maize. Phytochemistry 2003;62:181-9. Yakimova ET, Kapchina-Toteva VM, Woltering El. Signal transduction events in aluminum-induced cell death in tomato suspension cells. JPlant Physiol 2007; 164:702-8.

CHEMILUMINESCENCE DETERMINATION OF SPARFLOXACIN USING Ru(bipyh2+-Ce(IV) SYSTEM MM KARIM, 1 JH CHOI,2 SM ALAM, I SH LEE I JDepartment of Chemistry, Kyungpook National University, Taegu, 702-701, Korea 2Department of Chemistry, Andong National University, Andong, 760-749, Korea Email: [email protected]

INTRODUCTION Sparfloxacin (SPAX), 5-amino-I-cyclopropyl-7-(cis-3, 5-dimethyl-pi perazin-I-yl)-6, 8-difluoro-l, 4 -dihydro-4-oxoquinoline-3- carboxylic acid is a potent fluoroquinolone antibacterial agent. It is a broad spectrum antibacterial fluoroquinolone active against some microorganisms including Gram-positive and Gram-negative bacteria.' It demonstrates moderate activity against anaerobes and Mycobacteria, for which the quinolones in general have low activity.'·3 SPAX has been studied as a therapeutic drug. There is no official pharmacopeia method, so far been reported for the determination of SPAX. Analytical techniques to determine SPA X includes spectrophotometry,' microbiological assay,S and HPLC: Tris (2i -bipyridyl)ruthenium(II) has been used as the basis of CL detection of a wide range of compounds after oxidation to the ruthenium(III) complex. The analyte interacts with the ruthenium(III) complex reducing it to the ruthenium(II) complex in an excited state, which then emits CL as it returns to the ground state. In the present study, a flow injection procedure for SPAX determination with CL detection was proposed in which ruthenium(II) was oxidized by Ce(IV) solution. The CL emission intensity depended on the concentration of the analyte in the CL system. This work describes a relatively sensitive and rapid chemiluminescence method for SPAX determination based on tris(2i-bipyridyl)ruthenium(I1) without sample pretreatment process. EXPERIMENTAL Reagents. All solutions were prepared from analytical-reagent grade materials in twice-distilled water. SP AX was purchased from (Aldrich Co., Ltd. USA) and made up in distilled water. The Ru(bipYh 2+ solution (10. 2 moUL) was obtained by dissolving 0.3743 gm oftris(2,2' - bipyridal) ruthenium (II) (Fluka, Gillingham, Dorset, UK) in water and diluting to 500 mL. ·1 moUL Ce(JV) was made in I moUL H 2 S0 4 . Apparatus. A Spex (Edison, NJ, USA) Model FLIll spectrofluorimeter equipped with a coiled glass flow cell (1.0 mm i.d., 20 mm total diameter) was used for detecting and recording the CL intensity of the reaction product. The Spex OM 3000 program was used for data acquisition and data analysis. For the CL measurement, the light source of the spectrofluorimeter was switched off. The slit width of the emission monochromator was 0.25 mm. The high voltage for the photomultiplier tube (R 928, Hamamatsu, USA) was set to 950 V. Sample preparation. A total 20 t<;tblets of SPAX were accurately weighed, then 205

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ground and mixed well. An appropriate amount of SPAX equivalent to one tablet was accurately weighed and dissolved in water by sonication in a 200 ml volumetric flask and diluting with water to 250 ml. The dissolved sample was filtered through Whatman No.1 filter paper and diluted with water to volume to obtain the appropriate concentration for analysis. Right after the collection, 35 ml aliquots of urine samples from 5 volunteers were spiked with SPAX at variable concentration levels, in order to calculate the recoveries of the proposed method. From these pools, 1.0 mL aliquots were distributed to 0.5-ml Eppendorf and stored at -18°C until analysis.

RESULTS AND DISCUSSION Optimization of experimental variables. The influence of Ru(bipy) 3 2+ concentration on the sensitivity was studied with 3.0X 10-3 mollL ofCe(IV) and 1.0 x 10-2 mollL of H 2 S0 4, in the range of (2.0-8.0)X 10-3 mollL The results show that the CL signal increased with increasing ruthenium(II) concentration until4.0X 10-3 mollL and then decreased (Fig. I). Therefore, Ru(bipy)/+ concentration of 4.0X 10- 3 mollL was selected as optimum concentration. The influence of Ce(IV) concentration on the CL signal in the range of (1.0-8.0) x 10-3 mollL The results show that the sensitivity is increased with increasing Ce(IV) concentration up to 3.0X 10-3 moUL. The sensitivity is decreased at higher concentration. Then, 3.0xlO-3 mollL was selected as optimum concentration of Ce(IV). The influence of H2 S0 4 concentration on the sensitivity was studied in the range of 0.!-1.0xlO- 3 mollL H 2 S0 4 . The results show that the CL signal increased with increasing H 2 S0 4 concentration until LOX 10-2 mollL and then decreased. Hence LOX 10-2 mollL was chosen as optimal H 2 S0 4 concentration.

RU(biPY);Jl+ concentration, mM

Fig. 1. Effect of the concentration of Ru(bipyh 2+ on the CL intensity

Chemiluminescence Determination of Sparfloxacin

207

CL kinetic curve of the system. The CL intensity of Ru(bipy)/+-Ce(lV) system in the absence of and in the presence of SPAX were recorded batch wise with the emission monochromator using time base scanning, respectively and the obtained CL kinetic curves were shown in Fig. 2. The experimental results indicated that when Ce(lV) was added to the cell the reaction was initiated and that CL emission of the investigated system was weak but could be enhanced proportionally by the addition of SPAX into the ruthenium solution. The CL reaction for SPAX is faster and the intensity reached maximum at residence time of 47 s, after which the signal decreased slowly.

8-

.~ 5

.5

'.is.,0· b

ec

g

I

1.50.10'

C

'5

.2 .~

a

/\

1,00.'0'

~.oo.,o·

/

2.501110'

"

.

10

100

Time, second

Fig. 2. The CL kinetic curves of the systems. Ru(bipY)32+-Ce(IV)-H2S04 (a) and Ru(bipY)3 2+ -Ce(IV)-H 2S04-sparfloxacin

Analytical characteristics. Calibration curve was obtained for the determination of SPAX under the optimal experimental conditions. It gives a linear range from 7.0X 108 12 to 4.0X 10- mollL with the limit of detection (LOD) as defined by IUPAC, CLOD = 3 * Sb/m (where Sb is the standard deviation of the blank signals and m is the slope of the calibration graph) was found to be 1.2xIO- 12 mollL (s/n=3). The relative standard deviation of 1.0XlO-7 mollL SPAX was found to be 1.6% (n

=

11).

Analytical applications. The proposed method has been used to determine SPAX in urine samples from 5 volunteers. The samples were found to contain no SPAX, so they were spiked with SPAX at variable concentrations. Table 1 shows the results obtained in the determination of SPAX. As can be seen, the differences between the concentrations added and those found were small in all cases. Therefore, the proposed method can be reliably applied to real samples. The proposed method was applied to the determination of SPAX in pharmaceutical preparations. The results obtained and the labeled contents are given in Table 2. There were no significant differences between labeled contents and those obtained by the

208

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proposed method. Table 1. Analytical results in spiked urine Sample

Added (X 10-9 mollL) 2 4

1 2

Found (X 10-9 mollL) 1.98 3.82

Recovery (%) 99.0 95.5

Table 2. Determination of sparfloxacin in tablets Sample Sparfloxacin

Labeled amount (mg) 100 200

Amount found (mg) 98.09 199.92

Recovery (%) 99.09 99.96

CONCLUSION A simple and rapid chemiluminescence system was developed based on the enhancement effect of sparfloxacin on the CL peak from the reaction of Ru(bipyh 2+_ Ce(IV) for the determination of sparfloxacin. This proposed technique offering satisfactory dynamic range from 7.0X 10- 12 to 4.0X 10- 8 mol/L with the detection limit (30) 1.2Xl0- 12 mollL. This method was successfully used for the determination of SP AX contents in pharmaceutical formulations and in spkied urine. ACKNOWLEDGEMENTS This research was supported by Kyungpook National University Research Fund, 2007. REFERENCES 1. Goa K, Bryson H, Markham A. Sparfloxacin. A review of its antibacterial activity, pharmacokinetic properties, clinical efficacy and tolerability in lower respiratory tract infections. Drugs 1997;53:700-25. 2. Nakamura S, Minami A, Nakata K, et al. In vitro and in vivo antibacterial activities of AT-4140, a new broad-spectrum quinolone. Antimicrob. Agents Chemother 1989;33:1167-73. 3. Gidoh M, Tsutsumi S. Activity of sparfloxacin against Mycobacterium /eprae inoculated into footpads of nude mice. Lepr Rev 1992;63:108-16. 4. Marona H, Schapoval E. Spectrophotometric determination of sparfloxacin in tablets. J Antimicrob Chemother 1999;44:136-7. 5. Marona H, Schapoval E. Development of a microbiological assay for the determination of sparfloxacin in powder and tablets. InfTechnol 1998;9:251-4. 6. Marona H, Schapoval E. A high-performance liquid chromatographic assay for sparfloxacin. J Pharm Biomed Anal 1999;20:413-7.

FLOW INJECTION ANALYSIS WITH CHEMILUMINESCENCE DETECTION: DETERMINATION OF GATIFLOXACIN USING THE KMnOcFORMALDEHYDE SYSTEM MA KHAN, SM ALAM, SH LEE Department of Chemistry, KyungpookNational University, 702-701, Korea, Email: [email protected]

INTRODUCTION Gatifloxacin [1-cyclopropyl-6-fluoro-l,4 dihydro-S-methoxy- 7-(3-methyl-l-piperaz -inyl)-4-oxo-3-quinoline carboxylic acid], a new broad-spectrum S-methoxyfluoroquinolone antibacterial agent, has improved activity against Gram-positive and Gram-negative aerobic bacteria and atypical bacteria. I,2 It has activity against respiratory pathogens. Like other members of the family, it inhibits the bacterial enzymes DNA gyrase and topoisomerase IV. Gatifloxacin is excreted in urine. Several methods have been reported for the determination of gatifloxacin such as HPLC,3,4 fluorimetry,S spectrophotometry6 and HPTLC. 7 Chemiluminescence (CL) has become a powerful analytical tool due to its widespread application in various fields, high sensitivity, wide dynamic range and simple instrumentation. 8,9 We studied the sensitizing effect of gatifloxacin on the CL intensity emitted from the reaction of formaldehyde with acidic KMn04. A sensitizing effect of gatifloxacin on the CL emission is observed. Based on the sensitizing effect, a simple, rapid and sensitive flow injection CL method for gatifloxacin detection is proposed. To our best knowledge, this paper describes the first application of FIA-CL to the determination of gatifloxacin. This method is proved to be simple and less expensive in comparison to the above-mentioned techniques and at the same time, offers a good accuracy, high speed and precision. EXPERIMENTAL Instrumentation. FIA (Flow-injection analysis) assembly used in our work is shown in Fig.!. Two peristaltic pumps (Ismatec Model 404) were used to pump all solutions. Pump PI delivered carrier stream (H 20) at a flow rate of 2.5 mUmin which was incorporated with sample solution in a Rheodyne (Cotati, CA, USA) Model 7125 six-way injection valve with a loop, while pump P2 was used to pump all other CL reagents at an equal flow rate of 2.5 mUmin for each line. PTFE tubing (O.S mm id) was used throughout the manifold to carry all components. A Spex (Edison, NJ, USA) Model FLIll spectrofluorimeter equipped with a coiled glass flow cell. (1.0 mm id, 20 mm total diameter) was used for detecting and recording the CL intensity of the reaction product. The Spex DM 3000 program was used for data acquisition and data analysis. For the CL measurement, the light source of the spectrofluorimeter was switched off. The slit width of the emission monochromator 209

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Khan MA et al.

was 0.25 mm. The high voltage for the photomultiplier tube (R 928, Hamamatsu, USA) was set to 950 V. Origin version 6.0 has been used for data processing. Sample (Glltiflox\\(ill) \, CarrieI'

Injection valve

--+-~-+-----(

PI

TZ

FlowceU

KMlIO" Emission ~1ono('hromator

HC'HO

P

z FluOIimetel'

Fig. 1. Schematic diagram of the FIA CL manifold employed for the quantitative determination oflevodopa. PI, P 2: Peristaltic pumps; T" T 2: Y- pieces Reagents. All solutions were prepared from analytical-grade reagents. Gatitloxacin preparations were obtained from local pharmaceutical industries and were used as reference standards without further purification. Stock solution of KMn04 (0.01 mol/L) was prepared by dissolving 0.395 g of KMn04 (Merck) in water and diluting with water to 250 mL. The solution was kept in amber-coloured bottles in the dark. Working solution was prepared by diluting the stock solution in 1 xl 0- 3 mollL sulfuric acid. The solution of 1.0 x 10 -2 mollL sodium sulfite was prepared daily. RESUL TS AND DISCUSSION Kinetic characteristic of the CL reactions. The CL kinetic curves of KMn04-Na2S03-H2S04 and gatitloxacin-KMn04-Na2S03-H2S04 systems are shown in Fig 2. KMn04-Na2S03-H2S04 system has an ultra - weak CL. Adding gatitloxacin into the system enhances the CL intensity.

Flow Injection Analysis with Chemiluminescence Detection

211

1.0x10·

'"r:l. u .~

8.0x10'

.5I:j

6.0X10'

'"!.i

c I:j

~

4.0x10

-=.~

2.0x10'

's

l

..c

U

400

450

500

550

600

650

W 8velcngth, nm

Fig. 2. CL spectra ofKMnO c HCHO-H 2S0 4 (a) and gatifioxacin-KMnOcHCHO-H2S04 (b) Optimization of experimental variables. The effect of KMn04 concentration on the CL reaction was examined in the range of 1 x 10.3 -7x 10.5 mollL. The CL signal rapidly increased with increasing in KMn04 concentration up to 2.1 X 10.4 mollL. Larger concentrations resulted in a decrease of emission intensity. Therefore, 2.1 xl 0.4 mollL KMn04 was chosen for subsequent experiments. The effect of sulfuric acid has been examined in the range 1xl 0. 3-7x 10. 5 mollL. CL intensity was found to be maximum when the acid concentration was 5 x 10.3 mollL. Therefore, 5 x 10.3 mollL was chosen as sufuric acid concentration for subsequent experiments. The effect of the concentration of HCHO on the CL emission was studied in the range 2-18% (v/v). The peak heights increased with increasing formaldehyde concentration over the range 1-5% (v/v), above which the signal decreased gradually. Therefore, 5% (v/v) formaldehyde was chosen for further experiments. The influence of flow rate on determination was examined by investigating the signal-to-noise ratio (SIN) at different flow rates. A flow rate of 2.5 mllmin gave the highest SIN ratio and was then chosen based on the analytical precision. Analytical characteristics. Under the optimum conditions, the average value of relative CL intensity was linear with the concentration of gatifloxacin from 1.0 x 10- 8 to 3.0 x 10-5 mollL and three replicate injections were performed for each standard solution. The relative standard deviation for 11 repetitive determinations of 1 x 10.7 M gatifloxacin was 2.1 % showing a good reproducibility. The detection limit (3 0 ) of th is method is 1.1 x 10-9 mollL.

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CONCLUSION Based on the chemiluminescence reaction of gatifloxacin, formaldehyde and potassium permanganate in sulfuric acid medium, a simple, rapid FIA-CL method has been developed for the sensitive determination of gatifloxacin. ACKNOWLEDGEMENTS The support of this research by Korea Research (KRF-2004-005-C00009) is gratefully acknowledged.

Foundation

Grant

REFERENCES 1. Kearn SJ, Croom KF, Keating GM. Gatifloxacin: a review of its use in the treatment of bacterial infections in the US. Drugs 2005;65:695-724. 2. Perry CM, Barman lA, HM Lamb. Gatifloxacin. Drugs 1999; 58: 683-96.Able D, Campbell 1. eds. New instruments for light detection. New York: Special Publishers, 2001 :45-8. 3. Borner K, Hartwig H, Lode, H. Determination of gatitloxacin in human serum and urine by HPLC. Chromatographia 2000;52:S 105-7. 4. Liang H, Kays MB, Sowinski KM. Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin, trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin determination in human plasma. J. Chromatogr B 2002;772:53-63. 5. Lian N, Sun CY, Zhao He. Fluorimetric determination of gatifloxacin. Fenxi Ceshi Xuebao 2002;21 :79-80. 6. Amin AS, Ayman AE, Ragaa E, Faten Z. Spectrophotometric determination of gatitloxacin in pure form and in pharmaceutical formulation. Spectrochim Acta A 2007:67 A: 1306-12. 7. Shah SA, Rathod IS, SUhagia BN, Baldaniya M. A simple and sensitive HPTLC method for estimation of gatifloxacin in tablet dosage forms. Indian J Ph arm Sci 2004;66:306-8. 8. Zhang YF, Cai XL, Yu JS, Ju HX. Flow injection chemiluminescence nalysis for highly sensitive determination of noscapine. Photochem Photobiol A: Chern 2004;162:457-62. 9. Townshend A, Murillo Pulgarm lA, Alanon Pardo MT. Flow injection-chemiluminescence determination of propranolol in pharmaceutical preparations. Anal Chim Acta 2003;488:81-8.

DETERMINATION OF CIPROFLOXACIN IN PHARMACEUTICAL FORMULATION BY CHEMILUMINESCENCE METHOD MA KHAN, SH LEE, SMALAM, SM WABAIDUR, HY CHUNG Department o/Chemistry, Kyungpook National University, Taegu 702-701, Korea Email: [email protected]

INTRODUCTION Ciprofloxacin (CPLX) (a second-generation fluoroquinolone) is the most potent fluoroquinolone against Gram-positive and Gram-negative bacteria through inhibition ofNAD gyrase, a critical enzyme ino bacterial chromosome replication. It is used in a wide range of gastrointestinal, urinary, respiratory tract, ocular and skin infections and it is particularly active against Pseudomonas aeruginosa. I Therefore it is necessary to arrange sensitive and fast methods for determ ination of th is antibacterial agent. Numerous methods have been reported for the determination of ciprofloxacin using techniques such as spectrophotometry,' fluorimetry,'" high-performance liquid chromatography (HPLC),5.6 and chemiluminescence.' In this work we described a batch type chemiluminescence method for the quantitative determination of CPLX in pharmaceutical formulation. It was observed that CPLX could enhance the chemiluminescence (CL) emission Ru(phen)/+-Ce(IV) system and this enhancement effect was dependent on the concentration of CPLX, based on which, CL system was established for the determination of CPLX in pharmaceutical formulation. EXPERIMENTAL Materials CPLX (Aldrich Co., Ltd. USA) was directly dissolved in distilled water to prepare stock solution - final concentration of 5.00 X lO-3g/L and stored at 0-5 °c. The solution of Ru(phen)/+ (Aldrich, USA) (10-2 mollL) was obtained by dissolving 0.3743 g of Ru(phen)/+ (Fluka, Gillingham, UK) in water and diluting to 500 mL. CeS04 1 mollL (Aldrich) was made in I mollL H 2 S0 4 (Duksan Pure Chemical Co. Ltd. Korea). Apparatus A Spex (Edison, NJ, USA) Model FL111 spectrofluorimeter was used to accomplish the batch type chemiluminescence measurements. An Ismatec Model 404 peristaltic pump was used to convey the one CL reagent for initiation of the reaction. During the measurements, the light source of the excitation monochromator was switched off. Slit width of emission monochromator was fixed with 0.25 mm. The photomultiplier tube (PMT) used was a Hamamatsu Model R 928 (Hamamatsu, USA) powered at 950 V. Spectra data were collected by Spex OM 3000 spectroscopy computer. The instrument layout is shown in the Fig. 1. 213

214

Khan MA et al.

5

H.V

Fig. 1. Schematic diagram of batch chemiluminescence for ciprofloxacin 1. Peristaltic pump, 2. Light-tight housing, 3. Reaction cell, 4. Magnetic stirring bar,S. Emission monochromator, 6. Photomultiplier tube, 7. Amplifier, 8. Computer. RESUL TS AND DISCUSSION Effect of Ru(phenh2+ concentration on the CL intensity. With the solutions containing a variable amount of Ru(phenh 2+ from 2.0 x 10-4 to 1.0 x 10-3 mol/L, 4.0 3 4 x 10- mollL CPLX, and 2.0 x 10- mollL Ce(IV) (I mollL H 2 S0 4), (all cell concentrations) the effect of the concentrations of Ru(phen)3 2 + on the system was investigated by determining the CL intensity of Ru(phenh 2+_ Ce(IV) system (blank) and Ru(phen)3 2+_ Ce(IV)- CPLX. The experimental results showed that with the concentration of Ru(phenh 2+ increasing, the chemiluminescence intensity increased from 2.0 x 10-4 to 1.6 x 10-3 mol/L. The phenomenon may result due to the rapid chemiluminescence reaction kinetics because of the increased reagent to analyte radical ratio. The CL intensity then started to fall off from 1.6 x 10-3 to 4.0 x 10-3 mol/L with substantial increase in the blank value. Therefore the optimum concentration of Ru(phen)3 2+ is 1.6 x 10-3 mollL with best signal to background ratio. Effect of Ce(S04h concentration on the CL intensity. Ceric sulfate being a non luminescent and strong oxidizing agent was utilized as the oxidant in this CL system. The effect of Ce(S04)z concentration on CL intensity was studied over the range 6.0 x 10-5-1.8 x 10-2 mollL. Results shows that the maximum intensity was obtained when the concentration of Ce(S04)z was 2.0 x 10-3 mol/L Effect of H 2S04 concentration on the CL intensity. The chemiluminescence intensity depends on the concentration of H 2S04. The experiment was performed in the range of 1.6 x 10-2-0.2 mollL H 2 S04 (cell concentration) under the standard conditions mentioned. The maximum intensity reached at 0.1 mollL H 2S0 4. When the H 2 S04 concentration was above this level, the light intensity started to decrease up to 0.2 mollL H 2 S04 (cell concentration). Effect of mixing order of reagents on the CL intensity. In the batch system the chemiluminescence intensity was influenced by the mixing order of the reagents into the reaction cell. It has shown that the chemiluminescence intensity was the highest when Ru(phen)3 2+ and CPLX were added into the reaction cell at first, mixed well,

Determination ofCiprofloxacin in Pharmaceutical Formulation

215

and then Ce(IV) was injected after lOs interval. The major effect is caused by the oxidant. CL kinetic curves of the systems. The CL kinetic curves of Ru(phen)/+- CPLX -Ce(IV) s1':-stems were constructed with the recorder,. which are shown in Fig. 2. Ru(phenh -Ce(IV) system has an ultra-weak CL. Addmg CPLX into the system can largely enhance its CL intensity and the value of enhancement is proportional to the concentration of the substance added. By this property, CPLX can be determined sensitively with CL method. 3 $1111'

r

i

30x,,,'f-

f 1.~.10·

1t·

1.S.r10'

~

1.fh(10'

...

<.;

Time (.)

Fig. 2. CL time profile for Ru(phen)/+ -Ce(IV)-ciprofloxacin system Calibration and detection In order to obtain a calibration curve for CPLX, a series of standard solutions (N=IO) of CPLX were added to the Ru (phen)/+ -Ce(IV) system under the optimized experimental conditions: [Ru (phen)3 2+]=6.7 x 10-4 moUL, [Ce(IV)]=2.3 x 10-3 mol/L, 2 [H 2 S0 4]=3.3 x 10- moUL and the chemiluminescence were recorded. The experimental results show that under the optimum conditions noted above, the responses of CL intensity are linear to the concentrations of CPLX in the range of 7 1.0x10- to 1.0x10-4 mo\lL. The linear regression equation is Y = 37887.72 log X65701.13 (R=0.99732, where X is the concentration of CPLX expressed in /lg/mL and Y is the chemiluminescence intensity in cps unit). The limit of detection (LOD) as defined by IUPAC, CLOD = 3 * Sblm (where Sb is the standard deviation of the blank 8 signals and m is the slope of the calibration graph) was found to be 2.0x 10- mollL. 4 The relative standard deviation (RSD) for 10 repeated measurements of 1.5 x 10moUL CPLX was 1.30%. Analytical applications. The proposed method was applied to the determination of CPLX in pharmaceutical preparations. The results obtained and the labeled contents are given in Table I. There were no significant differences between labeled contents and those obtained by the proposed method. Recovery studies were also performed on each of the analyzed samples by recommended treatment. Recoveries ranged from 96.0 to 100.5%.

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KhanMAetal.

Table 1. Analytical results for the determination of ciprofloxacin in tablets (n=5) Sample

CPLX

Labeled (mg)

Found (mg)

Added (10- 7 mollL)

Found (10- 7 moI/L)

Recovery (%)

200.0

197.6

2.00

1.92

96.0

4.00

3.97

100.2

CONCLUSION The proposed chemiluminescence detection method has proved to be simple, rapid and sensitive for CPLX determination. The results indicated that the proposed chemiluminescence reaction system is not only appropriate for batch type system analysis but it may also be convenient for flow injection system due to intense chemiluminescence signal. Utilizing the proposed method, the CPLX content of commercial tablets as well as in urine sample can be determined with reasonable selectivity . ACKNOWLEDGEMENTS This research was supported by Kyungpook National University Research Fund, 2007. REFERENCES 1. Pascual M, Perez G, Molina A. A single spectroscopic flow -through sensing device for determination of ciprofloxacin. J Pharm Biomed Anal 2004;35:689-95. 2. Nagaralli B, Seetharamappa J, Melwanki M. Sensitive spectrophotometric methods for the determination of amoxycillin, ciprofloxacin and piroxicam in pure and pharmaceutical formulations. J Pharm Biomed Anal 2002; 29: 859-864. 3. Kommos M, Saleh G, Gizawi S, Elwafa M. Spectrofluorometric determination of certain quinolone antibacterials using metal chelation. Talanta 2003;60:1033-50. 4. Vilchez J, Ballesteros 0, Taoufiki J, Palencia G, Navalon A. Determination of the antibacterial norfloxacin in human urine and serum samples by solid-phase spectrofluorimetry. Anal Chim Acta 2001 ;444:279-86. 5. Maya M, Goncalves N, Silva N, Morais J. Simple high-performance liquid chromatographic assay for the determination of ciprofloxacin in human plasma with ultraviolet detection. J Chromatogr B 2001;755:305-9. 6. Vybiralova Z, Nobilis M, Zoulova J, Kvetina J, Petr P. High-performance liquid chromatographic determination of ciprofloxacin in plasma samples. J Pharm Biomed Anal 2005;37:851-8. 7. Wang L, Yang P, Li Y, Chen H, Li M, Luo F, A flow Injection chemiluminescence method for the determination of fluoroquinolone derivative using the reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticJes. Talanta 2007;72:1066-72.

CHEMILUMINESCENCE FLOW-THROUGH BIOSENSOR FOR HYDROGEN PEROXIDE BASED ON ENHANCED HRP ACTIVITY BY GOLD NANOPARTICLES DAN LAN, BAOXIN LI* Key Laboratory ofAnalytical Chemistry for Life Science ofShaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xi 'an 710062, China, E-mail: [email protected]

INTRODUCTION Hydrogen peroxide is not only the product of reactions catalyzed by a large number of highly selective oxidases, but also an essential mediator in food, pharmaceutical, clinical, industrial and environmental analysis. ' .2 Thus, the detection of hydrogen peroxide is of great importance. Enzyme-based biosensors are currently a major area for research. The immobilization of an enzyme is a key step for constructing an enzyme-based biosensor. Many methods have been used to immobilize enzymes. l . S In recent years, nanoparticles have been introduced for the immobilization of enzymes because of their specific features that differ from bulk materials: Metal nanoparticles have many unique properties: large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability. Metal nanoparticles have been used to fabricate many enzyme-based biosensors. Most work is now focusing on electrochemical biosensors, mainly attempts to fabricate third-generation biosensors based on the direct electron transfer between a protein and the electrode.'·9 Chemiluminescence (CL) analysis is becoming increasingly important in various fields because of the very low detection limit, rapidity and wide linear working range that can be achieved while using relatively simple instrumentation. Many enzyme-based CL biosensors have been reported, but to our knowledge, there is no report on CL enzyme-based biosensor using metal nanoparticles. In this paper, Au nanoparticles with large specific surface area and good biocompatibility'o are introduced to fabricate a new enzyme-based CL biosensor for H2 0 2 . 'O It is prepared by immobilizing horseradish peroxidase (HRP) and Au nanoparticles using the solgel method in a flow CL cell. In the presence of Au nanoparticles, the response of the biosensor is enhanced 50-fold. The proposed biosensor exhibited high sensitivity, easy operation, low cost and simple assembly. EXPERIMENTAL Chemicals and reagents. Luminol stock solution (2.5x I 0-2 mollL) was prepared by dissolving 4.43 g luminol (Shaanxi Normal University, China, >95%) in 20 mL of 0.10 mollL NaOH and then diluting to lL with water. HRP (Type VI, 330 U/mg) was obtained from Sigma (St. Louis, MO, USA). Chloroauric acid (HAuCI 4 ) was purchased from Shanghai Chemical Reagent Company (Shanghai, China). Tetraethyl orthosilicate (TEOS) were obtained from Tianjin Kermel Chemical 217

218

Lan D & Li B

Reagent Development Centre (Tianjin, China). All other chemicals were of analytical reagent grade and used without further purification. The water used was deionized and doubly distilled. Instrumentation. The flow system employed in this work is shown in Fig. 1. A peristaltic pump was used to deliver all flow streams at a flow rate of 3.0 mUmin (per tube). 120 ~L of mixture solution of sample and luminol was injected by a sixway injection valve into the carrier stream. The CL signal produced in the flow cell was detected and recorded with a computerized ultraweak luminescence analyzer (type BPCL, manufactured at the Institute of Biophysics, Academia Sinica, Beijing, China). The concentration of sample was quantified by the peak height of the CL intensity.

Fig.I. Schematic diagram of the flow-through biosensor for the determination of H 20 2 : (a) sample; (b) luminol; (c) buffer solution; P: peristaltic pump; V: injection valve; F: flow cell immobilized HRP and Au nanoparticles; D: detector; PC: personal computer. Preparation of Au nanoparticles. Au nanoparticles (~8 nm) were prepared by the sodium citrate reduction of HAuCl 4 - 100 mL of 0.01% HAuCl 4 was brought to reflux and then 4 mL of 1% sodium citrate solution was introduced while stirring. The solution was then kept boiling for another 10 min and left to cool to room temperature, and then the Au nanoparicles were stored in dark bottle at 4°C. The TEM image of the resulting gold colloids showed that the gold nanoparticles were spherical with a diameter distribution around 8 nm. Immobilization of HRP and Au nanoparticIes. The preparation procedure of the sol-gel stock solution was similar to that proposed by Parang et al. J J 2.2 mL TEOS, 0.7 mL H 20 and 50 ilL O.IM HCl were mixed in a glass vial. The mixture was stirred for 3 h and then a clear sol-gel stock solution was obtained. The stock solution was stored in a refrigerator at 4°C. The HRP solution was prepared by dissolving 5 mg HRP in 1 ml phosphate buffer (pH 7.4). After that, 0.2 mL sol-gel stock solution, 40 ilL Au colloids and 8 ilL HRP solution were simply mixed. By the spreading method, the mixture was coated on the inside surface of a U-type glass tube (length 100 mm, id 3.0 mm), then was allowed to polymerize and become a gel at room temperature. So, the sol-gel was stuck to the inner surface of the U-type glass tube to form an H2 0 2-sensitive surface. Conservation and storage (after gelation) was carried out at 4 °C in water, and both ends of the U-type tube were sealed to prevent cracking.

Chemiluminescence Flow-Through Biosensor for Hydrogen Pyroxide

219

RESULTS AND DISCUSSION Effect of Au nanoparticIes in this biosensor. The primary experiment showed that the response of biosensor could be increased in the presence of Au nanoparticJes during immobilizing HRP. We compared with Au nanoparticles with different particles size, and found the small Au nanoparticles showed good enhancement effect. In this system the 8 nm Au nanoparticles were used. We also investigated the effect of the amount of Au nanoparticles. When added 40 f.lL Au nanoparticles 4 (2.3xlO- mollL), the CL signal reached the maximal value, and the CL intensity increased 50-fold compared to the sensor without Au nanoparticles. It was obvious that Au nanoparticJes can increase the catalytic activity ofHRP. UV-vis absorption spectra. Au nanoparticles have large surface-to-volume ratio and high surface reaction activity, and can strongly adsorb HRP. We measured the UV-vis absorption spectra of HRP, Au nanoparticles, and HRP combined with Au nanoparticJes, respectively. HRP has a characteristic absorption at 393 nm and 277 nm, and Au nanoparticles have a characteristic absorption at 520 nm. When they were combined, the position of the characteristic absorption did not change. This indicated HRP was strongly adsorbed at the surface of Au nanoparticles, and retained its native character. Colloidal gold, provides an environment similar to that of a redox protein in a native system and allows the protein molecules more freedom in orientation. Performance of the sensor for H 20 2 measurements. Under the optimum conditions (5 x 10-4 mollL luminol and pH=8.5), the calibration graph was linear in 9 the range of 1 x 10-8 - 1 X 10-6 moUL, and the detection limit was 4 x 10- mollL. A complete analysis, including sampling and washing, could be performed in I min 6 with a relative standard deviation of less than 5% for 1 x 10- moUL H 2 0 2 (n = 9). The column of immobilized enzyme could be reused about 100-times over a period of4 h.

CONCLUSIONS We have used Au nanoparticles to fabricate a new CL enzyme-based biosensor. HRP was chosen as a model enzyme, and the enzyme bioactivity could be enhanced by 50-fold in the presence of Au nanoparticles. Hence, the amount of HRP could be decreased greatly, and the cost of the biosensor could be thus reduced. The system can be readily adapted to other analytes (such as glucose) by varying the enzyme.

REFERENCES 1.

2. 3.

Forzani ES, Rivas GA, Solis VM. Amperometric determination of dopamine on an enzymatically modified carbon paste electrode. J Electroanal Chern 1995;382:33-40. Kulys J, Wang L, Maksimoviene A. L-lactate oxidase electrode based on methylene green and carbon paste. Anal Chim Acta 1993;274:53-8. Xiao Y, Ju H, Chen H. Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode. Anal Biochem

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2000;278:22-8. Xu Y, Peng W, Liu X, Li G. A new film for the fabrication of an unmediated H 2 0 2 biosensor. Biosens Bioelectron 2004;205:533-7. 5. Yu J, Ju H. Preparation of porous titania sol-gel matrix for immobilization of horseradish peroxidase by a vapor deposition method. Anal Chern 2002;74: 3579-83. 6. Shi J, Zhu Y, Zhang X, Baeyens WRG, Garcia-Campana AM. Recent developments in nanomaterial optical sensors. Trends Anal Chern 2004;23:351-60. 7. Jia J, Wang B, Wu A, Cheng G, Li Z, Dong S. A method to construct a thirdgeneration horseradish peroxidase biosensor: self-assembling gold nanoparticles to three-dimensional sol-gel network. Anal Chern 2002;74:221723. 8. Xu S, Han X. A novel method to construct a third-generation biosensor: selfassembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres. Biosens Bioelectron2004;19: 1117-20. 9. Di J, Shen C, Peng S, Tu Y, Li S. A one-step method to construct a thirdgeneration biosensor based on horseradish peroxidase and gold nanoparticles embedded in silica sol-gel network on gold modified electrode. Anal Chim Acta 2005;553:196 -200. 10. Crumbliss, AL, Perine SC, Stonehuerner J, Tubergen KR, Zhao J, Henkens RW. Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition. Biotechnol Bioeng 1992;40:483-90. 11. Narang U, Prasad PN, Bright FV, et al. Glucose biosensor based on a sol-gelderived platform. Anal Chern 1994;66:3139-44.

4.

FLOW INJECTION CHEMILUMINESCENCE DETERMINATION OF THIAMINE BY THE ENHANCEMENT OF LUMINOL-K 3 Fe(CN)6 SYSTEM YH LI,I Y YANG,I JR LU 2 'School o/Science, Xi 'an Jiaotong University, Xi 'an 710049, China lSchool o/Chemistry and Materials Science, Shaanxi Normal University, Xi 'an 710062, China Email: [email protected]

INTRODUCTION Thiamine, a soluble vitamin, is an essential nutrient for humans and IS Important in carbohydrate metabolism, maintaining normal neural activity and preventing beriberi. Various analytical techniques have been reported for the determination of thiamine in pure form, in pharmaceutical preparations, or in biological fluids. Spectrophotometric methods suffer from poor sensitivity (mg/L detection limit).' Spectrofluorometric methods usually involve the conversion of thiamine to thiochrome.' High performance liquid chromatography requires a post-column derivatization step J and instrumentation for electrophoresis-based methods is expensive.' The use of chemiluminescence (CL) detection in pharmaceutical analysis for the assay of active components in dosage forms requires a fairly selective and sensitive technique with relatively simple instrumentation and has been frequently used for the analysis of pharmaceutical compounds.' However, the use of CL method for the determination of thiamine is uncommon. Grekas and Calokerinos 6 reported a continuous flow CL method for thiamine by its oxidation with K3Fe(CN)6 in alkaline medium, the detection limit of the method was 9.00xlO-6 mollL thiamine. Martinez Calatayud et at. proposed a CL thiamine detection method that combined UV irradiation of thiamine with acidic KMn04 oxidation, and this had a detection range of 0.05-84 mglL.' Du et at. also developed a FI-CL method for thiamine based on its enhancing effect on the luminol-H 20 2 reaction: the method detected 0.05-8.0 mg/L thiamine.' It was found that the CL emission generated from the oxidation of luminol with K3Fe(CN)6 could be enhanced significantly in the presence of thiamine. The experimental variables that affected the CL reaction were optimized and a simple and sensitive FI-CL method for the determination of thiamine was established. The proposed method was applied to the determination of thiamine in pharmaceutical preparations and the results compared well with those obtained by Chinese pharmacopoeia method.' MA TERIALS AND METHODS Apparatus. Figure 1 depicts the schematic diagram of the FI-CL system. PTFE tubing (id, 0.8 mm) was used to connect all components in the flow system. A HL-2 peristaltic pump was used to deliver the sample and reagent solutions through the flow system channels at a constant flow rate of 2.0 mLimin. Sample injection (50 ~L) was made by means of a six-way valve. The CL signal produced in the flow cell was detected with a CR-I05 photomultiplier tube (Beijing Hamamatsu Photo Techniques 221

222

Li YH et al.

Inc). Data acquisition and treatment was handled by a MCDR-A multifunction data processing system (Xi'an Remax Electronic Science-Tech Co. Ltd, China).

B

Thiamine Lummol KJFe(CN)6

-

Flow

-n

Valve Peristaltic pump

High voltage ...-P-ho-to-m.....u-lti-pl-'ier'-tu-be--.

~

Waste

Computer

Fig. 1. Schematic diagram ofCL flow system. Chemicals. All chemicals were of analytical reagent grade. Doubly distilled water was used throughout the experiments. A 500 mg/L thiamine stock solution was prepared by dissolving thiamine hydrochloride (Shanghai No.3 Chemical Factory) in water. Thiamine working solutions were freshly prepared by gradually diluting the stock solution with water. All thiamine solutions were stored in a refrigerator and protected from light. A 0.01 mollL luminol (Shaanxi Normal University) stock solution was prepared in 0.01 mollL of sodium hydroxide. A 0.01 mollL K3Fe(CN)6 (Xi'an Chemical Plant) solution was prepared in water and protected from the light. Procedure. Flow lines were connected with thiamine solution, luminol solution and K3Fe(CN)6 solution The luminol solution was firstly merged with K3Fe(CN)6 solution to give a stable base line. The CL signal was measured by injection 50 f-lL of thiamine solution into the merged stream of luminol with K3Fe(CN)6 by the six-way valve. The concentration of thiamine was quantified from the CL intensity. RESUL TS AND DISCUSSION Optimization. The oxidation of luminol with K3Fe(CN)6 emits CL in alkaline conditions. The alkalinity of reaction medium was controlled by the sodium hydroxide used to prepare the luminol solution. The effect of sodium hydroxide concentration was examined in the range of I x I 0- 3~2x I 0- 2 mollL. The signal-to-blank ratio was increased with increasing sodium hydrogen concentration up to I x I 0- 2 mollL. Higher 2 concentrations (> I x I 0- mollL) caused a decrease in the signal-to-blank ratio. Hence, 2 I x I 0- mollL sodium hydrogen was employed in subsequent experiments. The signal-to-blank ratio continued to increase as the luminol concentration was 7 increased from Ix10- mol/L to 8x10- 7 mollL. It reached a maximum at 8x10- 7 mollL 7 luminol and then decreased. Hence, 8x I 0- mollL luminol was employed in subsequent experiments. The signal-to-blank ratio rapidly increased with the increase in K3Fe(CN)6 concentration in the range of 5xI0-6~6xlO-5 mol/L. Above 6x10-5 mollL, the signal-to-blank ratio decreased slightly. Hence, 6x10-5 mol/L K3Fe(CN)6 was employed in subsequent experiments. Performance of the system for thiamine measurements. Under the selected experimental conditions, the CL intensity was linearly related to the concentration of

Flow Injection Chemiluminescence Determination of Thiamine

223

thiamine over the range of 0.03-1.0 mg/L. The linear regression equation was I (au) = 2 2.40e (10- mg/L) + 13.84 and the correlation coefficient was 0.9936 (n= 8). The detection limit (3s b) was 0.007 mg/L thiamine and the relative standard deviation was 1.1% for 0.5 mg/L thiamine solution (n=II). A complete analysis, including sampling and washing, could be finished in 40 s, giving a sample measurement frequency of 901h. Interference. The effect of some common ions, additives and excipients used in pharmaceutical preparations was studied on the determination of a 0.5 mg/L thiamine solution. A foreign specie was considered not to interfere if it caused a relative error less than ±5% in the peak height. No interference has been observed when including a 1000-fold Na+, K+, SO/-, cr, N0 3- and starch, IOO-fold Ca2+, Zn 2+, glucose and lactose, IO-fold Mg2+. Equal amount of Fe3+, Fe 2+, C0 2+, Cr3+ and Cu 2+ interfered with the determination of thiamine. Determination of thiamine in pharmaceutical prepartions. The proposed method was applied to the determination of thiamine in vitamin BI tablets and vitamin BI injections. The average tablet weight was obtained from the weight of20 tablets. They were finely powdered, homogenized and a portion of the powder, equivalent to about 50 mg of thiamine was accurately weighted and dissolved in water. The resulting mixture was filtered and the filtrate was diluted to 100 mL with water for further sample analysis. Vitamin BI injections were directly analyzed after appropriately diluting with water. The results are summarized in Table 1, which agreed well with those obtained by the Chinese pharmacopoeia method. 9 Tabl~

1. Results for the determination of thiamine in pharmaceutical prepartions

Samples

Nomina I content

Propose d meth 0d

RSD, n=5

Pharmacopoeia method

Tablet 1

10 mg/tablet

9.97 mg/ tablet

1.2%

10.16 mg/ tablet

Tablet 2

10 mg/tablet

10.15 mg/tablet

1.3%

9.74 mg/ tablet

Injection 1

50 mglinjection

46.7 mg/injection

1.2%

48.6 mg/injection

Injection 2

100 mg/injection

97.0 mg/injection

1.1%

98.5 mg/injection

_

Possible CL reaction mechanism. The CL spectra of luminol of K3Fe(CN)6 with and without thiamine were obtained using a modified RF-540 spectrophotofluorimeter. Both CL spectra showed a maximum emission wavelength at 425 nm, which suggested that the excited state of 3-aminophthalate ion JO was still the emitter in the present reaction. When the dissolved oxygen in all solutions were removed by the flow of nitrogen, the CL intensity of the reaction reduced significantly, which indicated that the dissolved oxygen played an important role in the reaction. It was well known that thiamine can

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easily convert to thiol-containing compound in alkaline solution. The thiol-containing compound produced can react with dissolved oxygen in alkaline solution to produce a superoxide anion radical intermediate." Superoxide anion radical then oxidize luminol to produce CL. 12 In the presence of K3Fe(CN)6, the CL signal from the reaction between superoxide anion radical and luminol was enhanced. 13

REFERENCES 1.

2.

3.

4. 5.

6. 7.

8.

9. 10. 11. 12.

13.

Ghasemi J, Abbasi B, Niazi A, Nadat E, Mordai A. Simultaneous spectrophotometric mUlticomponent determination of folic acid, thiamine, riboflavin and pyridoxal by using double divisor-ratio spectra derivative-zero crossing method. Anal Lett 2004;37:2609-23. Alonso A, Almendral MJ, Porras MJ, Curto Y. Flow-injection solvent extraction without phase separation fluorimetric determination of thiamine by the thiochrome method. J Pharm Biomed Anal 2006;42: 171-7. Bohrer D, do Nascimento PC, Ramirez AG, Mendonca JKA, de Cavalho LM, Pomblum SCG, Determination of thiamine in blood serum and urine by high-performance liquid chromatography with dried injection and post-column derivatization. Microchem J 2004;28:71-6. Mrestani Y, Neubert RHH. Thiamine analysis in biological media by capillary zone electrophoresis with a high-sensitivity cell. J Chromatogr 2000;871: 351-6 Mervartova K, Polasek M, Martinez Calatayud J. Recent applications of flow-injection and sequential-injection analysis techniques to chemiluminescence determination of pharmaceuticals. J Pharm Biomed Anal 2007;45:367-81. Grekas N, Calokerinos AC. Determination of thiamine by continuous flow chemiluminescence measurement. Talanta 1990;37: 1043-8. Wasielczuk A, Catala Icardo M, Garcia Mateo JV, Martinez Calatayud J. Flow-injection chemiluminescent determination of thiamine in pharmaceutical samp 1es by on-line photodegradation. Anal Lett 2004;37 :3205-18. Du J, Li Y, Lu J. Flow injection chemiluminescence determination of thiamine based on its enhancing effect on the luminol-hydrogen peroxide system. Talanta 2002;57:661-5. The Pharmacopoeia of People's Republic of China (Part II). Beijing: Chemical Industry Press, 2000:784-7. White EH, Bursey MM. Chemiluminescence of luminol and related hydrazides: the light emission step. 1 Am Chern Soc 1964;86:941-2. Shen 1M, Wu ZQ. Eds. Pharmaceutical Structure and Reagent. Beijing: Chinese Medicine Science and Technology Press, 1989:541. Ohno K, Arakawa H, Yoda R, Maeda M. Development of novel high-sensitivity chemiluminescence assay for luminol using thiourea derivatives. Luminescence 1999; 14:355-60. Du J, Li Y, Lu 1. Flow injection chemiluminescence determination of captopril based on its enhancing effect on the luminol-ferricyanide/ferrocyanide reaction. Luminescence 2002; 17: 169-172.

CHEMILUMINESCENT AND ELECTRON SPIN RESONANCE SPECTROSCOPIC MEASUREMENTS OF REACTIVE OXYGEN SPECIES GENERA TED IN WATER TREATED WITH TITANIA-COATED PHOTOCA T ALYTIC FIBERS C LIN,l,2 K. TANAKA,2 L TANAKA,2 T KAWANO l JGraduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan 2 K2R Inc., Kitakyushu 807-0871, Japan Email: [email protected].}p

INTRODUCTION Recently, a variety of ultraviolet-driven photochemically active catalysts designated as photo catalysts coated with titanium dioxide (Ti0 2) has been developed and applied for hygiene and antimicrobial purposes. The likely mechanism of such catalysts involves the generation of reactive oxygen species (ROS) on the surface of the catalyst as expected (not fully proven) from the previously proposed models. l However, no attempt to confer long-lasting chemical properties to the waters (e.g., preparation of waters rich in ROS) has been reported, despite of the increasing demands for the use of photocatalysts in various environments including the use in aqueous phase. In the present study, novel water conditioning photocatalytic apparatus (exPCA W-l, K2R Inc., Kitakyushu, Japan) equipped with sheets of TiOrcoated photocatalytic fibers were applied for THE preparation of ROS-containing water. We attempted to detect the generation of superoxide anion and hydroxyl radical as the key members of ROS generated in the water circulated in exPCA W-l by using the superoxide anion-specific chemiluminescent probe Cypridina luciferin analog (CLA), and a spin trapping agent, DMPO (5, 5-dimethyl-I-pyrroline-N-oxide) that readily forms an adduct with hydroxyl radical. Some other factors such as the effects of pH on the superoxide generation were also studied. METHODS Apparatus. For preparation of ROS-containing water, photocatalytic apparatus designated exPCA W-I (fabricated by K2R Inc., Kitakyushu, Japan) was used. It comprised an ultraviolet (UV-A) emitting bulb, ultrasonic wave (USW) generating devices, sheets of titania-coated fiber, a pumping system and gas (0 2 or NO) supply systems. As circulated in the exPCA W-I, water is treated with UV, USW and O 2, enabling the photo-catalytic oxidative conditioning of the water. Detection of superoxide anion. To detect superoxide generated and maintained in the water, a specific chemiluminescence probe, Cypridina luciferin analog (CLA; 2-methyl-6- phenyl-3,7-dihydroimidazo[I,2-a]pyrazin-3-one)2 purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan) was used. Water was circulated in the aparatus for at least 30 min and 0.5 mL was sampled from the reservoir (l L) and added onto CLA a containing reaction mixture (111M in 0.5 mL) in a glass cuvet placed insde the luminometer (CHEM-GLOW Photometer, AMINCO, Silver Spring, MD, USA) or 225

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Luminescencer (ATTO, AB-2200, Japan). Superoxide generation monitored as CLA-chemiluminescence was expressed as relative chemiluminescence units (rcu) as previously described. 3 Detection of hydroxyl radical. For detection of hydroxyl radicals, a spin trapping agent, DMPO was obtained from Sigma-Aldrich. 4 The water containing 500 JlM DMPO was circulated for 30 min in exPCA W-l ,and the water was sampled at 0, 1, 5, 10, 15, 20, 30 min of circulation and exposure to UV, USW and elevated O2 • The samples in a flat-shaped quartz ESR cell were analyzed with an electron spin resonance spectrometer (JEOL-TE200, X-band). DMPO-OH signal were collected with a sweep width of 5 mM, a 100 kHz modulation frequency, 60 s sweep time, a time constant of 0.1 s and microwave power of 10 mW, at room temperature of 20-25°C. RESULTS AND DISCUSSION The superoxide generation in the aqueous phase was monitored using CLA chemiluminescence and a photometer equipped with a pen recorder. The CLA luminescence in the glass cuvet increased following addition of processed water (treated with titania- coated photocatalytic fiber exposed to both UV-C and USW) after sampling from the aparatus. Despite its short life time, superoxide-dependent signal was shown to be long-lasting over 30-60 min even after isolation from the catalyst-equipped, photo-irradiating and USW generating apparatus (Fig. lA). In contrast, chemiluminescence could be no longer detected once Tiron, a superoxide scavenger, was added to the reaction mixture (Fig. 1B), supporting the view that the long-lasting nature of chmiluminescence can be attributed to the continuous generation of superoxide. In addition, the processed water-dependent superoxide generation showed pH-dependency, prefering nutral to alkaline conditions (Fig. 2) .

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Chemiluminescent and Electron Spin Resonance Spectroscopic Measurements

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35

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We also attempted to detect another key member of ROS, hydroxyl radical based on the formation of spin-trapped aduct (DMPO-OH) using ESR (Fig. 3). By circulating the spin trapping agent in the system, we observed the UV-dependent development of DMPO-OH signal with time. While we could detect the presence of hydroxy radicals in the circulating water, no signal could be detected in the water samples isolated from the photochemical reaction apparatus.

REFERENCES 1. Kagenishi,T. Yokawa K, Lin C, Tanaka K, Tanaka L, Kawano T. Chemiluminescent and bioluminescent analysis of plant cell responses to reactive oxygen species produced by newly developped water conditioning apparatus equipped with titania-coated photocatalystic fibers. In: Shen X, Yang X-L, Zhang X-R, Cui ZJ, Kricka LJ, Stanley PE eds. Light emission: Biology and scientiic appliations. Singapore:WorId Scientific. 2009:27-30. 2. Nakano M, Sugioka K, Ushijima Y, Goto T. Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a] pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-. Anal Biochem 1986; 159: 363-9. 3. Kawano T, Kadono T, Furuichi T, Muto S, Lapeyrie F. Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulations in tobacco BY-2 cells. Biochem Biophys Res Commun 2003; 308: 35-42. 4. Kawano T, Muto S. Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco suspension culture. J Experi Bot 2000; 51: 685-693.

A SENSITIVE MICELLAR-ENHANCED CHEMILUMINESCENCE METHOD FOR THE DETERMINA TION OF OFLOXACIN BY FLOW INJECTION ANALYSIS HONGY AN MA, Y ANTU ZHANG, LIXIAO MIAO, XUEHUA SUN

College a/Chemistry and Chemical Engineering, Yan 'an University, Yan 'an, 7J 6000, China Email: [email protected]

INTRODUCTION Ofloxacin is a member of the fluoroquinolone class of synthetic antibiotics.' It is widely used in the treatment of a wide range of infection, including respiratory tract, urinary tract and tissue-based infections. Several methods for the determination of ofloxacin have been reported in the literature including spectrophotometric,' fluorimetric,3 chromatographic' and electroanalytic. 5 Francis has reviewed the methodology for the determination of ofloxacin based on chemiluminescence (CL) detection. 6 But to the best of our knowledge, no flow injection CL analysis method for the determination of ofloxacin in the presence of sodium dodecyl sulfate (SDS) surfactant micelles has been described. We investigated the effects of surfactants on emission intensity in the luminol CL system due to the various advantageous properties of surfactants, which have been found to improve CL measurement efficiency,' and reported significant and useful improvements in relative intensity. The method possesses a good accuracy and precision and has been successfully used to determine ofloxacin in pharmaceuticals. EXPERIMENTAL Reagents. All reagents were of analytical grade and all solutions were prepared with doubly distilled water. Ofloxacin was obtained from Beijing Bio Life Science and Technology Co, Ltd, China, Ofloxacin injection was obtained from Ludi Pharmaceutical Ltd, Co" Jiangxi, China, Ofloxacin tablets were purchased from Nanchang Pharmaceutical Factory, Jiangxi, China, The stock standard solution of ofloxacin (I mg/mL) was prepared by dissolving 100 mg of ofloxacin in 0,001 mollL sulphuric acid solution and diluting to 100 mL with the same acid, Working standard solutions were prepared by appropriate dilution of the stock standard solution with water. Luminol was used as supplied to prepare a 0,03 mollL stock solution by dissolving 1.3258 g luminol (synthesized by Department of Chemistry, Shaanxi Normal University, China) with 0.1 mollL NaOH to 250 mL in an amber-colored measuring flask. SDS, H 2 0 2 , NaOH, NaAc-HAc solutions were prepared in water. Apparatus. The flow system used in this work (Fig, 1) consisted of two peristaltic 4 pumps, one of which delivered a sample stream (ofloxacin and 8,Ox 10- mol/L SDS in pH 4.65 NaAc-HAc medium) and H 20 2 stream at a flow rate of 3.4 mLimin, the 229

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other delivered NaOH carrier and luminol solution at the same flow rate. PTFE tubing (O.S mm id) was used to connect all components in the flow system. 100 ilL of a mixture of sample and H20 2 was injected into the NaOH carrier stream by a six-way injection valve and then mixed with luminol solution. The flow cell was a coil of glass tubing (1.3 mm id) spiraled to a diameter of 35 mm with five turns, located in front of the detection window of the photomultiplier tube. The emitted CL was collected with a photomultiplier tube (operated at -SOO V) of the BPCL Ultra Weak Chemiluminescence Analyzer (lnsistitute of Biophysics. Chinese Academy of Sciences, Beijing). The signal was recorded using a computer equipped with a data acquisition interface. Data acquisition and treatment were performed with BPCL WIN software. a

b

c

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Fig. 1. Schematic diagram for the determination of ofloxacin. Pj, P 2 . pump; V. six-way valve; F. flow cell; D. detector; Pc. personal computer; W. waste; a. sample solution (ofloxacin and S.OxI0-4 mollL SDS in NaAc-HAc buffer); b. H 2 0 2 solution; c. NaOH solution; d. luminol solution.

RESUL TS AND DISCUSSION Firstly, in order to obtain an understanding of the mechanism of the sensitized luminol-based CL reaction, the CL emission spectrum was obtained with a F-4500 fluorescence spectrophotometer. The results showed the maximum emission wavelength in the presence of ofloxacin was the same as that in the absence of ofloxacin and the maximum light emission was at about 425 nm. The emitter was 3-aminophthalate, the oxidation product of luminol. Furthermore, the preliminary investigations showed that the sample medium is a very important factor for the determination of ofloxacin with the proposed method. The form of ofloxacin existing in different pH media is different. The experiment indicited that: only when acidic ofloxacin solution was injected into the carrier stream was the enhanced CL signal observed. Thus, we concluded that the possible mechanism is: the ofloxacin may act as a energy source in the CL reaction, the presence of ofloxacin enhanced the luminol-H 2 0 2 CL quantum yield. The maximum CL intensity was obtained from pH 4.0-5.0. Thus, pH 4.65 NaAc-HAc buffer solution was selected as the sample medium. The principles of micellar enhancement including solubilization and solute organization, altering the local microenvironment and changing the light-emitting pathways that affect the quantum yield and reaction rate have already been discussed

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by Townshend et aI.' In order to investigate whether surfactant media enhanced effectively in the present CL reaction system, the characteristics of several different micelles including Tween 80, OP, Triton X-I00, CTMAB, TPB, SDBS and SDS were studied. The results showed SDS enhanced the signal dramatically. SDBS gave a somewhat enhanced signal whereas other surfactants were less effective. Thus, SDS was selected for the present work. The effect of SDS at various concentrations was studied in order to maximize the CL signal. The maximum CL intensity was observed using a concentration of 8.0 x 10-4 mollL SDS. Therefore, this was selected for the present work. The influence of luminol concentration on CL was examined from l.Ox 10- 7 to 6 2.0x 10- mollL. The result indicated that 8.0x 10- 7 mollL luminol gave the highest relative CL intensity and the sensitivity decreased on either side of this value. Therefore, 8.0x 10-7 mollL luminol was chosen for the subsequent experiments. The influence of sodium hydroxide concentration on the CL intensity was investigated at different concentrations from 0.005 to 0.2 mollL and the maximum CL intensity was obtained at 0.01 mollL. Therefore, 0.01 mollL sodium hydroxide was selected for the present work. The effect of H 2 0 2 concentration over the range of 2.0x 10- 4 to 2.0x 10-3 mollL on the CL emission was examined. The peak height increased steeply with increasing H 20 2 concentration up to 8.0xlO-4 mollL, above which CL intensity decreased. Therefore, 8.0x 10-4 mollL H 2 0 2 was used for subsequent work. Flow rate is an important parameter in CL detection. Too low or too high flow rates result in a decrease or even the absence of a CL signal in the flow cell. A rate of 3.4 mLimin was chosen as a suitable condition with superior sensitivity, precision and reduced reagent consumption. Under the selected experimental conditions, a linear calibration graph of oxfloxacin between 4.2x 10- 12 and 3.6x 10-9 g/mL was obtained. The calibration equation was 1=1.3 x 10 12 C + 190.4, r 2=0.9993, with a detection limit of 2.6x 10- 12 g/mL (S/N=3). The relative standard deviation (RSD%) for 11 determinations of l.Ox 10-9 g/mL was 2.0%. The sample measurement frequency was calculated about 60 sampleslh. Table 1. Determination of ofloxacin in a pharmaceutical formulation (n=5) Sample Tablet Injection

Label (mg) 100.0 200.0

Official method (mg) 99.4 197.6

Proposed method(mg) 99.7 198.4

RSD (%) 2.2 2.3

Added (mg) 50.0 50.0

Recovery (%) 98.5 101

The interference of foreign species were tested by analyzing a standard solution of 9 l.Ox 10- g/mL ofloxacin. The tolerable concentration ratios for interference at 5% level were over 5000 for glucose, sucrose, 1000 for magnesium stearate,

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hydroxypropyJcellulose, starch, lactic acid, cr, Ca2+, Mg2+, respectively. Finally, tablets and injections were analyzed by the proposed method. Injectable preparations of ofloxacin were directly diluted with water so that final concentration was in the working range. Ten tablets were weighed to obtain the mean weight, then ground to homogenized powder and an accurately weighed portion of powder corresponding to 100 mg was diluted with 0.001 moUL sulphuric acid fot., the quantitative analysis (see Table 1); the results agree well with those obtained by an official method.' The recoveries varied from 98.5 %-101 %. .

REFERENCES 1.

Schaeffer AI.

The

expanding

role

of fluoroquinolones.

Am

I

Med

2002;113:45-54. 2.

3.

4.

5.

6. 7.

8.

Issa YM, Abdel-Gawad FM, Abou Table MA, Hussein HM. Spectrophotometric determination of ofloxacin and lomefloxacin hydrochloride with some sulphonphthalein dyes. Anal Lett 1997;30:2071-84. Gong QJ, Qiao IL, Du LM, Dong C. Recognition and simultaneous determination of ofloxacin enantiomers by synchronization first derivative fluorescence spectroscopy. Talanta 2000;53:359-65. Halkar UP, Ankalkope PB. Reverse phase high-performance liquid chromatographic determination of ofloxacin and tinizadole in tablets. Indian Drugs 2000;37:585-8. Wu J, Zhao H, Wei L, Ai TZ, Dong XZ. Preparation and application of a poly(vinyl-chloride) membrane coated glass electrode-based ofloxacin ISE. Chin J Anal Chem 2001;29 : 11 06-9. Francis PS, Adcock JL. Chemiluminescence methods for the determination of ofloxacin. Anal Chim Acta 2005; 541:3-12. Townshend A, Youngvises N, Wheatley RA, Liawruangrath S. Flow-injection determination of cinnarizine using surfactant-enhanced permanganate chemiluminesence. Anal Chim Acta 2003;499:223-33. Editorial Committee of the Pharmacopoeia of People' Republic of China. The Pharmacopoeia of People' Republic of China (Part II). Beijing: Chemical Industry Press, 2005 :606-8.

EXCESSIVE EXTRACELLULAR CHEMILUMINESCENCE AND NECROSIS OF NEUTROPHILS IN BOVINE NEONATES AND POTENTIALLY SUPPORTIVE ROLE OF VITAMIN C J MEHRZAD,1 M MOHRI,2 C BURVENICH3 1Department of Pathobiology, F erdowsi University of Mashhad, Mashhad, Iran 2Department of Clinical Science, Ferdowsi University of Mashhad, Mashhad, Iran 3Department of Physiology and Biometry, Ghent University, Merelbeke, Belgium E-mail: [email protected]

INTRODUCTION Neutrophils are the most critical part of the innate immune defense in dairy cOWS. t,2 Their quality in the blood circulation and tissue is crucial during early life of neonatal calves. Vitamin C is one of the most important water-soluble protective agents in mammalian cells. 3 Bovine neonates are unable to synthesize vitamin C. Substantial evidence suggests a link between vitamin C and immunity.t,3 Bovine neutrophils have a potential to produce a substantial amount of reactive oxygen species (ROS) to kill engulfed bacteria. t.6 These ROS production can be both extracellular and intracellular. 2,4,7.9 The neutrophils ROS production and its kinetics can be measured following stimulation with soluble agents, e.g., phorbol 12myristate 13-acetate (PMA) or with particles e.g. zymosan, bacteria, latex beads, using chemiluminescence (CL) assay,2,3,7.9 which was first described by Allen et aJ.1 The different responsiveness of blood neutrophils to PMA stimulation during physiological conditions could result from differences in protein kinase C, NADPHoxidase and myeloperoxidase (MPO) activities. 2,6.9 As these enzyme activities reflect intracellular and extracellular reactions, changes might offer some evidence about the neonates' susceptibility to infections. For example, in dairy cows the maximal animal susceptibility for environmental pathogens coincides with the minimal neutrophil ROS production capacity.l,2,6 Furthermore, dietary vitamin C in dairy cows somehow improves the quality of milk neutrophils 3. Retrospectively, there have been no investigations on the issue of "neutrophils ROS production and antioxidants versus neonates" in cows. Therefore, to obtain a clearer insight into the oxidationreduction reactions of the neutrophils in bovine neonates, the kinetics of PMA stimulated luminol-enhanced CL of blood neutrophils, their viability and the role of vitamin C were investigated in calves immediately after birth. MATERIALS AND METHODS Calves and experimental plan. In total twenty Holstein early neonates were selected; they all were clinically normal. Two separate studies were conducted. In the first study, simultaneous analyses of CL kinetics and viability of blood neutrophils within 12 hours after birth in the healthy calves (n = 10; from Ghent 233

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University dairy farm) were measured. In the second study, at 10 days before anticipated calving, ten healthy Holstein dairy cows from Mashhad University dairy farm were divided in two groups. The cows were fed diets that provided 0 (n = 5) or 50 (n = 5) gm/d of supplemental vitamin C. Within 12 hours after birth, blood samples were collected for isolation of neutrophils, their viability and superoxide anion production capacity. In both studies, isolation of pure blood neutrophils was carried out as described previously.2,4-6 Chemiluminescence, O£ production and viability of blood neutrophils. In the first study luminol-enhanced PMA-stimulated CL was used to assess blood neutrophils CL kinetics. Briefly, CL was performed using a luminometer (LB96P; EG&G Berthold, Germany) at 37°C. A total volume of 200 J.1L was prepared for CL kinetics determination. Immediately after addition of PMA (final concentration of 200 ng/mL) and luminol (final concentration of 0.3 mmoVL) to lOS neutrophils into microtiter plates, CL was measured. The area under the curve (AUC) was determined for registered impulse rate (counts/min) over the entire measurement period of 30 min as previously described. 2,4.6 Viability of neutrophils was evaluated by means of flow cytometry (FACSScan, Becton Dickinson Systems, CA, USA), using propidium iodide exclusion. 2.4.6 In the second study, the superoxide anion (Oi) was measured using superoxide dismutase inhibitable cytochrome C reduction assay. Briefly, after incubation for 30 min the optical density at 550 nm was determined in a Microplate reader, the results were converted to nanomoles of cytochrome C reduced using the extinction coefficient Esso nm = 2.1x104/moVcm. The viability was quantified with microscopic observation of neutrophils using trypan blue dye exclusion. In both studies whole blood and isolated neutrophils were microscopically examined on slides, as described previously.2.4 For statistical analyses of the parameters, the SAS Version 9.1 with analysis of variance was used. Hypothesis testing was done at the 5 % significance level. RESULTS AND DISCUSSION In general, the CL of blood neutrophils in early neonates revealed something interestingly different from their adult counterparts. The CL kinetics of PMAstimulated neutrophils was monophasic pattern. This pattern was more noticeable during day 1 of birth (Figure 1). However, when the neonates drink more colostrum the CL tends to switch towards a biphasic form (Figure 1). Furthermore, microscopic examination of the neutrophils showed the presence of both immature and apoptotic neutrophils in the prepheral blood (Figure 1). From our data in the first study we can conclude that the intensity of neutrophil CL was always lower in early neonates than in the adults. 2.4.6 This discrepancy seems to be somehow related to the existence of immature neutrophils and excessive extracellular ROS in the blood stream. All of this shows the lower effectiveness of the oxygen-dependent intracellular bactericidal mechanism of neonatal neutrophils. 2,4-9 Moreover, as the luminol-dependent system requires hydrogen peroxide (H20 2), 8,9 it is likely that the intracellular H 20 2

Excessive Extracellular Chemiluminescence and Necrosis of Neutrophils

235

pnXitlctlon is low in calves immediately after birth. The absence of a second in blood neutrophils of bovine neonates warrants further study.

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vitamin C potentially improves neonatal neutrophils functions; left is the viability and right panel is the superoxide anion production of Bars represent means and the standard error of the means of 10 calves. In the second stduy, we observed that dietary vitamin C somewhat burst activity or OZ" production (Figure 2; right panel) and 2; left panel) in bovine neonates. Compared with non-vitamin sUJ'plem.enlted group, feeding vitamin C to the pregnant cows around ",,,.tHr,ft neutrophil functions in their neonates. We assume that the

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supplemental vitamin C appears in the colostrum or milk. When the neonates drink this milk the vitamin C is absorbed via gastrointestinal tract into bood circulation. Although statistically the improvement was slight (due to limited number of calves) but immunobiologically this slight imptovement could be vital for the neonates. Overall, these findings suggest that the subsequent intracellular MPO-H 20 2 system is impaired, and may represent an immunosuppressed condition in the very early bovine neonates. Conversely, the slight increase of neutrophil functions with vitamin C application could lead to a better protection of neonatal calves from infectious pathogens. Further studies are in progress to explain these findings.

ACKNOWLEDG EMENTS This study was financially supported by Ferdowsi University of Mashhad (Grant No. 36900) and the Flemish Institute for the Encouragement of Research in the Industry (lWT-grant No. 030784). The authors wish to thank K. Demeyere and A. Shavalian for their excellent technical assistance. REFERENCES 1. Burvenich C, Van Merris V, Mehrzad J, Diez-Fraile A, Duchateau L. Severity of E. coli mastitis is mainly determined by cow factors. Vet Res 2003; 34:521-62. 2. Mehrzad J, Dosogne H, Meyer, E, Heyneman R., Burvenich C. Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation. J Dairy Res 2001; 68:399-415. 3. Weiss WP, Hogan JS. Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows. J Dairy Sci 2007; 90:731-9. 4. Mehrzad J, Dosogne H, Vangroenweghe F, Burvenich C. A comparative study of bovine blood and milk neutrophils functions with luminol dependent chemiluminescence. Luminescence 2001; 16: 343-56. 5. Mehrzad J, Duchateau L, Py6rala S, Burvenich C. Blood and Milk Neutrophil Chemiluminescence and viability in primiparous and pluriparous dairy cows during late pregnancy, around parturition and early lactation. J Dairy Sci 2002; 85:3268-76. 6. Mehrzad J, Duchateau L, Burvenich C. Viability of milk neutrophils and severity of bovine coliform mastitis. J Dairy Sci 2004; 87:4150-62. 7. Allen RC, Stjernholm RL, Steele RH. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 1972; 47: 679-84. 8. Briheim G, Stendahl 0, Dahlgren C. lntra- and extracellular events in luminoldependent chemiluminescence of polymorphonuclear leukocytes. Infec lmmun 1984;45: 1-5. 9. Lind J, Merenyi G, Eriksen TE. Chemiluminescence mechanism of cyclic hydrazides such as luminol in aqueous solutions. J Am Chern Soc 1983; 105:7655-61.

CHEMILUMINESCENCE OF 9-BENZYLIDENE-IO-METHYLACRIDANS WITH ELECTRON-DONATING GROUPS BY CHEMICALLY GENERATED SINGLET OXYGEN - APPLICATION TO METAL ION SENSING USING AZACROWNED COMPOUND J MOTOYOSHIYA, T TANAKA, M KUROE, Y NISHII Diivision o/Chemistry and Materials,Faculty o/Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan, [email protected]

INTRODUCTION The thermal decomposition of a 1,2-dioxetane into two carbonyl compounds, one of which is formed in the excited state, often produces chemiluminescence. I The singlet oxygenation of 9-benzylidene-IO-methylacridans to produce chemiluminescent 2 acridan dioxetanes has been occasionally investigated. ,3 The thermal decomposition of the acridan dioxetanes gives the corresponding aldehydes and a fluorescent Nmethylacridone (NMA), the latter of which is the emitter (Scheme I). Although electron donating-substituents attached to the phenyl group of the benzylidene moieties increases chemiluminescence efficiency, the dimethylamino group drastically inhibits the chemiluminescence. 4,5 Such a peculiar effect of the amino group prompted us to investigate this system in more detail and apply it to a metal cation recognition system, Here we report the chemiluminescence behavior of 9-benzylidene-IOmethylacridans bearing electron-donating groups (e,g" alkoxy and amino groups) and describe the potential application to metal cation sensing using an azacrowned compound. {7'/

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4.0 X 10-5 M), a blue-light flash emission was observed, and the chemiluminescence spectrum was completely in agreement with the fluorescence spectrum of NMA, indicating that the excited NMA was generated from this oxidation reaction. The fluorescence spectrum of the spent solution also agreed with that of NMA and no other fluorescent product was found. A different way to generate singlet oxygen by a combination of alkaline hydrogen peroxide-acetonitrile, a metal ion free system,6 was also carried out [aqueous solution containing hydrogen peroxide (lmL, 1 x 10-5 M) 2 and tetrabutylammonium hydroxide (1 mL, 1 x 10- M) added to acetonitrile solution 7 of la (1 mL, 1 x 10- M)] and this produced a blue-light emission. Table 1. Data of chemiluminescence reaction of benzylideneacridanes (1).

R

:Ea+

1a

p-OMe

-0.27

Ib

m-OMe

0.12

Ie

2.4-(OMe)2

Id

3,4.5-(OMe)3

Ie

p-NMe2

a C

b

0.91

0.33

0.83

0.44

2.7

1.0

3.0

-0.03

4.3

0.99

4.8

-0.83

0.094

1.00

0.1

Measured by photon-counting method. b Determined by UV spectrum. Calculated by
-:.0 . . . - - - - - - - - - - - ,

The results of the chemiluminescence reactions of la-d are presented in Table 1. The chemiluminescence quantum yield of la was the highest of all, but that • p-OMe -3.0 of Ie was extremely low. The Hammett • >I!-OMe relationship was found to hold for la, I b and Id, except for Ie, as shown in Figure 1. The lower
Chemiluminescence of9-Benzylidene-10-Methylacridans with Electron-Donating Groups

239

oxygen or the fluorescence quenching of the emitter, i.e., the excited NMA. The former assumption was readily ruled out because the dioxetane formation from Ie was apparent from the detection of both p-dimethylaminobenzaldehyde and NMA as the decomposition products. The fluorescence quenching rate constant of pdimethylaminobenzaldehyde was reported to be high enough to act as a quencher of the emitting species in the sphere of influence of the quenching moiety. This fluorescence quenching effect became clear later using the azacrowned compound. The remarkable inhibition of chemiluminescence due to the amino group led to the idea that if the electron-donating ability of the amino group is modulated by metal chelation, the chemiluminescence efficiency will be controlled in the presence of metal cations. The azacrowned 2 benzylidenacridan (2) seems to be a suitable one that plays such a role, because there are some examples in which the interaction between the azacrown cavity and metal cations modulates the fluorescence intensity of the fluorescent azacrowned compounds. Before the chemiluminescence measurements, we completed a spectral study to reveal whether the azacrown moiety acts as a metal cation recognition site. The drastic increase in the fluorescence intensity of 2 at the high calcium ion concentration was observed, but not for Ie, indicating that the incorporation of the calcium ion into the azacrown moiety inhibited the intramolecular fluorescence quenching because the benzylidenacridan is highly polarized in the excited states, in which the azacrown moiety behaves as an electrondonor site. The binding constants (Ks) of metal cations and 2 in acetonitrile were 2 estimated and it was found that log Ks of the divalent metal cations, Ba + (3.30), Mg2+ 2 100 (3.01) and Ca + (2.71), were higher than those of the univalent ones, such the alkaline metal cations, Lt (l.98), Na+ SO (l.l7), and K+ (2.03). The pronounced 2 interaction of Ca + in the aza-15-crown-5 is known to increase the fluorescence 7 intensity of some fluorophores. The effect of several metal cations on the chemiluminescence efficiency of 2 was ~ 40 investigated using an alkaline hydrogen peroxide-acetonitrile system for the singlet cO oxygen generation. The treatment of 2 with tetrabutylammonium hydroxide and aqueous hydrogen peroxide in acetonitrile a produced only a slight light emission. However, the addition of calcium Fig.2 Otemilmrune,eence quanttul1 yield (c:0 perchlorate led to an enhancement of the of 2 in the presence of metal cations. emiSSIOn intensity in contrast to the absence of such an effect for Ie, which support the fact that the incorporation of the

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metal cation and the azacrown moiety also works well in the chemiluminescence system. The enhancement became greater with the increasing metal cation concentration, and Mg 2+ and Ca2 + were much more effective than the others (Figure 2) as expected from the binding constants between the metal cations and 2 (vide supra). The enhancing effect on the chemiluminescence efficiency by the metal cations is dependent on the change in the fluorescence lifetime of the NMA, the emitter, because the addition of an excess amount of calcium ion to the mixture of NMA and the azacrowned benzaldehyde, the fragments from the intermediate 1,2-dioxetane, led to prolongation of the fluorescence lifetime ofNMA. In conclusion, we observed a remarkable quenching effect of amino groups on the chemiluminescence of 9-benzylidene-IO-methylacridans bearing electron-donating groups at the benzyliden moieties. This effect was applied to establish a metal cation recognition system using the benzylideneacridan with an azacrown moiety. Chemiluminescence intensity was enhanced in the presence of a few divalent matal cations, and this provides the basis of a metal cation sensing system.

ACKNOWLEDGEMENTS The authors are thankful for the financial support by a Grant-in-Aid for the GCOE Research and the basic research (19550137) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. REFERENCES I. Adam W. Four-membered ring peroxides: 1,2-dioxetanes and a-peroxylactones. In: Patai S. Ed. The chemistry of peroxide. New York: Wiley. 1983 :829-920. 2. McCapra F, Beheshti I, Burford A, Hann R. A, Zaklika K. A. Singlet excited states from dioxetan decomposition. JCS Chern Commun 1977;944-6. 3. Chris L, Singer LA. Structural effects on the intramolecular electron transfer induced decomposition of a series of 1,2-dioxetanes derived from 9-alkylidene10-methylacridans. J Am Chern Soc 1980;102: 3823-9. 4. Perkizas G, Nikokavouras J. Substituent effect on the chemiluminescence quantum efficiency of some acridan derivatives. Monatsh Chern 1983;114:3-11. 5. Perkizas G, Nikokavouras J. Substituent effect on the chemiluminescence quantum efficiency of some acridan derivatives. Chemiluminescence quenching. Monatsh Chern 1986;117:89-95. 6. McKeown E, Waters W A. Chemiluminescence as a diagnostic feature of heterolytic reactions which produce oxygen. Nature 1964;203:1063. 7. Crochet F, Malval J-P, Lapouyade R. New fluoroionophores from aniline dimer derivatives: a variation of cation signaling mechanism with the number of amino groups. JCS Chern Commun 2000;289-90.

EFFECTS OF 1,4-BUTANEDIOL DIMETHACRYLATE ON HL-60 CELLS METABOLISM G NOCCA,I P DE SOLE,! F DE PALMA,! GE MARTORANA,! C ROSSI,! P CORSALE,! M ANTENUCCI, 1 B GIARDINA, 1,2 A LUPI,2 I Biochemistry and Clinical Biochemistry Institute, School of Medicine, Catholic University, Rome, Italy: 2Istituto di Chimica del Riconoscimento Molecolare, CNR, Rome, Italy Email: [email protected]

INTRODUCTION The polymerization of methacrylates present in dental composite resins is never complete l and when monomers such as 1,4-butanediol dimethacrylate (BDDMA) urethane dimethacrylate (UDMA) or others are converted to the high-molecular-weight networked solid, residual unreacted molecules remain trapped into the structure 2 Therefore such incomplete conversion causes, together with a reduction of mechanical strength,3 the release of monomers that may implement adverse effects in the organism, i,e, allergic reactions,4 systemic toxicity, cytotoxicity, estrogenicity and mutagenicity, 1 Since the intracellular mechanisms of the aforesaid effects are still not completely clear, we were interested and stimulated to investigate the biochemical interactions between methacrylates and human cells, In the last years, a few researches about the effects of some methacrylic monomers (TEGDMA, Bis-GMA) on cellular parameters like lipid metabolism, glutathione (GSH) concentration, reactive oxygen species (ROS) production, cell cycle and mitochondrial activity have been reported,5 So far, very little information is however available on the effects of UDMA and BDDMA on cell metabolism. The present work is therefore concerned about the effects of UDMA and BDDMA on cellular energetic metabolism (oxygen consumption rate, glucose consumption, and lactate production). The well characterized promyelocytic HL-60 cell line has been here employed because of its sensitivity to cytotoxic, metabolic and differentiating agents which may be also present in dental materials,6 During chemically induced differentiation, for example by all-trans retinoic acid (ATRA),7 HL-60 cells stop growing and acquire the ability to respond to antigenic stimulation through a respiratory burst, a process assayed in this study by means of a chemiluminescence technique, MATERIALS AND METHODS Preparation of all-trans retinoic acid and methacrylate solutions. Stock dimethyl sulfoxide (DMSO) solutions of UDMA (from 27,5 mmollL to 220 mmollL), BDDMA ( 0.2 mollL and 0.4 mollL) and ATRA (1,00 mmollL) were prepared immediately before use. As a general procedure, one of the above-mentioned solutions (1.0 ilL) was added to 200,000 HL60 cells in 1,0 mL of RPMI 1640 medium. Cell viability. Exponentially growing HL-60 cells, 15 x 106 in 75,0 mL of RPMI 1640, were incubated with either A TRA, or UDMA or BDDMA for five days, The total cell number was determined every day using the trypan blue dye exclusion test. Both the cellular proliferation - as area under curves (AUC) - and cellular mortality were calculated,

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Differentiation assay. The respiratory burst of methacrylate-treated cells was assessed through chemiluminescence technique (CL). Exponentially growing HL-60 cells, 15 x 10 6 in 75.0 mL RPMI 1640 were incubated (5 days) with ATRA (1.00 x 10-3 mmol/L) or UDMA (27.5 x 10-3 mmol/L and 55 x 10-3 mmol/L ) or BDDMA (0.2 mmmollL and 0.4 mmolfL). Experiments were performed as already reported. 6 • 6 Oxygen consumption rate in intact cells. Exponentially growing HL-60 cells (20 x 10 ) in 3 RPMI 1640 (100.0 mL) were incubated with ATRA or UDMA (55 x 10- mmollL ) or BDDMA (0.4 mmolfL) (I h at room temperature), washed (PBS solution with neither Ca++ nor Mg++). Experiments were performed as already reported 6 Determination of cellular glucose consumption. Exponentially growing HL-60 cells (1 x 106 ) in RPMI 1640 (5.0 mL) were incubated with either ATRA or UDMA or BDDMA (0.2 mmmollL and 0.4 mmolfL). Cellular glucose consumption was evaluated as already reported 6 . Determination of cellular lactate production. Lactate concentration changes were evaluated on culture medium by lH nuclear magnetic resonance analysis (NMR Gemini 300 spectrometer, Varian, Palo Alto, CA). Experiments were performed as already reported. 6 Statistical analysis. All results were expressed as means ± standard error of the mean (SEM) taking into consideration at least three different experiments performed in duplicate. The means were compared by analysis of variance (ANOVA), p<0.05 was considered significant. RESULTS Cell viability. UDMA induces a fall in the cellular proliferation rate compared to control at a 3 concentration of 27.5 x 10- mmolfL (p
Cellular proliferation in presence of UDMA Cellular proliferation in presence of BDOMA

2500,--------~

--Control 2000

- - UDMA 27.5 J.lmoVL UDMA 55 J.lmoVL - - UDMA 110 J.l.mollL -+- UOMA 220 J.lmollL

1000

-Control _ATRA

2000

~ 1500

- - BDDMA 0.2 mmoVL BDDMA 0.4 mmoVL

soo

days

Days

Fig. 1. Cellular proliferation in presence of monomers.

Effects of 1,4-Butanediol Dimethacrylate on HL-60 Cells Metabolism

243

Differentiation assay. Chemiluminescence analysis showed that all monomers induced a comeback of oxidative burst in HL-60 cells. This differentiating effect is present at 55 x 10.3 mmollL UDMA and 004 mmol/L BDDMA (Fig. 2)., i.e. at the same concentrations at which the monomers increase cell mortality (as above).

B =Butanediol DMA U =Urethane DMA

Fig. 2. CL analysis of monomers treated cells. measurements in intact cells. Methacrylic monomers unlike ATRA cause a significant reduction of oxygen consumption rate by cells (p<0.05) (Fig. 3).

3. Oxygen consumption rate in intact cells. Glucose and lactate determination. After 24 h incubation, no evidence of glucose consumption modification was present (data not shown), while after 48 h cells treated with monomers showed a significant increase of glucose consumption (p< 0.01) (Figo4). HL-60 cells treated with monomers showed a statistically significant increase of lactate production (p
244 Nocca G et al. lactate production

Glucose consumption (48 h)

ATf\A

aDDMA

OUDMA

4. Glucose consumption and lactate production metabolic modifications due to a maturation process induced by monomers. The recovery of ROS production capability by cells following antigenic stimulation has been further utilized as a differentiation marker. The alterations of the energy metabolism of treated cells were detected analysing both the decrease of oxygen consumption and the increase of that is easily understood considering the close relationship between the two UDMA and BDDMA monomers induce an increase in and on alteration of glucose metabolism in HL-60 cells. This study can therefore to understand the biochemical mechanisms responsible for the cytotoxicity of the considered and underlines the importance of assessing such knowledge to the biocompatibility field in order to promote the development of better and safer dental materials.

REFERENCES

2.

3. 4. 5. 6.

7.

Geurtsen W. Biocompatibility of resin-modified filling materials. Crit Bioi 2000;11:333-55. Elliott JE, Lovell LG, Bowman CN. Primary cyclization in the of bisGMA and TEGDMA: a modelling approach to understanding the cure of dental resins. Dent Mater 2001; 17 :221-9. Peutzfeldt A. Resin composites in dentistry: the monomer system. Oral Sci 1997;105: 97-116 Hensten-Petterssen A. Skin and mucosal reactions associated with dental materials. Eur Or Sc 1998;106:707-12. Lefeuvre M, Amjaad W, Goldberg M, Stanslawki L. TEGDMA induces mitochondrial and oxidative stress in human gingival fibroblasts. Biomaterials 2005;26:5130-7. Nocca G, De Palma F, Minucci A, et al. Alterations of energy metabolism and resins. glutathione levels of HL-60 cells induced by methacrylates present in Dent 2007;35: 187-94. Collins SJ, Gallo RC, Gallaher RE. Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 1977;270:347-9.

DETERMINATION OF PYROGALLOL BY IMIDAZOLE CHEMILUMINESCENCE ENHANCED WITH HYDROGEN PEROXIDE

OSAMU NOZAKI,I MOTONORI MUNESUE,2 HIDEYUKI MOMOI,3 MOTOHIRO SHIZUMA,4 HIROKO KA WAMOTO,5 TADASU IKEDA 5 1Dept

of Clinical Lab Med, Kinki Univ School of Med. Osaka-Sayama 589-8511, Japan, 2 Chemco Scientific Corporation Limited, Osaka 530- 0016, Japan,

3Sun Plastics Co. Ltd. Higashi-Osaka, 577-0065, Japan, ~Technochemistry Department, Osaka Municipal Technical Reserach Institute, Osaka, 536-8553, Japan, 5Tottori Univ. School of Med, Yonago 683-8503, Japan;

E-mail: [email protected] INTRODUCTION Pyrogallol (1,2,3-trihydroxybenzene) is a plant polyphenol that has antioxidant activity. Boronic acids have been used as the enhancers for assaying pyrogallol by chemiluminescence (CL).I The flow injection (FI) horseradish (HRP) catalyzed imidazole CL was studied for assay of hydrogen peroxide (H 20 2),z The aim of this study is to investigate the assay of pyrogallol using FI-imidazole CL, and enhancement of the pyrogallol CL with H 2 0 2 . The principle of the assay of pyrogallol by imidazole CL is as follows. a) pyrogallol + imidazole + dissolved oxygen

-->

imidazole hydroperoxide

b) imidazole hydroperoxide + HRP ....... light emission. MA TERIALS & METHODS HRP embedded monolith reactor. The HRP embedded monolith reactor was prepared by irradiating it with UV light (360 nm) for 30 min at room temperature with the mixture of 2-hydroxy-2-methy I-l-pheny Ipropan-l-one- 3-methacryloxypropyl -trimethoxysilane, ripoxy SP-1507, sorbitan and horseradish peroxidase (HRP) in the Hematocrit glass tube. Determination of pyrogallol. Pyrogallol was assayed by HRP catalyzed imidazole chemiluminescence coupled to the micro-flow injection system at room temperature. Pyrogallol specimens (50 ilL) were injected using an autosampler (AS-950, JASCO, Tokyo, Japan) every five min into a stream of water (I 00 uL/min) using a HPLC pump (PU-980, JASCO), and the other mobile phase (imidazole 100 mmollL in the Tricine buffer 50 mmollL, pH 9.3) was delivered at 100 ilL/min. The light emitted from the reactor tube was detected with a 245

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Nozaki 0 et al.

photomultiplier in the CL monitor (825-CL, lASCO), and the light intensity (relative light unit; RLU) was calculated with a data processor (LCSS-905, lASCO).

RESULTS AND DISCUSSION Chromatogram of pyrogallol, H 2 0 2 and mixture of pyrogallol and H 2 0 2 • Pyrogallol specimens (0.1 mollL, 50 J!L; #1-3), H2 0 2 (9.7 mmollL, 50 J!L; #4, 5, 6) and the mixture of the same pyrogallol and H2 0 2 specimens #1-6 (50 J!L, v/v = 50: 50; #7-9) were assayed by consecutive injection every five min without prior incubation of the specimens. The results are shown in Fig. 1. All of the specimens showed light emission, and the peaks of #3 (pyrogallol) and #4 (H 2 0 2 ) overlapped partially. The overlaping light emissions were caused by prolonged reaction of pyrogallol to produce imidazole hydroperoxide in the HRP reactor and prompt light emitting reaction of H 2 0 2 . In addition, the light intensity of peak #4 (H 20 2) was 6 times higher than the light intensity of peak #5 and 6. The light intensities of peak #7-9 (mixture of pyrogallol and H 20 2) were 4.7 times and 10 times higher than the light intensity of the peak of#1 and 2 (pyrogallol) and #5 and 6 (H 20 2), respectively. The shapes of the peaks of # 7-9 were more regular than the shapes of the peaks of #1 and 2. This meant that the mixture of pyrogallol and H2 0 2 were more promptly converted to imidazole hydroperoxide in the HRP reactor than pyrogallol alone.

7

8 9

4

1 23

M

Tirn.e (rn..ir1)

Fig. 1. Chromatogram of pyrogallo I , H 20 2 and the mixture of pyrogallo I and H2 0 2 • (#1,2,3: pyrogallol, #4,5,6: H 2 0 2 , #7,8,9: the mixture of pyrogallol and H2 0 2).

Determination of Pyrogallol by Imidazole Chemiluminescence

247

Influence of H 2 0 2 on enhanced chemiluminescence of pyrogallol. The mixtures of pyrogallol (2.5, 5.0, 7.5, 10, 12.5 !lmoIlL) and H20 2 (0.48, 0.97, 4.9, 9.7 mmollL) (v/v = 1:1, 50 !lL) were assayed by the FI-imidazole CL (Fig. 2). Specimens with a H 20 2 concentration < 0.97 mmollL showed no peaks, and the specimens of H20 2 (4.9 mmollL and 9.7 mmollL) showed peaks corresponding to the dose amounts. The peaks obtained covered the background light from the reaction of HRP, imidazole and H 20 2. The regression equation of the pyrogallol with addition of H 20 2 (9.7 mmol/L) was Y = -21.7 X2 + 472X + 216 (R2 = 0.949), and the regression equation of pyrogallol with H 20 2 of 4.9 mmollL was Y = -3.60 X2 + 212X + 178 (R2 = 0.945), where Y was light intensity (RLU) and X was pyrogallol. The increasing rate of light emission from pyrogallol of 12.5 !lmollL v.s 2.5 !lmollL when added H 20 2 was 9.7 mmollL was 1.6, and that the increasing rate of light emission was 2.3 when added H 20 2 was 4.8 mmollL. This meant that addition of 4.8 mmollL H 20 2 was most effective in increasing the light intensity of pyrogallol.

3.000

2.500

S

g 2.000

G' .;; c::

1.100

-5,

1.000

o /l202-9. 7mM o H202-4.85mM

.!l .5

... H202-0.97mM

:3

X 11202-0.48 mM

SOO 0 2

10

12

14

Pyrogallol (umolfL)

Fig. 2. Influence of concentration of H 2 0 2 on enhancement of detection of pyrogallol by imidazole chemiluminescence.

CONCLUSION Pyrogallol showed enhanced light emission in the presence of H20 2 • The reason was determined to be that H 2 0 2 promoted the oxidation of pyrogallol with imidazole.

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REFERENCES 1.

2.

Nozaki 0, Ji X, Kricka LJ. New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin. J Biolumin Chemilumin 1995; 10: 151-6. Nozaki 0, Munesue M, Kawamoto H. Determination of serum glucose by horseradish peroxidase-catalysed imidazole chemiluminescence coupled to a micro-flow-injection system. Luminescence. 2007;22 :40 1-6.

CHEMILUMINESCENCE STUDY ON THE REGULATION OF NADPH OXIDASE ACTIVITY BY THIOREDOXIN REDUCTASE IN VASCULAR ENDOTHELIAL CELLS XUN SHEN and ZHEN-BO LIU

Institute

0/ Biophysics, Chinese Academy a/Sciences, Beijing 100101, China

INTRODUCTION The endothelium, which consists of a single layer of endothelial cells, lines the inner surface of all blood vessels and the heart, forming an important interface with circulating blood. The vascular endothelial cells have well-established roles in cardiovascular homoeostasis and the initiation of the inflammatory process such as artherosclerosis. It is now accepted that the expression of some inflammatory genes, regulated by reactive oxygen species (ROS), is considered as the molecular mechanism of the early atherosclerosis. A major source of ROS in vascular cells is 1 the NAD(P)H oxidase, which consists of the membrane subunits gp91 phox and p22phox and the cytosolic subunits p67phox, p47phox, and the small GTPase racl. Mechanisms that control activity of this multisubunit enzyme complex are incompletely understood. Thioredoxin reductase (TrxR) is an antioxidant enzyme that participates in thioldependent cellular reductive processes. 2 Since the enzyme regenerates reduced thioredoxin that serves as reducing equivalent and may also scavenge ROS, TrxR has been considered as an enzyme to reduce the ROS level in cells including vascular endothelial cells. However, we studied the effect of TrxR on the NADPH oxidase activity (using a chemiluminesence method) of either overexpressing TrxRl or knockdown the endogenous TrxRl in the an endothelial cell line, and found that TrxR actually promotes ROS generation by upregulating the activity of NADPH oxidase. MATERIALS AND METHODS Cell culture and transfection. The endothelial-like cell line EA.hy926 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 UlmL penicillin and 100 g/mL streptomycin at 37°C in a 5% CO 2 -incubator. The cells overexpressing TrxRI were established by stably transfecting the TrxRexpressing vector pIRESne02-TrxRI, which contains the neomycin resistance gene and the wild-type human TrxRl gene, and selected with G418 for 4 w. The cells expressing only neomycin resistance gene were used as control. In the experiments with the TrxRI-cells, the cells and their controls were incubated in the medium supplemented with 40 nM sodium selenite for 48 h before usage. The Trxlknockdown cells were obtained by transfection of 150 nM TrxRI siRNA (5'AACACGTGCTTGTGGACATCA-3') for 72 h. The cells transfected with a scramble RNA were used as control. The cells having their endogenous p22phox 249

250

Shen X & Liu Z-B

knocked

down

were

obtained

by transfecting siRNA

for

"A'''"''~ (HRP)-catalyzed chemiluminescence detection of the intracellular ROS,3 The HRP-Iuminol solution was prepared by an of horseradish peroxidase (HRP) in luminol (0.1 The 6 0".lt"'nm,a 2 mL of cell suspension (10 cells/mL) was in a homemade counter, and then, the photon emission from the cell was recorded before and after addition of the HRP-Iuminol solution. The emission reached after addition of HRP-Iuminol solution rp,rlrP":p,nt,, level in cell "'U"IJIO"'"l'''

RESULTS AND DISCUSSION and in cells. To know if any role in regulation of NADPH oxidase in vascular EAhy926 cell line was established and is referred as cells. The activity of TrxR 1 in the stably transfected cells was determined as a DTNB reduction rate after subtracting the contribution of other PtH\nr,(\TPlnC in the presence of the TrxRl inhibitor gold thioglucose. As lA the activity of TrxRl in EAhy-TrxRI cells (the was 34% than that in control (the cells expressing resistance gene, and referred as EAhy-neo cells). The Western blot cells also showed a 33% higher expression of TrxRl in the and

TrxRl p-actin

Time (min)

and expression of TrxRl in TrxRl-overexpressing cells. (A) The of TrxRl was determined by reduction rate of DTNB, the substrate of TrxR. The relative TrxR activity in corresponding cell extracts after correction for the DTNB reduction in the presence of gold thioglucose. >I< indicates P
The and

Chemiluminescence Study on the Regulation ofNADPH Oxidase Activity 251

the controls the cells transfected with pIRESne02) was recorded It is clear that the enhanced chemiluminescence is very sensitive to the ROS in a cell and overexpression of TrxRl enhanced intracellular generation of ROS in endothelial cells. The TrxRI-enhanced EeL almost disappeared when the inhibitor of NADPH oxidase, was added into the cell suspension, indicating that the TrxRI-enhaced ROS generation might be connected to activation of NADPH oxidase.

Time(s)

2. chemiluminescence from the cells overexpressing TrxRl and their controls (expressing only neomycin resistance gene). HRP-Iuminol solution was added in cell suspension 100 s after the chemiluminescence was recorded. At 400 s, the NADPH oxidase inhibitor, was added into the mixture. of the NADPH oxidase subunit p22phox TrxR1. Increased ROS generation in TrxRl-overexpressing cells only suggests a possibility that thioredoxin reductase might upregulate the activity of NADPH oxidase. To know if and how thioredoxin reductase regulates NADPH oxidase activity, the level of the membrane subunit of NADPH oxidase, p22phox, in either _,n,pr{'vnrpccm,a or TrxRl-knockdown cells was determined. Western blot showed that thioredoxin redactase 1 upregulates the expression of the NADPH oxidase subunit (see Fig. 3). It was found that over-expression of TrxRl led to while knockdown of the endogenous TrxRl down-regulated the

Protein level of p22phox in TrxRl-overexpressing cells (A) and the cells their TrxRl knocked down (B). The cells transfected with gene (EAhy-neo cells) and the cells transfected with a scramble RNA (Mock RNA) were used as control respectively.

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expression of p22phox in the endothelial cells. It was interestingly noticed that the upregulation of p22phox in TrxRl-overexpressing cells can be abolished incubation of the cells with inhibitor of thioredoxin reductase, It suggests that the expression ofp22phox is dependence ofTrxRl in the p22phox-knockdown cells. To that the increased ROS generation in TrxRI-overexpressing cells is due to increased of siRNA for p22phox was transfected into EAhy.926 cells to knock down endogenous p22phox, and to see if ROS generation is reduced. The results are shown in 4. It can be seen that knockdown of p22phox reduced intracellular ROS generation detected by chemiluminescence. Based on the observation nr<>ccir," of TrxRI upregulated p22phox expression and intracellular fi""H>r.Htrm as well as knockdown p22phox expression reduced ROS it is reasonable to suggest that thioredoxin reductase 1 ROS by upregulation of, at least, of the

IIIIII~II~

II-actin

Times (8)

Chemiluminescence detection of the ROS knockdown CONCLUSIONS Thioredoxin reductase upregulates activity of NADPH oxidase expression of its subunit p22phox. Enhanced chemiluminescence as a sensitive method to detect intracellular ROS generation. REFERENCES 1. Shah, AM. NADPH oxidase and endothelial cell function. 09:217-26. 2. A. Thioredoxin. Annu Rev Biochem 1 3. Kricka, Moseley SR, Whitehead TP. Phenols as enhancers of the chemiluminescent horseradish peroxidase-Iuminol-hydrogen reaction: application in luminescence-monitored enzyme Chern 1 31:1335-41.

QUANTITATIVE DETECTION OF SINGLET OXYGEN WITH A CHEMILUMINESCENCE PROBE DURING PHOTODYNAMIC REACTIONS Y ANCHUN WEI, DA XING', SHIMING LUO, WEI XU, QUN CHEN MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou, China, E-mail: [email protected]

INTRODUCTION Chemiluminescence is often used to detect qualitatively or quantitatively, microelements, free radical and other biologic or pharmacologic molecules.'·' Active oxygen species, especially singlet oxygen e02), are important cytotoxins.' 10 2 can depress cell activity and even induce cell death by oxidizing lipid, proteins and DNA; for example, in most photodynamic therapies (PDT) 102 is produced as the main cytotoxin; 4 cells often respond to wounding or stress by presenting abnormal 10 2 levels. s Thus 10 2 often needs to be detected as an important measure of the harm to a cell. There are many methods to evaluate whether 10 2 is present and how much was produced. A 102- CL method which uses a chemiluminescence probe molecule to chemically interact with 10 2 and results in the production of excitated energy state products has been reported as a method of 10 2 detection.' However, the CL method also has shortcomings, such as the inefficiency of CL reactions. We have considered ways to improve CL detection. Thus during 10 2 detection with CL, correcting CL was considered. To implement this concept, 102 was measured by FCLA in a photodynamic reaction to achieve correction. We have previously reported that the CL probe, FCLA, which can selectively detect singlet oxygen and superoxide. 7 Here CL is measured and analyzed at different probe concentrations and a method of making the detection precise is discussed. MATERIALS AND METHODS ROS specific chemiluminescence probe FCLA (Tokyo Kasei Kogyo Co., Tokyo, Japan) (100 IlmollL pH7.0) Photosensitizer Protoporphyrin IX disodium salt (PPIX, Aldrich Chemical Co., Milwaukee, Wisconsin) was prepared according to the manufacturer's directions to a concentration of200 IlmollL. The irradiation source for the photosensitization reaction is a custom-built, gated diode laser system (lOOmW, 635 nm, LDC 2000, ThorLab; TEC2000, Wavelength Electronics, USA); and filters (FF500/646Beamsplitter, Semrock Co. USA and 510 nm and 530 nm band-pass filter, Oriel Co., USA.) were used to protect the PMT from scattered irradiation light. The fluorescence was measured using a photon multiplier tube (MP952, PerkinElmer Optoelectronics, Germany) with a counter (PCL-836, Advantech Co., Ltd. Taiwan). The irradiation and fluorescence system is synchronized and controlled by Labview (Labview version 6.1 National Instruments, USA). The laser system is controlled by the TTL level of the counter. 253

254

Wei Y et al.

FCLA was prepared in various concentrations for quenching experiments. PPIX 2 concentration was 10 !AomollL. The laser (10m Wfcm ) irradiated the two reagents at once after mixing them. Signal was record simultaneously. Quenching of FCLA excitation state by FCLA oxide. The photodynamic reaction was performed with PPIX 5 !AomollL, FCLA 2 !AomolfL and a laser setting of 20 mwfcm 2 . Fresh FCLA was used for the first experiment, and then the same reaction was done with added oxidized FCLA 2 !AomollL in solution. The experiment was repeated three times. 2000

1200

400 ~~r-~r-~~~~-'~~~

~EL. ~ '.0

d '~..

20

••

40 S

50

/, ""'"

0.0010.20.30.4 0.5 246810121416182022

FCLA ("M)

Fig. 1. Relationship of chemiluminescence and probe concentrations in the photodynamic reaction. Fig.2A: CL with 0.075 and 20 !AomollL FCLA; Fig.2B: initial CL with different FCLA concentrations (0.05- 20 !AomollL). RESULTS AND DISCUSSION Fig.lA shows the different probe concentration have distinctive luminescence courses. Both the primary intensity and the decline course are different. In Fig. 1B the CL intensity influenced by FCLA concentrations is shown. The results indicates that the CL declined when the reagent concentration was > 1 !AomolfL. Thus the CL is influenced by the concentration of the FCLA reagent. In other experiments the light absorption at 532 nm and 635 nm was measured. The values were very low although the concentration increased. So the absorbtion influences CL relatively little. As the probe concentration increased the odds of molecular collision increased, thus more energy was transferred intramolecularly. This is the reason for the decline in CL intensity. Fig. 1 indicates fresh FCLA can quench luminescence; but FCLA oxide can also quench CL (Fig. 2). The figure indicates that oxidized FCLA retained its quenching characteristics. Considering the similar molecule structure, the quenching ability of both must be same and the quenching rate should still be stable during the FCLA depletion. Thus the self-quenching to CL can be ignored if using an appropriate proportion to reflect singlet oxygen.

Quantitative Detection of Singlet Oxygen with a Chemiluminescence Probe

CL

corrected.

255

by FCLA oxide in solution. (means±S.E)

the reaction rate which is related with the probe concentration must be Lineweaver-Burk equation, the formulas of correcting CL was ) can be corrected as

and [FCLA]o is t and 0 time FCLA concentration; It is t time CL is solution volume and t> is system proportion coefficient; k is FCLA constant; NA is Avogadro' number. Using the equation, the detected CL was corrected. 3 shows CL and its corrected CL. Comparing the direct and the latter falls slowly with the probe concentration depletion been The result indicates that direct detected CL signal was incorrect due to depletion and could not be precisely related to singlet oxygen. Here the CL accumulation is corrected from 2.96 x 106 to 4.57 X lOti. In conclusion, CL can reflect singlet oxygen in photodynamic reactions; but some factors will affect the CL, including light absorption, self-quenching and concentration

1(JO

150

time(s}

3. FCLA 6Jl mol/L, PPIX 10 Ilmol/L, laser 10 mWlcm 2 . In the correction, the

256

Wei Yet al.

constant k is 10 6 morl-L and the coefficient -L is 1.4 NAV

X

10- 11 mollL

depletion. So some measures have to be taken to correct for these factors. The result indicates that with the correction, more precise singlet oxygen detection can be made to improve CL monitoring.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30470494; 30627003) and the Natural Science Foundation of Guangdong Province (7117865).

REFERENCES 1. 2.

3.

4. 5.

6.

7.

Dodeigne C, Thunus L, Lejeune R. Chemiluminescence as a diagnostic tool. A review. Talanta 2000:51 :415-39. Wang J, Xing D. Detection of vitamin C-induced singlet oxygen formation in oxidized LDL using MCLA as a chemiluminescence probe. Acta Biochim Biophys Sin 2002:34: 11-5 Oleinick NL, Morris RL, Belichenko I. The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 2002:1:1-21. Sharman WM, Allen CM, van Lier JE. Photodynamic therapeutics: basic principles and clinical application, Drug discovery today 1999:4:507-17. Chen WL, Xing D, Tan S C, Tang YH, He YH. Imaging of ultra-weak bio-chemiluminescence and singlet oxygen generation in germinating soybean in response to wounding. Luminescence 2003;18:37-41. Qin YF, Xing D, Zhou J, Luo SM, Chen Q. Feasibility of using fluoresceinyl cypridina luciferin analog in a novel chemiluminescence method for real-time photodynamic therapy dosimetry, Photochem Photobiol 2005:81: 1534-8. Wei YC, Zhou J, Xing D, Chen Q. In vivo monitoring of singlet oxygen using delayed chemiluminescence during photodynamic therapy. J Biomed Opt 2007:12:1-7.

FLOW-INJECTION CHEMILUMINESCENCE DETERMINATION OF HUMAN SERUM ALBUMIN BASED ON FLUORESCEINYL CYPRIDINA LUCIFERIN ANALOG- 10 2 REACTION WEI XU, YANCHUN WEI, DA XING,SHINGMING LUO, QUN CHEN MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510631, China, E-mail:[email protected]

INTRODUCTION The concentration of human serum albumin (HSA) is an important biomarker and quantitative analysis of HSA in urine can provide critical information for early diagnosis and treatment of nephrosis. The most commonly used methods for analysis of micro-concentrations of HSA, are the Lowry, CBBG-250,'" el ectrochemiluminescence, spectrophotometry / fluorospectrophotometry,' Ray leigh light scattering methods,,6 and chemiluminescence.' Among them, chemiluminescence (CL) especially coupled with flow injection analysis (FIA) is considered as the most sensitive and versatile analytical technique. It is characterized by high sensitivity, a large dynamic range, minimum background interference, and good reproducibility. The conventional methods used a quenching effect of proteins for quantitative measurements.' Our proposed technique based on CL enhancement effect effectively determined low concentrations of HSA. In comparison to the CL quenching technique, the method significantly improves the detection sensitivity (ca. 1DO-fold higher). The purpose of this work is to use the CL enhancement technique coupled with FIA to determine HSA in a fluoresceinyl Cypridina lucifer in analog (FCLA)_I02 system, and is based on previous work that CL from the FCLA- 102 system can be strongly enhanced in the presence ofHSA. A simple and fast flow injection analysis has been developed for the determination of HSA, which has been satisfactorily applied to analyze clinical urine samples. METHODS All reagents were of analytical grade or the best grade available. Stock solution of 1 x 10-4 moUL FCLA purchased from Tokyo Kasei Kogyo Company (Tokyo, Japan) was prepared by dissolving 1 mg FCLA in 15 mL water deoxygenated by N2 bubbling and stored at refrigerator (-20°C). The schematic of the flow system used in this work is shown in Fig. 1. There are two peristaltic pumps (A and B). Pump (A) was used to deliver the flow streams of FCLA and hydrogen peroxide, and pump (B) was used to deliver merged stream of either sample or standard of HSA and sodium hypochlorite. FCLA solution (75 JlL) was injected into the carrier stream through an eight-way injection valve equipped with a 75 JlL sample loop, and then it was merged with the mixture solution of sample and sodium hypochlorite, finally reached the flow cell to produce CL emission. The CL signal produced in the flow cell was detected and recorded with a computerized luminescence analyzer MPI-B purchased from Remax ElectronicScience and Technology Company (Xi'an, China). 257

258

Xu Wetal.

Fig. 1. Schematic diagram ofCL flow system. (a) FCLA solution; (b) hydrogen peroxide solution; (c) sample solution or standard solution;(d) sodium hypochlorite; PMT photomultiplier tube; PC personal computer; HV high voltage power

RESULTS Interference studies. We examined the potential interference by basic amino acids, glucose, certain ions commonly found in urine samples (Table 1). The acceptable deviation of the measurement result should be less than 5% of that with HSA only. The results indicated that, up to 20-fold NaCI, MgS04 , NaH 2P0 4 , oxalate, 50-fold glucose, IO-fold KN03 and some amino acids up to the clinically observable maximum concentrations, had no practically significant influence on the CL measurement results. Calibration curve and detection limit. A representative calibration curve characterizing the relationship between CL and HSA concentration is shown in Fig. 2. The calibration curve obeyed a second order equation: M CL = 262.39 + 38.69 IgCHsA + 1.84 (lgC HSA)2 The regression coefficient was R2>0.99. However, the linear dynamic range was 1 x 10- 10 to 1 x 10- 8 mol/L and was expressed by the first-order equation: MCL=116.51 + 5.84 IgC HsA (R 2=0.99, n=7), with a detection limit of 4.5 x 10- 11 mollL (SIN = 3). R.S.D for the consecutive CL detection of without HSA was 3.28%(n=11).

=!

~

T

70

~

TIl .i. .1.

1 "" 1

Log concentration of HSA {mol {.")

8

2

Fig. 2. Calibration curve for HSA. Conditions: FCLA, I x 10- moUL; NaCIO, 3.5 x 10mollL; H 2 0 2 , 1 x 10-2 mollL; PBS 7.4; negative high-voltage, -800 V; flow rate, 2.4 mLimin; injection volume 75 flL.

Flow-Injection Chemiluminescence Determination of Human Serum Albumin

259

Table 1. Effect of common substances in urine samples on CL measurement Interfering substance

Concentration IO·lrnoi/L

Change ofCL (%)

Na'H,PO;

511.8

-4.4%

Na+Cr NH:S,O,'Oxalate

226.7 533.2 1.2 300 200 b 18.5 50 b 480 b JOO b 25 b 8b

-8.0% 4.3% 2.4% 3.1% -4.0% -3.5% -3.3% 2.7% -4.1% 1.2% 4.9%

K~O;

Uric acid Glucose L-Arg L-G1y L-Val L-Tyr L-Trp a

Ion concentration

in urine(IO" mol IL)

PO/ 8.5 Na+ 86.7 cr 113.3 NH; 13.3 0.06 K+34 -------

0.4 -------------

--.-----

---------------

R.S.O' 4.6% 2.5% 3.7% 3.5% 1.0% 3.4% 4.6% 2.8% 4.4% 3.2% 2.5% 4.4%

Each expenment was repeated 4 tImes,. b ,ug/mL

Mechanism of CL enhancement. According to Forster's theory, energy transfer is a distance dependent interaction between the different electronic excited states of molecules in which excitation energy is transferred from one molecule (donor) to another molecule (acceptor). Fig. 3 shows that fluorescence of HSA at 348nm decreased with increasing FCLA concentration. The energy from excited-state HSA maybe be transferred to FCLA or be lost in a non-radiative manner.

(al

800

:i

~ :;;c

GOO

~

5

.;;; 400

.

"c "

III

~ u::"

200

300

350

400

450

soo

550

Wavelengthlnm

Fig. 3. HSA (2 xl 0-6 mollL) fluorescence spectra as a function of FCLA concentration (Aem =280 nm). Curves 1->5 : 0, 2 ,4,12, and 16x 10-6 mollL FCLA. Slit width are both 5 nm.

260

Xu Wet al.

Fig. 3 shows fluorescence intensity of FCLA in the presence of HSA (..l.ex=288 nm). Intensity increased greatly in the presence as compared to the absence of HSA in the same conditions as for the studies shown in Fig. 3. This provides strong evidence for the occurrence of Forster type energy transfer from the tryptophan moiety (donor) in HSA to the FCLA molecule (acceptor). We know that the distance from bonded-FCLA to Trp214 is less than 5 nm. Therefore, efficient energy transfer can exist between HSA (donor) and FCLA (acceptor).

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30470494; 30627003) and the Natural Science Foundation of Guangdong Province (7117865) and the US NIH grant POI-43892 REFERENCES 1. Marion MB. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72; 248-54. 2. Flores R. A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue. Anal Biochem 1978;88;605-11. 3. Chun Q, Ke AL, Tong SY. Spectrophotometric micromethod for protein determination with tetrachloro tetraiodofluorescein. Anal Lett 1998;31; 1021-36. 4. Li N, Li K A, Tong S Y. A novel protein assay method using tetraphenylporphin tetrasulfonate (TPPS4). Anal Lett 1995;28; 1763-74. 5. Li Y, Dong L, Wang W P, Xingguo Chen. Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample. Anal Biochem 2006;354;64-9. 6. Feng P, Huang CZ, Li YF. Direct quantification of human serum albumin in human blood serum without separation of y-globulin by the total internal reflected resonance light scattering of thorium-sodium dodecylbenzene sulfonate at water/tetrachloromethane interface. Anal Biochem 2002;308 ;83-9. 7. Huang CB, Zhang K, Ci YX. Sensitization of surfactants on the chemiluminesecence reaction of fluorescein isothiocyanate labeled proteins. J Biochem Biophys Methods 2007;70;341-7. 8. Zhou J, Xing D, Chen Q. Enhancement of fluoresceinyl Cypridina lucifer in analog chemiluminescence by human serum albumin for singlet oxygen detection. Photochem Photobiol 2006;82; I 058-64.

CHARGE-TRANSFER-INDUCED LUMINESCENCE (CTIL) MECHANISMS OF CHEMI- AND BIOLUMINESCENCE REACTIONS K YAMAGUCHI, H ISOBE, S YAMANAKA, M OKUMURA

Dept of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan Email:[email protected] INTRODUCTION Chemi- and bio-Iuminescence phenomena have been attractive not only from the scientific interest conceming with their molecular mechanisms but also from analytical, clinical, and for other various useful applications. Many theoretical studies have been performed to elucidate detailed mechanisms of chemi- and bio-Iuminescence reactions. In past decades, our group has carried out both broken-symmetry (BS) and symmetry-adapted (SA) molecular orbital (MO) studies to clarifY the electronic mechanisms of these reactions. Here, our basic concepts and methodologies, together with computational results, are briefly summarized.

THEORETICAL BACKGROUNDS Symmetry and stability analysis. The semi-empirical unrestricted Hartree-Fock (UHF) method was used for symmetry and stability analysis of chemical reactions at early stage of our theoretical studies. I ,2 The BS MOs for CT diradicals are also expanded in terms of composite donor and acceptor MOs to obtain the Mulliken CT theoretical explanations of their electronic structures. Instability in chemical bonds followed by the BS ab initio calculations is one of the useful approaches for elucidating electronic structures of active reaction intermediates and transition structures. 2 The concept is also useful to characterize chemical reaction mechanisms in combination with the Woodward-Hoffman (WH) orbital symmetry criterion,3 as illustrated in Figure 1. According to the Woodward-Hoffmann rule,3 there are two types of organic reactions: orbital-symmetry allowed and forbidden. On the other hand, the orbital instability condition is the other criterion for distinguishing between nonradical and diradical cases. 2 The combination of the two criteria provides four different cases: (i) allowed nonradical (AN), (ii) allowed radical (AR), (iii) forbidden nonradical (FN), and (iv) forbidden radical (FR). The charge and spin density populations obtained by the ab initio BS MO calculations are responsible for the above classifications as shown in Fig. I, No charge and spin separations appear in the case of AN because of closed-shell character, but spin separation (SS) (1 D is significant for FR case, although the charge separation (CS) (ffi 8) is rather weak, as in the case of homolytic diradical. On the other hand, the zwitterionic (ffi 8) CS is remarkable for FN case, and both SS and CS (1 +, -!) become important for AR case such as electron-transfer diradical reactions. In recent papers, we have performed the symmetry-stability analyses of chemi- and bio-Iuminescence reactions from these theoretical view points. 4,5 261

262

Yamaguchi K et at.

Allowed Radical (AR) CS and SS

:§

:eo

Forbidden Radical (FR) SS but no CS

----_ .. _--------- ----, -----------.-----------_ ..

Allowed Nonradical : Forbidden Nonradical (AN) : (FN) CS but no SS no CS and no SS j

Orbital Symmetry

Fig. 1. Classification of chemical reaction mechanisms by symmetry and stability criteria of molecular orbitals Molecular oxygen reactions. In accord with the classification in Fig. 1, we may expect four characteristic reaction mechanisms of singlet molecular oxygen (10 2) with C-C double bonds to generate dioxetane and dioxetanone, as illustrated in Fig. 2.1 The perepoxide (or 2s + 2a) type cycloaddition of 102 to olefin is regarded as one of the AN reaction paths. On the other hand, 1,4-zwitterion path (B) is regarded as FN, whereas 1,4-diradical path (D) is characterized as FR. The electron-transfer diradical path (C) corresponds to the AR case in our terminology. It has been demonstrated that these mechanisms are very useful for systematic understanding of chemical reactions of singlet molecular oxygen e02) with various olefins. The computational results have been already summarized in one chapter of the book. 1

I

8°'-......0

?X"/C;'/ :

'i>

I

"" "/c--c:;:::

--<';... ··0

0=0 "/c···~-···c:;:::

.°'-......0

I

. "/c--c:;:::

I (A) AN (8) zw (e) AR (D) FR Fig. 2. Four different reaction paths for the cycloaddition of 102 to olefin 4

Very recently, Isobe et al. ,5 have shown that the charge-transfer (CT) configuration in Fig. 2C play an important role of the intersystem crossing from triplet (T) molecular oxygen e02) to the singlet one via the spin-orbit (SO) interaction in the course of dioxetane (DO) (dioxetanone (DON» formation reactions.

The SA CAS CI calculations have been carried out to evaluate the SO coupling parameter at the curve crossing point between the T and S states.

MECHANISM OF CHEMI- AND BIO-LUMINESCENCE REACTIONS Decomposition reactions of dioxetane. Our BS MO calculations have shown that the least motion decomposition of dioxetane skeleton to generate directly the singlet

Charge-Transfer-Induced Luminescence Mechanisms

263

excited state of carbonyl fragmene is energetically unfavorable,' and alternatively the homolytic diradical species are generated thermally as shown in Fig. 3A. The heterolysis of the 0-0 bond was found to be feasible by the participation of metal ions in Fig. 3B.' The inter- and intra-molecular charge transfer (CT) diradical mechanisms become favorable if the electron donating property of donor (0) exceeds a certain limit.' Recently, Isobe et al. 4 ,5 located the transition structures (TS) of these CT diradical reactions and found that the activation barriers (OEt) for CT TS are significantly reduced, as compared with the homolytic diradical TS in Fig. 3A. Table 1 summarizes the OEt values for homolytic diradical and CT diradical TSs of several donor compounds. ~; o

(j)

eo

\ / -?c--c", (A) FR

(8)ZW

M(+)

0 .. :.... 0

0=0

-?c-c",

-;C-cv-

I -; I

(C) AR (inter)

I -; I

D+

"-

(0) AR (intra)

Fig. 3. Four different reaction paths for the decomposition of dioxetane Meta-para selection rules for chemiluminescence. Accumulated experimental results for efficiency of chemiluminescence have clearly demonstrated that meta-substituted aromatic rings emit efficiently like luciferin, but para-substituted isomers do not exhibit such a remarkable property. Meta-para selection rules for efficiency of chemiluminescence reactions have been explained by the orbital interaction modes; LUMO-LUMO interaction for meta Isomers and the LUMO-HOMO interaction for para isomers. 5 ,6 The selection rules were also confirmed by natural orbital analysis of the BS solutions. Thus it is concluded that the BS and SA MO theoretical calculations are very useful for theoretical illustration of the electronic mechanisms of chern i- and biD-luminescence reactions. Table 1. Activation energies and related quantities in the gas phase at the B3LYP/6-31+G(d) level ilEt+ ZPC roo YHOMO Molecule [%] [kcal mor'] [A] Dioxetanes 0-0

I

I

W,C-C.- H H H 0-0

I

I

H c-,c-q-CH:; R,C

I

c ..,c-q-CH

R,C

1.975

16.2

HD

21.7

2.068

25.5

HD

21.6

2.040

27.4

HO

CH, 0-0

I

H

16.4 b

Br

CH,Br

264

Yamaguchi K et al.

Tablel (continued).

~ ~H'

0-0 e (AMPPD)

fJ::::7

C

2.187

45.8

HD

1.822

4.6

CT

2.060

14.7

HD

19.5

2.126

24.2

HD

18.1

2.055

18.0

HD

0.9 d

1.657

0.4

CT

4.0

1. 713

1.0

CT

25.7

8.9

Dioxetanones

0-0

;....r--PhOH

ON:(H

00PJN=

Ph

(coelenterazine)

oblH'X)~(fi . ) 'N Ire fl y IUCI'fienn a

Reaction mechanism. b 6-31I+G(d). c 6-3IG(d). a 6-3I+G(d,p)//6-3IG(d).

ACKNOWLEDG EMENTS This work has been supported by Grant-in-Aid for Scientific Research from the Promotion of Scienece (JSPS).

REFERENCES J. Yamaguchi, K. Theoretical calculation of singlet oxygen reactions. In: Frimer AA 2.

3. 4.

5.

6.

Ed. Singlet Oxygen vol. III. Boca Raton:CRC Press, 1985;chapter 2. Yamaguchi K, Fueno T, Fukutome H. A molecular-orbital theoretical classification of reactions of singlet ground-state molecules. Chern Phys Lett 1973; 22: 461-5. Woodward RB, Hoffmann R. The conservation of orbital symmetry. Angew Chern IntEd 1969;8:781-853. Isobe H, Takano Y, Okumura M, Kuramitsu S, Yamaguchi K. Mechanistic insights in charge-transfer-induced luminescence of 1,2-dioxetanones with a substituent of low oxidation potential. J Am Chern Soc 2005; 127:8667-79. Isobe H, Yamanaka S, Kuramitsu S, Yamaguchi K. Regulation mechanism of spin-orbit coupling in charge-transfer-induced luminescence of imidazopyrazinone derivatives. J Am Chern Soc 2008;130:132-49. Takano Y, Tsunesada T, Isobe H, Yoshioka Y, Yamaguchi K, and Saito I. Theoretical studies of decomposition reactions of dioxetane, dioxetanone, and related species. CT induced luminescence mechanism revisited. Bull Chern Soc Jpn 1999;72:213-25.

A NOVEL SYNERGISTIC ENHANCER FOR HRP-LUMINOL-H 2 0 2 BASED CHEMILUMINESCENCE AND ITS APPLICATION IN IMMUNOASSAY XIAOLIN YANG, 1 XUDONG SUN I

2

People's Hospital of Peking University, Beijing100044, China 2China Medical Technologies inc. Beijing 100176, China Email:[email protected]

INTRODUCTION Para-substituted phenols and phenylboronic acids and derivatives of benzothiazole are the well-known enhancers of the HRP-luminol-H 2 0 2 chemiluminescence,1,2 and are widely applied in immunoassay as an essential component of the chemiluminescence substrate 3 for sensitive detection ofHRP labeled antibodies and antigens. However, no synergism for enhancement among them has been found so far, i.e., enhancement by a mixture of enhencers is greater than the sum of the enhancement by the enhancers alone. We have now discovered several compounds that play a synergistic role with common enhancers of the HRP catalyzed luminol--H 2 0 2 chemiluminescence reaction. MATERIALS AND METHODS Prostate specific antigen (PSA) chemiluminescence immunoassay kits, microwells and the luminometer were supplied by China Medical Technologies Inc. (Beijing, China). The enhancers, HRP, luminol, antipyrine and its derivatives (Fig. 1) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). All other reagents were AR grade commercial products. The enhancers and other reagents were diluted in a universal chemiluminescent solution - 20 mmollL Tris-HCI pH 8.7, I mmollL luminol, 1.5 mmoliL H 20 2 . HRP, and 100 ilL of enhanced chemiluminescent solution with different synergitic compounds was added into each microwell, and then 10 ilL of HRP (diluted in PBS) was added. The signal was detected for I second per well by a luminometer at different times. The chemiluminescent immunoassay was processed according to manufacture's instruction, except the enhanced chemiluminescent substrates was substituted by a substrate containing the synergistic reagent. RESULTS AND DISCUSSION In the experiments with HRP, the light emission was ~2-3 times higher in the presence of I-50 IlmollL aminopyrine (4-dimethylaminoantipyrine) (Fig. 2A), 'and also increased light emission in the presence of 4-phenylphenylboronic acid as the enhancer (Fig. 2A). Further experiments revealed that aminopyrine also intensified 265

266

Yang X & Sun X CH,

CH'F1,OH

ell,.

-:

CH" /N'N)-ZO

A.Antipyrine

F.3,4-DIMETHYL-1PHENYL-3-PYRAZOLIN -5-0NE

6

NCH,

CH/YO

B. 4-Hydroxyantipyrine

C.4-Amlnoantipyrine

G,4,4-DICHLORO-3METHYL-1-PHENYL -3-PYRAZOLlN-5-0NE

H,2,3-DIMETHYL-4(1-HYDROXY-2,2,2TRICHLOROETHYL)1-PHENYL-3-PYRAZOLlN5-0NE

D.4-Antlpyrinecarboxaldehyde

E. 4-Dimethylaminoantipyrine

4-(2-CHLOROACETYL)ANTIPYRINE

J. 3-(3-AMINOPHENYL)-2-

t

ETHYL-1-PHENYL-3PYRAZOLlN-5-0NE

CI

K,2,3-DIMETHYL-4(4-HYDROXYBENZYLIDENEAMINO)1-PHENYL-3-PYRAZOLlN-5-0NE

N,4-(2,4-DICHLOROBENZYLIDENEAMINO) ANTIPYRINE

L. 2,3-DIMETHYL-4(4-METHYLBENZYLIDENEAMINO)1-PHENYL-3-PYRAZOLlN-5-0NE

0, 4-CINNAMYLIDENEAMINO-2,3-DI METHYL1-PHENYL-3-PYRAZOLlN-5-0NE

M, 4-(4-CHLOR08ENZYLIDENEAMINO)ANTIPYRINE

P N,N'-METHYLENEBIS (4-(METHYLAMINO) ANTIPYRINE)

Fig. 1. Structures of aminopyrine and its derivatives

A Novel Synergistic Enhancer for HRP-Luminol-H202 Based Chemiluminescence

267

B. 50 Jl mal/L 4-Phenylpheny loboricacid

A Aminopyrine only

Counts/S

Counts/S

3000

1~~~~ /~ _~.~~ 6000 : 4000

2500 2000 1500

/-~.-~".

'-...

1000

2000

o

' o

"

,

500

1. 56 3. 12 6.25 12.5 25

50

100

o

2.5 4.9 9.8 18.737.5 75

Aminopyrine (u nol/L)

150 300

Aminopvrine( J.1mol/L)

Fig. 2. Dose-response of aminopyrine on chemiluminescence initiated by HRP. A. Aminopyrine (signal at 30 min after HRP addition). B. Aminopyrine + 50 IlmoIlL 4-phenylphenylboronic acid (signal at 100 min after HRP was addition). -+-- aminopyrine Counts/S

180000 -

::::' 100000 80000 60000 40000 20000

o

C

t

~ooro

1

~

:---"",--""

I

~~

__

~

__

~

__

- L_ _- L_ _~~

Fig. 3. Time-course of enhanced chemiluminescence reaction with aminopyrine. Chemiluminescent solution was mixed with 50 IlmoIlL 4-phenylphenylboronic acid as the enhancer, and then 20 IlmollL aminopyrine was added). the chemiluminescence for 4-hydroxycinnamic acid and 4-phenylphenol as the enhancer, but depressed it for 4-iodophenol, 6-hydroxybenzothiazole and tetraphenylboron sodium as the enhancer respectively (data not shown). Aminopyrine also modified the kinetics of light emission as shown in Fig. 3. The chemiluminescent solution with aminopyrine was tested for detection of both free HRP and HRP conjugates in immunoassay. As shown in Fig. 4 for HRP in solution or HRP conjugates bound to the surface of a solid phase, the slopes of the calibration curves were higher when aminopyrine was added, thus improving the sensitivity of detection offree or immobilized HRP.

268

Yang X & Sun X A. Free HRP

B. IIDJIlunoassay

Count sis

180000 160000 140000 120000 100000 80000 60000 40000 20000

Counts/S

200000 -+--Aminopyrine

--+--Aminopyrine

150000

- -.- - Contra 1

/

. ...0""'. /

...•

100000 50000

~....•....

,.

O~~--~-------L----~

50

100 HRP(pg!ml.)

150

10

15

20

25

30

35

40

PSA (~glL)

Fig. 4. Calibration curve for detection of free (A) and immobilized HRP (B) by enhanced chemiluminescence (measured at 60 min) (Aminopyrine: 20 f.lmollL aminopyrine plus 50 f.lmollL 4-phenylphenylboronic acid as the enhancers. Control: 50 IlmollL 4-phenylphenylboronic acid as the enhancer). To discover the relationship between structure and the synergism, several derivatives of aminopyrine were also tested (Fig. I) but none showed the synergism found with aminopyrine (compound E. 4-dimethylaminoantipyrine). In contrast, compound K. 2,3-dimethyl-4-(4-hydroxybenzylideneamino)-I-phenyl-3pyrazolin-5-one acted as an enhancer; it enhanced chemiluminescence alone but depressed it in the presence of an enhancer (data not show). We also found another substance with a totally different structure, 10-methylphenothiazine, acted similarly to aminopyrine (data not show) and further study of the mechanism of the synergistic effect will be needed.

REFERENCES 1.

2. 3.

Thorpe GHG., Kricka LJ. Enhanced chemiluminescent reactions catalysed by horseradish peroxidase. Methods Enzymol 1986;133,331-53. Kricka LJ. Chemiluminescent enhancers. U.S. Patent 1997:5,629,168. Yang X. An improvement of enhanced chemiluminescence and its application to immunoassay. In: Roda A, Pazzagli M, Kricka LJ, Stanley PE eds. Bioluminescence and Chemiluminescence-Perspectives for the 21 st Century. Chichester:Wiley,1999;138-41.

SEPARATION AND DETECTION OF AMINO ACIDS WITH A NOVEL CAPILLARY ELECTROPHORESIS CHEMILUMINESCENCE SYSTEM DG YINY CJ XIE,' Z LI,' BH LIU,' MH WU'* JCollege

0/ Environmental and Chemical Engineering, Shanghai University,

Shanghai 200444, PRo China. 2Department o/Chemistry, Jackson State University, Jackson, MS, USA

INTRODUCTION A major area of study in electrophoresis (CE) is the development of a sensitive, simple, efficient and universal detection method and system. Chemiluminescence (CL) should be a very ideal spectroscopic detection technique for CE. l -' Compared with other CL systems, the peroxyoxalate CL system has advantages of high luminescence intensity and it does nor require a catalyst or enhancer, thus making it a useful reaction for CE-CL. In the present study, a novel peroxyoxalate CE-CL system was developed to achieve high signal stability and sensitivity based on a new interface design including a new mixing mode and a new grounding electrode mode. Experimental results show this new system is effective for separation and detection of amino acids with high stability and resolution. MATERIALS AND METHODS Reagents and apparatus. All reagents and chemicals were commercially available and analytical grade and were purchased from Sigma-Aldrich (Jackson, MS, USA). All solutions were prepared with Milli-Q. Buffer solution used for CE was filtered through 0.22-~m hydrophobic PTFE filters. CE-CL separation and detection were performed using a laboratory-built system (Fig. I). A high-voltage supply (O-30kV; Glassma High Voltage, Inc. USA) was used to drive the electrophoresis. A 50 cm x 75 ~m 10, two 75 cm x 250 ~m 10 uncoated fused-silica capillaries (Polymicro Technologies Inc. USA) and a 60 mm x 1.5 mm 10 glass tube (Kimble, USA) were used for CE, delivering chemiluminescence reagents (DCR), and reaction cell as well as detection window, respectively. The reaction cell was placed in front of the photomultiplier tube (PMT, R374 equipped with a C1556-50 DA-type socket assembly; Hamamatsu, Shizuoka, Japan). The end of CE capillary was inserted into a stainless steel needle tube (40 mm x 1.2 mm 10, Becton Dickinson & CO. NJ, USA), and the capillary outlet was put on the outlet of the needle tube where the capillary outlet was exposed to the detection window. The metallic needle tube loaded CE capillary, and two capillaries for DCR were fixed to the reaction cell. The outlet of needle tube was inserted into the upper end of the cell and the ends of two DCR capillaries, one for delivering TCPO and another for H2 0 2 , were inserted into the lower end of the cell. A platinum wire connected to the cathode of high voltage source was attached tp the metallic needle tube (it was used as grounding electrode). Distance between the outlets of the CE and DCR was 2 mm. In order to facilitate

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mixing of DCR and CE solution, the cell was maintained full of the solution throughout the CE-CL process so that bubbles of air escaped more quickly. This mixing mode was designed with the outlet of the reaction cell 1 cm higher than the other end that was sealed. The DCR were injected to the reaction cell by two syringe micropumps (Bioanalytical System Inc, USA), one for injecting TCPO and another for HzO z. The CL emission detected by the PMT was recorded and processed by a computer using an in-house software program. In order to collect the most intense CL signal, the detection window was situated just in front of the PMT. The whole CL detection system was held in a light-tight box to exclude stray light. Precolumn derivatization. Derivatization of amino acids by NDA was carried out as follows: a 250 ftL sample solution in a 2 mL glass bottle was mixed sequentially with 750 ftL 0.1 mol/L borate buffer (pH 9.25), 250 ftL 2 mmol/L NDA methanol solution and 250 ftL 20mmol/L KCN aqueous solution. The mixture was vortexed for 1 min and allowed to stand for 30 min at room temperature. The resulting mixture was subjected to analysis. CE separation and CL detection conditions. Separation of dansyl-amino acids was performed at 2.2 kV using a 50 cmx75 ftm ID capillary and a CE running buffer of 0.1 mol/L borate buffer (pH 9.0). Separation of dansyl-amino acids was carried out at 1.85 kV in 25 mmol/L borate buffer (pH 9.2) containing 25 mmol/L SDS and 7.5% methanol in a 50 cm x 75 ftm ID capillary. All samples were injected into the capillary by hydrodynamic flow at a height difference of 20 cm for 10 s. The new capillaries were rinsed sequentially with 1 mol/L NaOH, water and running buffer overnight, 30 min and 30 min, respectively. The CL reagents, 5 mmol/L TCPO in ethyl estate and 0.5 mol/L HzO z in acetone, were individually injected into the reaction cell by two syringe micropumps with flow rates of 10 ftLimin. The CL detection was conducted using the laboratory-built system shown in Fig. 1.

RESULTS AND DISCUSSION Design of CE-CL interface. As discussed above, there are a number of challenges for the peroxyoxalate CL detection system when coupled to CEo The design of the interface of the CE-CL system is a crucial factor for achieving good separation, good peak profile and high sensitivity. Before the final CE-CL system (Fig. 1) was established, many reported interfaces were tested but without success. Most of the previously interfaces used for CE-CL system were post-column," where a four-way Plexiglas tee-joint held the separation capillary and reaction capillary in place. The CE capillary end was inserted into the reaction capillary, and the grounding electrode was either put inside one joint of the tee that is before the reaction capillary or put after the reaction capillary. Experimental results showed that bubbles were a serious problem for this kind of interface, which resulted in a unstable current and irreproducible peak positions. Moreover, the CE process was easily disturbed. Other kinds of interfaces were also tested but the same problems were encountered. I' However, these problems could be solved to a great extent with the presented apparatus. We propose that bubbles could easily interrupt the contact

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271

of and grounding electrode causing the current becoming unstable. To overcome these problems, we designed a new grounding electrode mode, where the CE end was inserted into a metaiiic needle tube which worked as the grounding electrode. Using such a configuration, the contact of electrolyte and grounding electrode are uninterrupted and lead to a stable current. Based on the new interface combing this new grounding electrode mode and new mixing mode described as above, a total novel CE-CL system was established. Compared to other CE-CL this new system could be used as a universal method for analysis of amino acids with stability, resolution and sensitivity.

Schematic diagram of the CE-CL system. power (HVP); (2) Pt electrode; (3) running buffer rp~,prV()1 capillary; (5) metallic needle tube; (6) Pt electrode; (7) cathode of power source; waste solution reservoir; (9) reaction cell; (10) recorder; (II) for delivering TCPO; (13) capillary for delivering (14) pump (15) pump for injecting H20 2 • of

conditions. In order to obtain optimum of voltage, injection time, running buffer concentration, value were investigated. It was observed that CL emission increased with increasing running buffer concentration. The increases of buffer concentration results in a large difference between the of the sample solution and that of the carrier electrolyte, therefore efficiency and signal intensity are produced. However, considerable louie from the larger electrophoretic current will also be V'H''''''''', which will lead to bubble generation. Additionally, the migration time of increases with increasing electrophoretic buffer concentration due to the decrease of electroosmotic flow. Different borate concentrations of 50 and 100 mmol!L were tested. It was found that 25 mmol!L was optimum in terms of stable current and reproducibility. SDS can improve the separation. But it was observed that at concentrations of SDS that more bubbles were produced. Moreover, concentrations of SDS led to a long migration time and a broad peak. The effect of the of the running buffer was evaluated. It was noted that optimum resolutions were obtained with running borate buffers having pH values

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from 8.7 to 9.2. The effects of sample injection time and applied voltage were also investigated. The results indicated that with increasing sample injection time, the signal intensity increased but the separation efficiency decreased which is attributed to the decrease of theoretical plate number. Higher voltages can shorten migration time and sharpen peaks, but it also leads to a high current and a high louIe heating. From the above studies, the separation conditions of dansyl-amino acid and NDAamino acids were selected as described above and produced efficient separations. Analytical figures of merit. To investigate the linearity of the CL response, continuous injection of a series of dansyl-Asp and dansyl-Leu standard mixture solutions was performed. Results show that a good linear response was obtained. Linear regression analysis of the results yielded the following equation: Y=l.36C2 l.8(r =0.991) for Leu and Y=0.655C-0.37(r2 =0.997) for Asp where Y is the peak height and C is the concentration of the amino acid in IlM. From these calibration curves, the limits of detection (signal/noise=3) were estimated to be l.1 nM for Leu and 2.0 nM for Asp. The reproducibility was investigated by injecting a standard mixture solution of dansyl-Asp and dansyl-Leu 8 times and recording the migration times and peak heights. The relative standard deviations of peak height and migration time were in the ranges of 2.3-3.8 % and 1.2-1.5 %, respectively.

ACKNOWLEDGEMENTS We express our appreciation to Dr.Liu Yiming for his direction and support on this paper. Financial support from Shanghai Pujiang Program and Shanghai Nano Program is highly acknowledged. REFERENCES I. Liu BF, Ozaki M, Utsumi Y, Hattori T, Terabet S. Chemiluminescence detection for a microchip capillary electrophoresis system fabricated in poly(dimethylsiloxane). Anal Chem 2003 ;75 :36-41. 2. Bednar P, Aturki Z, Stransky Z, Fanali S. Chiral analysis of UV nonabsorbing compounds by capillary electrophoresis using macrocyc1ic antibiotics: Separation of aspartic and glutamic acid enantiomers. Electrophoresis 2001 ;22 :2129-3 5. 3. Liu YM, Cheng lK. Ultrasensitive chemiluminescence detection in capillaryelectrophoresis. J Chromatogr A 2002;959:1-13.

A NOVEL CHEMILUMINESCENT IMMUNOASSA Y OF TOTAL THYROXINE USING THE ACRIDINIUM ESTER 2',6'-DIMETHYL-4'-(NSUCCINIMIDYLOXYCARBONYL) PHENYL-IO-METHYL-ACRIDINIUM -9-CARBOXYLATE METHOSULFATE AS LABEL DG YIN, 1.2 YF HE,2 YB LIU,2 DC SHEN,2 SQ HAN2, ZF LUO,2 CJ XIE,I LZHANG,I BH LIU,I MH WU I* 1College

of Environmental and Chemical Engineering, Shanghai University,

Shanghai 200444, PRo China; 2 Department of Isotope, China Institute ofAtomic Energy, Beijing 102413, China

INTRODUCTION Thyroxine is an iodine-containing hormone produced and secreted by the thyroid gland. It has an important action on the regulation of metabolism. Measurement of total thyroxine is important in the diagnosis of thyroid diseases and in the evaluation of therapeutic effects. Many methods of chemiluminescent immunoassay for total thyroxine have been reported. '.2 Here we describe a novel chemiluminescent of total thyroxine using an acridinium ester, immunoassay 2' ,6' -dimethyl-4' -(N-succinimidyloxycarbonyl)phenyl-l O-methyl-acridinium -9-carboxylate methosulfate (DMAE'NHS) as label. Streptavidin-biotin separation and enhancement techniques were applied in this assay. This is the first report of a chemiluminescent immunoassay of total thyroxine using a DMAE·NHS label combined with streptavidin-biotin system. Because of high luminescent signal of DMAE'NHS, and the tetrameric binding feature of streptavidin and the high affinity between streptavidin and biotin, the detection sensitivity was increased and assay time was shortened. Moreover, biotinylating the hapten-protein conjugate rather than biotinylating a detecting second antibody, simplified the assay process and lead to the assay time becoming much shorter. This approach provides a potential general method for chemiluminescence immunoassay of various analytes.

EXPERIMENTAL Instrumentation, reagents and chemicals. Chemiluminescence was measured on a Wallac Victor 1420 Multilabel Counter (Wallac, Finland). All reagents and chemicals were analytical grade and purchased from Sigma-Aldrich. Human serum samples with T4 values measured by the Ciba Corning chemiluminescence immunoassay were kindly provided by 301 Hospital, Beijing, China. DMAE'NHS was synthesized in our laboratory. 273

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The coating buffer was O.OS mol/L sodium carbonate buffer (pH 9.S). The blocking buffer was O.OS mollL phosphate buffer (pH 7.4), containing 0.9% NaCI, 1% BSA, 0.04% NaN 3 . The assay buffer was 0.1 mol/L Tris-HCI (pH 8.6), containing 0.06% ANS, 0.2% sodium salicylate, 0.9% NaCI, 0.1 % BSA, O.OS% NaN 3 , 0.03% Tween-20, O.OS% NMS. The washing buffer was 0.01 mollL phosphate buffer (pH 8.0), containing 0.9% NaCI, O.OS% Tween-20. The labeling buffer was 0.1 mollL NaHC0 3 (pH 9.0). The purifying buffer was 0.1 mol/L phosphate buffer (pH 7.0), containing 0.9% NaCI. Preparation of DMAE·NHS-SA. 6.S mmollL DMAE·NHS solution was prepared by dissolving DMAE·NHS in DMF. 90 ~g SA, 200 ~L labeling buffer and 10.S ~L 6.SmmollL DMAE·NHS were mixed well in a 2 mL brown glass bottle. After a 2 h incubation at room temperature, the reaction was terminated by adding 100 ~L 10 giL lysine and allowing it to stand for IS min at room temperature. The unreacted DMAE·NHS was separated from the DMAE-NHS-SA conjugate by size-exclusion chromatography on a 1 x 2S-cm column of Sephadex G-SO, eluting with purifying buffer and monitoring the protein peak at 280 nm with Shimaedzu LC-6A High Pressure Liquid Chromatograph. Chemiluminescence intensity and protein concentration were determined on multi label counter and UV- Visible spectrophotometer, respectively. Fractions having high chemiluminescence intensity and protein concentration were pooled and stored at -20°C after adding 1% BSA. Molar ratio of DMAE·NHS to SA were determined to be 4.2 (amount of DMAE·NHS conjugated to SA divided by the amount of SA). Preparation of B·NHS-T4-BSA. 167 ~L 6 mg/mL T4-BSA was first dialyzed against labeling buffer overnight. The T4-BSA was then transferred to a clean brown glass bottle. Then, 70 ~L 2.84 mg/mL B·NHS in DMF solution was added in the bottle and the mixture was incubated for 2.5 h at room temperature, with mixing by vortexing. Saturated ammonium sulfate solution was then added to precipitate the B·NHS-T4-BSA conjugate. After centrifuging and decanting, purifying buffer was added to dissolve the precipitate. Then, the solution was dialyzed against purifying buffer overnight and stored at -20°C after adding 1% BSA. Preparation of surface antibody. In each microwell, ISO ~L coating buffer containing 1.2 ~g Anti-T4 monoclonal antibody was added and incubated for 24 h at 4°C. After twice washing with washing buffer, the microwells were blocked by incubation with 200 ~L of blocking buffer for 2 h at room temperature. After decanting the solution, the microwells were allowed to thoroughly dry, placed in plastic bags, vacuum sealed tightly, and stored at 4 Dc. Serum TT4 eLlA. After careful optimization, the serum total T4 CLlA based on acridinium-ester-labeled streptavidin was performed as follows: B'NHS-T4-BSA and DMAE·NHS-SA were first diluted with assay buffer to the ratio of 1 :3S0 and 1: 100, respectively. Duplicate SO ~L of T4 standards or serum samples and 100 ~L B'NHS-T4-BSA diluted solution were pipetted into the microwells coated with anti-T4 monoclonal antibody. The mixture was incubated for 20 min at room

Novel Chemiluminescent Immunoassay a/Total Thyroxine Using the Acridinium Ester 275

temperature with slow shaking. The microwells were washed 4 times with washing buffer. Then, 150 flL DMAE'NHS-SA diluted solution was added to each well and incubated for 10 min at room temperature with slow shaking. After washing 6 times, the chemiluminescence was measured for 1 s on Wallac Victor 1420 MuItilabel Counter. TT4 concentrations of serum were calculated from the standard curve. RESULTS AND DISCUSSION Standard curve and detection limit. A typical standard curve is shown in Fig. 1. The B/BO value decreases with increasing T4 concentration in the T4 range of 15-240 ng/mL. The detection limit of the assay was 0.56 ng/mL, defined as the T4 concentration corresponding to the mean chemiluminescence reading of zero standard (n=24) minus two times the standard deviation.

Fig. 1. Typical standard curve of the present T4 assay Precision. The within-run precision of the assay was determined by assaying three control sera, corresponding to different levels of T4 (mean T4 concentrations of 15, 59.3, and 122 ng/mL), in 12 replicates in a single assay. For the determination of the between-run precision, duplicate measurements of these control sera were performed in 12 different runs. The within-run CVs were 4.5, 5.6, and 4.0% (mean), and the between-run CVs were 5.4,10.3, and 5.9%, respectively. Specificity. The specificity of the assay was evaluated by determining the cross-reaction values for a number of chemically related compounds or metabolites of T4. The cross-reactivity for each of these compounds was expressed as the percentage ratio of the amount of T4 which corresponded to 50% of the zero standard binding divided by the amount of the compound that corresponded to the same level of binding. 3,5-diiodo-L-thyronine, 5,5-diphenylhydantoin and triiodothyronine (T3) were tested and they exhibited cross-reactivity values less than 0.02% except T3 which had a crossreactivity of 1.5%. Recovery. The recovery was assessed by analyzing human serum samples before and after the addition of known concentration of exogenous T4 (20, 80, and 240 ng/mL). The measured increase in the T4 concentration of the sample, expressed as percentage of the expected increase, was determined as the recovery of the assay. The recovery of added exogenous T4 was found to be 104.7, 97.6, and 112.2%, respectively.

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Correlation. For comparison, 56 clinical samples were assayed by the present method and by commercially available Ciba Corning CLIA T4 kit. The values obtained by present method were in good agreement with those obtained by the commercial Ciba Corning CLlA, as shown in Fig. 2. The linear regression equation is as follows: yz 0.998x-3.02, rzO.962.

::i

200

Y

'00

Ciba Corning TT. ellA (ng/ml)

Fig. 2. Correlation between the present method and Ciba Corning CLIA. 17 samples from healthy people, 30 samples from hyperthyroid patients, and 9 samples from hypothyroid patients.

ACKNOWLEDGEMENTS We express our appreciation to Professor Borong Bao for his helpful suggestions on this paper. Financial support from Isotope Department of China Institute of Atomic Energy, Shanghai Pujiang Program, and Shanghai Nano Program is gratefully acknowledged. REFERENCES 1. Kemppainen RJ, Birchfield JR. Measurement of total thyroxine concentration in serum from dogs and cats by use of various methods. Am J Vet Res 2006;67:259-65. 2. Waseem A, Yaqoob M, Nabi A. Determination of thyroxine in pharmaceuticals using flow injection with luminol chemiluminescence inhibition detection. Luminescence 2006;21: 174-8.

DETERMINATION OF ASCORBIC ACID BY A FLOW INJECTION CHEMILUMINESCENCE METHOD WITH A NOVEL RHODANINE YU JINGHUA,* ZHANG CONGCONG, TAN YUN, GE SHENGUANG, DAI PING, ZHU YUANNA

School a/Chemistry and Chemical Engineering, University 0/Jinan, Jinan 250022 E-mail:[email protected]

INTRODUCTION Ascorbic acid is important in nutrition and overdose of ascorbic acid will cause abdominal pain, diarrhea, even diabetes and kidney stones.

Many methods have

been used for its determination including iodometry, I spectrophotometry,2 spectrofluorimetry,3 HPLC,4 and electrochemical methods. 5 However, they often suffer from a variety of limitations. Rhodanine and its derivatives are important organic reagents mainly used in spectrophotometry,6 but recent studies have shown that they are also fluorescent reagents,7 however, rhodanine derivatives for chemiluminescence reactions have not been reported.

We found that 2NRASP

(3-(2'-nitrophenyl)-5-(2'-sulfonophenylazo) rhodanine) could be used as a chemiluminescent reagent in the 2NRASP- KMn04-HCI system, and the CL intensity decreased when ascorbic acid was added. Based on this, a new, simple and rapid flow

injection chemiluminescence method was established for the

determination of ascorbic acid. Furthermore, the method has been used for the determination of ascorbic acid in tablets and fruit with satisfactory results. MATERIALS AND METHODS All chemicals were of analytical reagent grade and were used without further purification. All solutions were prepared with distilled deionised water. Stock solution of 2NRASP (2.000x 10-4 mollL) (synthesized by our own laboratory) was prepared in dehydrated alcohol (Laiyang Reagent Ltd., China). Standard solutions were prepared by diluting stock solution with water. Stock solution of ascorbic acid (Tianjin North Tianyi Chemical Reagent Company, China) (100 Ilg/mL) was stored 277

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in a refrigerator. Working standard solutions were prepared daily from the stock solution by appropriate dilution immediately before used. Potassium permanganate and hydrochloric acid were purchased from Yao Shun Import & Export Co, Ltd. and China Shanghai Lianshi Reagent Ltd., China, respectively. The flow system used in this work is shown in Fig. 1. The fluorescence spectrophotometer (P.E Company, USA) and UV-Vis spectrophotometer TU-1901(Beijing Puxi instrument Ltd., China) are used for the mechanistic study. a

~

b

d

w Fig.1.

~

~~~

c

Flow-Injection CL system.

w (a) Sample or blank solution; (b) 2NRASP;

(c) KMn04; (d) HCI; (P) Peristaltic pump; (V) injection valve; (F) CL flowcell; (PMT) photomultiplier tube; (AMP) ultra-weak chemiluminescence analyzer; (PC) personal computer; (NHV) negative high voltage. Procedure. Channel (a) and (b) delivered ascorbic acid sample or blank solution and rhodanine solution respectively at 4.5 mUm in; while channel (c) and (d) was used to deliver potassium permanganate solution and HCI solution respectively at 4.5 mUmin. The negative high voltage was -400 mv. The measuring chamber was kept at a constant temperature of 25°C. RESULTS AND DISCUSSION CL intensity spectrum. Fig.2 shows that after sampling 3.5 s, chemiluminescence signal is produced and 2.8 s later, chemiluminescence signal peaks. Then after 4.2 s, the signal returned to baseline value. After adding ascorbic acid to potassium permanganate-hydrochloric acid, a weak CL signal was detected. The system 2NRASP- KMn04-HCI produced a very strong signal. However, the CL signal became much weaker when ascorbic acid was added in a dose dependent manner

Determination of Ascorbic Acid by a Flow Injection Chemiluminescence Method

279

and this formed the basis of a new flow injection chemiluminescence method for determining ascorbic acid.

4000 3000 >--.

2000 1000 0

o ") Fig.2

CL intensity kinetics.

4

(i

8 10 12 14 16

Is I :HCI+KMn04 2:HCI+KMn04+Vc (10 flg/mL) 3:

2NRASP+HCI+KMn04+Vc (10 flg/mL) 4: 2NRASP+HCl+KMn04+Vc (5 flg/mL) 5:2NRASP+HCl+KMn04 Optimum conditions.

In this study, the following oxidants were tested in 1.0

moUL HCl: KMn04 (2.5 x 1O·4mo llL), Ce(S04)Z (0.01 mollL), KzSzO g (0.01 mollL), and K3Fe(CN)6 (0.01 moUL) was tested in 0.1 moUL NaOH. KMn04 (HCl) was chosen for subsequent use. The concentration of HCl was investigated from 1.2 to 3.0 mol/L in the flow injection system. 1.5 moUL was selected for further

experiments. The effect of KMn04 concentration on the CL intensity was studied from 1.0 x 10-4 to 2.5 x 10-4 moUL - 2.0 x 10-4 moUL was selected. By varying 2NRASP concentration, it was observed that the luminescence intensity increased until 6.0 x 10-5 moUL - 6.0 x 10-5 moUL was selected for the further experiments. The effect of flow rate was tested in the range of 2.5-6.0 mUmin for each stream and the best signals were obtained at 4.5 mUmin flow rate of each stream. Method performance. A linear calibration curve was obtained in the range 0.2-10 flg/mL [M= -315 + 206 P (p:flg/mL; correlation coefficient of 0.995 (n = 5). The

detection limit (3m}) is 0.02898 flg/mL., relative standard deviation (n

=

II) for 5

flg/mL ascorbic acid standard is 1.5%.

Interference study. Analysis of a standard solution of I flg/mL ascorbic acid containing increasing amounts of interfering species revealed that the tolerable

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concentration ratios for interference at 5% level were> I 000 for sucrose, lactose, K+, Na+,

cr, F, N0 3 -, 500 for glucose, starch, cyclodextrin, Cd 2+, 300 for fructose,

40 for Ca2 +, C0 2 +, 30 for Cs +, AI 3 +, tartaric acid, respectively. Applications. The proposed method was applied to determine ascorbic acid in a sample of Yinqiao vitamin C tablets (0.0500 g ground up tablets (20) dissolved in water and filtered and diluted to 500 mL). The recovery rates of the method is 96.6% and 96.3 %; RSD was <±3.00% which indicated the results were quantitative and (-tests assumes there was no significant differences between recovery efficiency and 100 % at confidence level of 95 %.

ACKNOWLEDGEMENTS This study was supported by the Natural Science Foundation of Shandong province (No. Y2007B07). REFERENCES 1. Douhui B, Li G. The improvement of measuring vitamin C by using iodometry. Agr Res Arid Areas 2004:1. 2. Luque-Perez E, Rios A, Valcarcel M. Flow injection spectrophotometric determination of ascorbic acid in soft drinks and beer. Anal Bioanal Chern 2000;8:857-62. 3. Lun W, Li Z, She S, et al. Direct fluorimetric determination of ascorbic acid by the supramolecular system of AA with /3-cyclodextrin derivative. Acta A: Mol Biomol Spectrosc 2005;11-12: 2737-40. 4. Giovanni B. Development of a quantitative method for the analysis of total I-ascorbic acid in foods by high-performance liquid chromatography. J Chromatogr A 2007;1-2:97-102. 5. Shujuan L, Jianguo M, Daofeng P. The determination of ascorbic acid in food with polymeric film of derivative of /3-cyclodextrine modified electrode. Food Sci Technol, 2006;8:227-30. 6. Zij un L, Jiaomai P, Jian T. Highly sensitive and selective spectrophotometric method for determination of trace gold in geological samples with 5-(2-hydroxy-5-nitrophenylazo) rhodanine. Anal Bioanal Chern 2003;3 :408. 7. Jinghua Y, Qingyu 0, Tao L. Synthesis of a new reagent 3-(4' -fluorophenyl)-5-(2' -carboxylphenylazo)-rhodanine and the fluorescent determination of bismuth. Chin J Anal Chern 2004;5:644-6. Note: a version of this paper is http://jpkc.ujn.edu.cn/fxhxlkfsy/yjbg/Ol.pdf.

published

on-line

at

STUDY OF SUPERWEAK LUMINESCENCE IN PLANTS AND APPLICATION TO SALT TOLERANCE IN ALFALFA

ye

ZHOU HE, I YANG QIJIAN,2 LIU Institute of Grass land Science, China Agricultural University, Beijing, 100094, PR China, E-mail:[email protected] 2 Department of Biotechnology, Beijing Agricultural College, Beijing, 102206, PR China, E-mail:[email protected]

1

INTRODUCTION Living cells emit light in the UV, visible and infrared (180 ~ 800 nm). It has low intensity, ranging from a few to hundreds of photons/s/cm 2, and is called " superweak luminescence" (SL).1-7 It is known that SL exists in all animal and plants and provides important information on metabolism and energy transformation of a living biological organism. SL of plants is closely related to the environment. Under stress conditions, plant SL has obvious changes, and is one of the indicators for evaluating and identifying of plant resistance. Alfalfa (Medicago sativa L.) is an important cultivated plant. Evaluation of salt-resistance of alfalfa, requires a quick and reliable method. We have studied SL as a comprehensive metabolic indicator, and report our results of the comparison of SL intensity at the seed germination stage in alfalfa. METHODS Plant materials. 5 varieties of alfalfa were supplied by the Institute of Animal Husbandry, Chinese Academy of Agricultural Science. Duoye and Zahua were tolerant varieties, Xinfu and Yuxian were medium tolerance varieties, and Yongji was a sensitive variety. Experiment Methods. Seeds of each alfalfa variety were soaked in distilled water for 2 h, blotted dry with filter paper to remove excess water, and then respectively transferred to distilled water, 0.5 % NaCI and 1 % NaCI solution. Seeds were germinated in duplicate at 25 ± 1 'C. SL was measured at 2, 4, 24, 48, 72, 96, 120, and 144 h. The germination rate of alfalfa seeds were >95 % in the pre-experiment. SL was measured using a single photon monitor, Beckman-5801 (liquid scintillation counter) at constant temperature in the absence of light for 30 s. RESULTS Characteristic SL curve of germinating alfalfa seed. The SL of germinating alfalfa seeds in distilled water showed no significant difference (P> 0.05) among 5 281

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alfalfa varieties. There were two peaks during the seed germination period. Dry seeds emitted high luminescence when they absorbed water and this corresponded with the first peak value. But 24 h after germination SL increased to give a second peak at 96 h. The luminescence value at 96 h was as much as 14-times of that in the dry seed. Plant seeds increased their luminescence value when they encountered water. The main characteristic of the initial seed germination period is the dissolution of the nutriment. When non-saturated fatty acids are oxidized they generate free radicals that are then responsible for the SL. With the growth of the seed and the cell activity the second peak of SL is related to mitosis.

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Hours j:igure 1 Dynamic curvc of supcrweak luminescence of germinating seeds in alfalfa

This peak value is a characteristic value of metabolism in the germinating plant seed, and the SL time curve of a germinating seed is characteristic of the seed metabolism in each plant. The high SL values in alfalfa make it a good system to study SL. Comparison of SL between two varieties of alfalfa seed in distilled water and NaCI. The physiological action of the salt stressed germinating alfalfa seeds was restricted to some extent. SL of alfalfa seeds in NaCI was lower than in distilled water. In NaCl, the SL value of the seed decreased markedly, and the characteristic SL peak appeared 24 h later than for others treatments (Fig. 2).

Superweak Luminescence in Plants and Application to Salt Tolerance in Alfalfa

283

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dis L i j I od waler

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-.-10 1% ;\aCl

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a Duoye Alfalfa Fig. 2.

Comparison of SL in Duoye alfalfa in distilled water and NaCI solution.

There was a concordant luminescence tendency but large differences in SL intensity among the different varieties of alfalfa. Results for the different varieties indicated that Duoye, a tolerant variety, had little change in germinating metabolism and growth rate under salt stress. SL values of germinating seed in 0.5% and 1% NaCI were similar to that in distilled water. However, for a sensitive variety such as Yongji, 0.5 % and 1 % NaCI solution seriously affected the SL intensity. The peak value at 96 h decreased 26 % in 0.5 % NaCI and 65 % in I % NaC!. Thus, there was a significant difference between two varieties Yongji and Duwye. Comparison of salt tolerance of different variety of alfalfa. As the previous result, the SL value of the seed among the 5 alfalfa varieties showed no significant differences during the germination period in the distilled water, but had large differences under salt stress (Fig 3a). Under 0.5 % NaCI stress, the difference appeared in 48 h after seed germination. At 72 h after seed germination, the two salt tolerance varieties, Duoye and Zahua had higher luminescence values than the other three varieties. At 72 h the luminescence value ofXinfu. Yuxian and Yongji were identical. After 96 h, the sensitive variety, Yongji, emitted slightly higher SL than the two medium salt tolerant varieties. But after 120 h, they had the same SL value. Comparing the SL curve of germinating seeds in I % and 0.5 % NaCl, there

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Superweak Luminescence in Plants and Application to Salt Tolerance in Alfalfa

285

was a significant lag phenomenon in the former condition (Fig. 3b). In 1 % NaCl, the difference appeared 72 h after the seed germination among the 5 varieties. The peak value appeared at 120 h. At this time, the SL value of the tolerant variety was significantly higher than the other 3 varieties.

DISCUSSION SL is a unique method that allows continuous measuring of plant metabolism without destroying the regular growth of plant in biological research work. The mechanism of emission of has not been elucidated. Generally, when an electron in biological system is excited, and the excess energy is released as photons and the system returns to the ground state. Both biological oxidation and cell mitosis may be the source of plant SL and we hypothesize that biological oxidation and cell mitosis co-existed, each with its own effect on SL of the seed. The two peaks in the SL curve of germinating alfalfa seeds supports our hypothesis. There was a significant lag in luminescence of germinating seeds in 1% NaCI compared to distilled water and 0.5%NaCl, and this agrees with the observation that germination of the alfalfa seed under salt stress increases with increase in salt concentration. Under the same saIt stress, the germinating seed of different varieties of alfalfa emitted different SL, and this difference allowed ranking of the degree of salt tolerance of the alfalfa. Our data was the same as the culture experiment data, except that there was some difference in the rank of the sensitive variety and the medium tolerance variety. This may be related to the "flashing phenomenon" of the sensitive variety. Under adverse conditions, especially extreme conditions, some sensitive plants may have disordered metabolism, that is destructive and induces a rise in luminescence that appears as the "flashing phenomenon". In order to find out the salt tolerance limitation of the different varieties, we can increase the salt concentration. This method may be regarded as a useful attempt in researching the "plant flashing phenomenon" under the extreme condition.

ACKNOWLEDGEMENT Supported by Beijing Science Foundation (2007 [N] 16)and Foundation of National Science committee (NQCR-l 0-28) REFERENCES l. Popp FA, Li KH, Qu Q. Eds. Recent advances in biophoton research and it's application. Singapore:Worid Scientific. 1992. 2. Gurwitsch AA. A historical review of the problem of mitogenetic radiation. Experientia 1988,44:545-50.

286

Zhou H et al.

3. Inaba H. Photonic Sensing technology is opening new frontiers in biophotonics. Opt Rev 1997;4:1-10. 4. Beloussov L, Popp FA, Voeikov VI, Wijk RV. Biophotonics and coherent systems. Moscow:Moscow University Press, 2000:439. 5. Yang Q, Yu T. Superweak luminescence of pea seedling under Na-salt stress. In: International symposium. Plants under environmental stress, Moscow, Russia. 2001:319-20. 6. Yang Q, Zhou H. The superweak luminescence and its oxidative reaction of plant under nonbiotic stress. 1st Asia conference on photobiology, Japan. 2002:91. 7. Zhou H, Yang Q. A study on the superweak luminescence of different plant seeds at the stage of germination. Acta Biophys Sin 1996,12: 157-60.

DEVELOPMENT AND OPTIMIZATION OF A QUANTITATIVE WESTERN BLOT AND DOT BLOT PROCEDURE FOR THE DETERMINATION OF RESIDUAL HOST CELL PROTEINS PRESENT IN INACTIVATED POLIO VACCINE USING A GZll BASED SIGNAL REAGENT

G ZOMER, M HAMZINK, A DE HAAN, G KERSTEN, K REUBSAET Unit Research and Development, Netherlands Vaccine institute, PO Box 457, 3720AL Bilthoven, The Netherlands Email: [email protected] INTRODUCTION Inactivated polio vaccine (lPV) contains as the active ingredient protective D-antigens. Because the polio virus is grown on a host cell system, residual host cell proteins (HCPs) may be present in the bulk product. During poliovirus cultivation the Vero host cells lyse. Proteins are released into the medium and cell fragments detach from the micro carriers. Cell fragments and host cell proteins are removed during down stream processing (DSP). DSP consists of filtration and chromatographic purification steps. Traces of host cell proteins (HCPs) however will remain in the monovalent pools. We investigated the amount of residual host cell proteins and the effect of the filtration and purification steps on the removal of the host cell proteins. In order to quantify these proteins two approaches were used. Firstly, the HCPs were separated on gel and after incubation with rabbit polyclonal antiserum directed against the HCPmix (capture antibody) followed by incubation with detecting antibody (HRP-labeled mouse anti-rabbit) subsequently visualized and quantified using a GZ 11 based signal reagent. Secondly, a dot-blot approach was followed using the same two incubation steps and detection with GZII signal reagent. MATERIALS AND METHODS Rabbit HCP-mix antiserum. The polyclonal rabbit anti-HCP serum was obtained by a cascade immunization of rabbits with a HCP mix from Vero cells. Three days before immunization blood was obtained from the rabbits. This pooled serum serves as preimmune rabbit serum. The Hcr mix, a blank culture of Vero cells is three times frozen and thawed to induce cell lysis. The culture supernatant was pooled and filtered. The pore size of the filters was comparable with those used during the IPV Vero production process. The HCP mix was divided in small aliquots, rapid frozen using dry ice and stored at -70 °c until use. Rabbit anti-HCP serum: Three rabbits were immunized with the HCP-mix and received a booster with the same HCP mix after 28 days. On day 42 blood samples were collected. Using the antibodies as an affinity column the immuno-dominant proteins were removed from the HCP-mix. The treated HCP-mix was used to immunize the 3 rabbits again on day 56. The same procedure was performed with serum of day 77 to immunize at day 85. The rabbits were given a booster on day 121 followed by collection blood on day 135. The pooled serum of day 135 was used in this study as anti-HCP serum.

287

288

Zomer G et al.

Dot blot procesure. Suitably diluted (in PBS) samples or standards (50 ilL) were applied to a nitrocellulose membrane using an ELI FA (Pierce) Unit. After washing with 200 ilL of PBS buffer, the membrane was incubated with polyclonal anti-HCP (1 :300 in assay buffer) for 1 h with gentle shaking. After washing with wash buffer (PBST, PBS containing 0.1 % Tween20), the membrane was incubated with 2nd Ab.HRP (l: 100,000 in assay buffer) for 1 h with gentle shaking. The membrane was washed (4x) with wash buffer. GZll signal reagent (a gift from ZomerBloemen b.v.) was added to the membrane, and after incubating for 5 min, decanted. The glowing membrane was measured in a chemiluminescent imager (Fluorchem 8900) and images were stored as TIF-files and analyzed using Image-l software. Western blot analysis. A suitable amount of sample was first precipitated using a 72% w/w trichloroacetate (TCA)-solution. The samples were thoroughly vortexed followed by centrifugation. The supernatant was carefully removed and 15 ilL of reducing buffer was added. The samples were thoroughly vortexed and placed in a thermostat at 100 DC during 5 minutes. After this the samples were centrifuged for a few seconds, only to let the liquid in the top of the sample container to be spun down. The slots of the precast gel were loaded with 15 ilL of sample and MW -markers (10 ilL). After electrophoresis the gel was washed with water and blot buffer. Via a cold tank transfer in blotting buffer a Western Blotting was performed. After washing with PBS buffer, the membrane was incubated with polyclonal anti-HCP during one hour with gentle shaking. After washing with wash buffer, the membrane was incubated with 2 nd Ab.HRP (1:100,000 in assay buffer) during one hour with gentle shaking. The membrane was washed (4 x) with wash buffer. GZll signal reagent was added to the membrane, and after incubating for 5 minutes, decanted. The glowing membrane was measured in a chemiluminescent imager (Fluorchem 8900). Resulting images were stored as TIF-files and analyzed using Image-l software. Samples. Down stream processing samples that were analyzed consisted of the fraction after concentration (2.1), after size exclusion chromatography (3.1), and after ion exchange chromatography and sterile filtration (5.1). RESULTS AND DISCUSSION An example of a western blot together with a residual protein staining blot is shown in Fig 1. Clearance of the host cell proteins during DSP using a combination of size exclusion and ion exchange chromatography is successful. During the first chromatographic step HCPs are cleared about 50-fold, while after the second chromatographic step no more HCPs can be detected using western blot. Also with respect to the concentration of D-antigen, the vaccine active ingredient, HCPs are cleared efficiently resulting in an increase of specific activity of D-antigen with respect to total protein. From the protein stained blot clearance of HCPs can also be inferred; in the 5.1 fraction only virus related proteins are observed with no indication of HCPs. Although useful in detecting residual HCPs western blot analysis gives only semi-quantitative results (when using serial dilutions of samples, results not shown). I

Ue 1vell)plrlem and Optimization of a Quantitative Western Blot and Dot Blot Procedure

289

Left Western blot stained with HCP polyclonal antibodies. Lane 1 contains mw markers 40,50,60,80, 120,220 kD), lane 2 is a sample of production fraction 1 diluted 250 times, lane 3 contains a sample of production fraction 3.J diluted 10 lane 4 contains the HCP-mix diluted 10 times, lane 5 contains an of production fraction 5.1. Lane 6 contains normal rabbit serum as a control. residual protein staining blot (right, lane 1 mw lane lanes 3-9 correspond with lanes 1-6 of the western blot) on a Because dot blot analysis allows for more samples to be membrane and because dot blots are more easily quantified this technique was also used to assess HCP clearance during DSP. An example is shown in 2. As can be seen from this dot blotting can be performed with reasonable reproducibility and The lowest concentration of the calibrator that can be quantified is about 100 ng/mL. This allows for dilution of 2.1 (6,000-50,000) and 3.1 ~'mll'II'~ (200-2,000) while 5.1 samples were run undiluted. To obtain reliable results were diluted using sample weights instead of sample volumes. Within reproducibility was better than 10% while day to day reproducibility was better than 30%.

290

Zomer G et al.

• •• ••

••• •••

2. Dot blot analysis (top left) Dot blot stained with HCP polyclonal antibodies. The top two rows are calibration standards ofHCP mix in duplicate (7 dilutions from 3.8-0.19 /lg/mL plus blank, from left to right). The third and fourth rows are 8 dilutions in duplicate (4&,000-6,000, left to right) of a 2.1 IPV production fraction while the fifth and sixth rows are &dilutions in duplicate {2,000-250, left to of a 3.1 IPV production fraction. Corresponding profile data of the dot blot are shown on the right with the calibration curve (bottom

REFERENCES 1.

Zomer Smitsman C, Arts R, Hamzink M, Kooijman M. blotting using a GZ-ll based chemiluminogenic signal reagent. In: Hill Pl Kricka LJ Stanley PE, eds. Proceedings of the International Symposium on Bioluminescence and Chemiluminescence. World 2006:171-4.

DEVELOPMENT AND OPTIMIZATION OF A FAST AND SENSITIVE ELISA FOR POLIO D-ANTIGEN USING A GZll BASED SIGNAL REAGENT G ZOMER, M HAMZINK Unit Research and Development, Netherlands Vaccine institute, PO Box 457, 3720AL Bilthoven, The Netherlands Email: [email protected]

INTRODUCTION The potency of inactivated polio vaccine (IPV) traditionally is determined by the measurement of the protective D-antigens present in the vaccine. Mostly, a sandwich ELISA test is used for this purpose. l During production of the polio virus and during down stream processing of the vaccine there is a need for a reliable indication of the concentration of D-antigens that is present in the different sample types. Customarily, the ELISA test involves several incubations: Firstly, the binding of the antigens using a type specific antibody coated micro titerplate. Secondly, the antigen is bound by a type specific monoclonal antibody, and thirdly the monoclonal antibody is bound by an HRP-labeled 2nd antibody (conjugate). Each incubation step is followed by washing steps making this assay rather time consuming. In order to speed up the assay the second and third incubation step were combined. Moreover, the incubation steps were performed at 37°C on a shaking incubator resulting in a faster assay. Furthermore, because of the high sensitivity of detection of the HRP-conjugate using the GZ-ll signal reagent much less antibodies (coat, monoclonal, and 2 nd antibody) could be used resulting in a rapid (within 2 hours) and sensitive ELISA. MATERIALS AND METHODS ELISA plates (Greiner, white, high-binding) were coated with type-specific caprylated bovine antiserum diluted 1: 1600 in PBS, overnight at 4°C. Coated plates can be stored dry at -80°C. The plates were washed with wash buffer (PBST, PBS containing 0.1 % Tween20). Samples and standards (100 [lL, diluted in assay buffer (wash buffer containing 0.5% Protifar (Nutricia)) were added to the wells. The sealed plate was incubated at 37°C on a shaking incubator during 30 minutes and washed two times using wash buffer. Incubation with 100 [lL of a mixture of type specific suitably diluted monoclonal (capture antibody) and HRP-Iabeled goat anti mouse (detection antibody) was performed at 37°C on a shaking incubator during 30 minutes and washed four times using wash buffer. GZll based signal reagent (a gift from ZomerBloemen b.v.) was added (100 [lL Iwell) and the glowing plate was placed into a plate luminometer (Berthold Centro LB960). From the raw data calibration curves were constructed using a four parameter fitting routine (MS-Excel) from which unknowns were calculated. Two different protocols were compared during the study (please refer to Table 1 for details). 291

292

Zomer G & Hamzink M

Ta bilL e ayouto f two-steJ Three-step protocol Incubation with antigen (step 1) Wash Incubation with capture antibody (step 2) Wash Incubation with detection antibody (stefl 3) Wash Addition ofOZll signal reagent Measurement

an d tree-step protoco h Two-step protocol Incubation with antigen (step 1) Wash Incubation with capture and detection antibody (step 2) Wash Addition ofOZll signal reagent Measurement

RESULTS Effect of coating dilution. The effect of using different concentrations of coating antibody in the range 1 :200 to 1: 1600 is very small (please refer to Fig. 1 for details). In the final assay a dilution of 1: 1600 was used. 25.0 20.0 -+-200 : ....... 400

15.0 E!'l

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........ 800

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5.0 0.0 0

10

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20

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type I (DElmL)

Fig. 1. The effect of coating dilution on calibration curve for type 1 D-antigen using sequential incubations with monoclonal antibody and conjugate. Effect of shaking. Shaking the micro titerplate during the first incubation (with antigen) results in a much faster establisment of equilibrium (see Fig 2 for details). Equilibrium was reached after shaking the plate during 30 minutes, and this time period was adopted for all incubation steps.

Development and Optimization of a Fast and Sensitive ELISA for Polio D-Antigen

293

the effect of shaking on 1st incubation 100%

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Effect of two-step vs three-step incubation In order to combine the incubations with capture and detection antibody the effect of diluting the capture antibody concentration was studied. Figure 3 shows the results which clearly indicate that diluting the capture antibody greatly improves the specific binding. When more concentrated capture antibody concentrations are used the detection antibody binds less to the sandwich resulting in a lower signal. 45000 40000 35000 30000 =0 ..l

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Fig. 3. Effect of diluting the monoclonal antibody in the two-step protocol results in improved assay performance (detection antibody dilution - 1 :500,000) When the capture antibody is further diluted, down to 1:50,000 the calibration curves for the two-step protocol and three-step protocol became virtually superimposable (please refer to Fig 4 for details).

294

Zomer G & Hamzink M

60000 50000 40000 :> ..J

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....... twostep

30000

...... three step

20000 10000 0 0.01

0.1

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Fig. 4. Comparing two-step vs three-step protocol using 1:50,000 dilution of capture antibody (detection antibody dilution 1:500,000) DISCUSSION In this study we have shown that by combining two incubation steps and by performing all incubations at 37 DC on a shaking incubator together with the use of the GZl1 signal reagent a sensitive and rapid ELISA method has been developed. The use of GZll signal reagent with its inherent great sensitivity for HRP detection allowed for the dilution of coating, capture and detection antibodies, resulting in much less aspecific binding. The signal reagent is a two-component stable formulation which when mixed 1:1 can be used at least during one working day. REFERENCES 1. Rezapkin G, Dragunsky E, Chumakov K. Improved ELISA test for the determination of potency of Inactivated Poliovirus Vaccine (lPV). Biologicals 2005;33:17-27. 2. Zomer G. Development of a chemiluminescence immunoassay for clara cell protein at sub-pM levels. In: Case JF, Herring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE, ed. Proceedings of the 11 th International Symposium on Bioluminescence and Chemiluminescence. Singapore:World Scientific, 2001: 377-80.

PARTS APPLIED ELECTROLUMINESCENCE

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DETECTION OF XANTHOMONAS ORYZAE Pv. ORYZICOLA BY ELECTROCHEMILUMINESCENCE POLYMERASE CHAIN REACTION METHOD JIE WEI, LINGRUI ZHANG MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631,China Email: [email protected]

INTRODUCTION

Xanthomonas oryzae pv. Oryzicola (Xoo) is a gram-negative, rod-shaped bacterium. It is emerging as an important pathogen of rice and is a recognized biosecurity threat

to most of the rice growing countries. Thus, a highly sensitive, yet simple and safe approach for Xoo detection is required. The electrochemiluminescence polymerase chain reaction (ECL-PCR) method has high sensitivity in nucleic acid analysis, which we have described in our previous articles. '4 We have now developed a new ECL-PCR method and used it to detect Xoo for the first time. The principle of this ECL-PCR method is shown in Fig. I. 16-23s rDNA of Xoo was amplified by PCR. At the end of 3' terminal of the primers, a pair of universal sequences is added so that all PCR products contain these sequences. These PCR products are used to hybridize with a TBR-probe and biotin-probe, which is complementary to the universal

sequence.

streptavidin s.7 ,

Through the

specific

interaction

between

biotin

and

the hybridized products are captured by magnetic beads that are

coated by streptavidin. After magnetic separation, the samples are mixed with TPA and detected by ECL. This method is simple and highly sensitive; it can significantly reduce costs by employing the universal probes. EXPERIMENTAL The forward primer was 5'-TAACTGAATAGACTAAGACGCATGACGTCAT CGTCCTGT -3'.

The

reverse

primer

was

5'-CTAATCAACGACCTTGTATCCTC GGAGCTATATGCCGTGC-3'. The TBR probe was 5'-TBR-TAACTGAATAGA CTAAGAC-3'. The biotin probe was

5'-biotin-GATACAAGGTCGTTGATTAG-3'.

297

The

cetyltrimethyl

298

Wei J & Zhang L

ammonium bromide (CT AS) method for sample extraction and purification reported by Lipp. et aZ. was used in this study.8 The amplification protocol consisted of 2 min at 94°C for initial denaturation, 30s at 63°C for primer annealing and I min at 72 °c for extension. After amplification, biotin-probe and TBR-probe were added to. The mixtures were incubated for 5 min at 94°C and I h at 63°C. Then, streptavidin coated magnetic beads were added. The mixture was then shaken at room temperature for 30 min. After washing and removing the supernatant, the samples were added to the detection cell of ECL analyzer (built in our lab).9

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Fig. 1. The principle of this EeL-peR method for detection of Xanthomonas oryzae pv. Oryzicola RESULTS AND DISCUSSION

Fig. 2a shows the EeL detection results of both healthy and infected samples. From this figure we can see that, the EeL intensity of infected samples and un infected samples has such a striking contrast that we can clearly distinguish them. In order to verify the feasibility of this method, 1% agarose gel electrophoresis analysis for peR products was performed in the experiment (Fig. 2b). condition is 1% agarose gel at 80Y for an hour.

The electrophoresis

Lane I and lane 2 are all the peR

products of infected samples. Lane M represent markers (100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp). Lane 1-2 has a band between 400 and 500bp which was consistent with the expected peR product of size

oryzae pv. Or,vzicola by Electrochemiluminescence peR Method

Detection

299

electrophoresis are consistent with the results of ECL

The results of detection.

In

we have developed a sensitive ECL-PCR method for Xoo detection. to the traditional detection methods, this ECL-PCR is a

low

noise and specific technique. Due to its sensitivity and simplicity, this will have an enormous potential for detecting plant pathogenic bacteria.

time(s)

(b)

ECL detection results of both healthy and infected samples and the agarose electrophoresis analysis results of the PCR products.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China 307001

the National High Technology Research and

of China (863 Program) (2007 AA lOZ204», and the Natural Science Foundation of

,imm2:00IH!

Province (7005825).

REFERENCES . Liu

Shen XV, Zhu DB.

Detection of genetically modified

electrochemiluminescence PCR method. Biosens Bioelectron Shen XY, Liu JF. A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. Biochem Biophys Res Commun

300

Wei J & Zhang L

3. Liu JF, Xing D, Shen XY. Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms. Anal Chim Acta 2005;537:119-123. 4. Zhu DB, Xing D, Shen XY, Liu JF, Chen Q. High sensitive approach for point mutation detection based on electrochemiluminescence. Biosens Bioelectron 2004;20:448-53. 5. Blackburn GF, Shah HP, Kenten JH. Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin Chem 1991;37:1534-39. 6. Jong MD, Weel JFL, Schuurman T, Quantitation of Varicella-Zoster Virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence. J Clin MicrobioI2000;38:2568-73. 7. Collins RA, Ko LS, Fung KY. A method to detect major serotypes of foot-and-mouth disease virus. Biochem Biophys Res Commun 2002;297:267-74. 8. Schutzbank T, Smith 1. Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe, J Clin MicrobioI1995;33:2036-41. 9. Zhu DB, Xing D, Shen XY, Yan GH. High sensitive detection of presenilin-l point

mutation

2003 ;48: 1741-44.

based

on

electrochemiluminescence.

Chin

Sci

Bull

A NOVEL ELECTROCHEMILUMINESCENT SENSOR BASED ON CATiONIC POLYMER/CHITOSAN FOR ULTRASENSITiVE DETECTiON OF HYDROGEN PEROXIDE XIAOPING WU,* YOUMEI WANG, HONG DAI, GUONAN CHEN Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety and Department of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China; *E-mail: [email protected]

INTRODUCTiON Electrochemiluminescence (EeL) is the most sensitive detection technique for EeL-based sensors have emerged as powerful tools for ultrasensitive analysis. I ,2 The electron transfer rate between the electrode and as well as the reactivity and interaction of the target analyte with modified are key considerations for biosensor fabrication. the the modified and selectivity of biosensor largely depend on the formation and immobilization of a functional modified film on the base surface. have gained interest in industrial applications and because of their macromolecular size exclusion effect and We found that poly(diallyldimethylammonium chloride) used cationic polyelectrolyte,4 could enhance the EeL intensity of luminol. Therefore an EeL-based sensor - PDDA-chitosan modified glassy carbon electrode was developed and applied for ultrasensitive analysis (Fig. 1), of peroxide via reaction with luminol (detection limit of 0.85 nmoI/L). The combination of highly specific biological reaction and the sensitive EeL detection provides a powerful analytical tool for clinical application. MATERIALS AND METHODS Luminol, poly(diallyldimethyl ammonium chloride) and chitosan were obtained

1. EeL generation on the PDDA-chitosan modified GeE. 301

302

WuX etal.

from Sigma. ECL detection was performed by using a BPCL Ultra-Weak Chemiluminescence Analyzer (Institute of Biophysics, Chinese Academy of Sciences) with a CHI 620B electrochemical analyzer (Shanghai Chenghua Instrument Co., China) as potential controller. A three-electrode system was used, including a PDDA/chitosan modified GCE as the working electrode, a platinum wire as the counter electrode and Ag/AgCI (sat. KCI) electrode as the reference electrode. Electrode preparation. A 0.5 % w/w chitosan solution was prepared according to Burchardt et al. 4 The PDDA-chitosan film modified electrode was prepared by immobilizing 5 /-lL of PDDA-chitosan solution [60 /-lL PDDA (10 % w/w) and 40 uL chitosan (0.5 % w/w)] on the surface of glassy carbon electrode. After drying at room temperature, it was used directly for ECL detection. RESULTS AND DISCUSSION Electrochemical chracterization of PDDA-chitosan modified GCE. Fig. 2 shows cyclic voltammograms (CYs) of 1.0 mmoliL ferricyanide at differently modified electrodes. Compared with the response at bare GC (curve a), the peak current obtained at the chitosan modified electrode was increased (curve b), owing to the absorbtion of the negatively charged ferricyanide to the highly positively charged chitosan on the electrode surface. When the electrode was modified with PDDA-chitosan solution, the continuing increase of peak current became obvious (curve c). That can be attributed to the replacementofa part of chitos an by PDDA, which has highly positive charge density and permselectivity. The PDDA-chitosan modified film is a better mass transfer layer, which is useful as an ion-to-electron transducer. 120 80

-= ~

8

___

b

40

0 -40

-120 ' 0 - - - - - - , - - - - - , - - - - - - - - - , - - - - - - - . ' 0.50 0.40 0.30 0.20 0.10

Potential I V

Fig. 2. Cyclic voltammograms offerricyanide at different electrodes: (a) bare GCE; (b) Chitosan modified GCE; (c) PDDA-chitosan modified GCE. Supporting electrolyte, 1.0 mmollL Fe(CN)63- + 0.2 moliL KCI; scan rate, 50 mY·s- 1 .

A Novel Electrochemiluminescent Sensor Based on Cationic PolymerlChitosan

303

Cyclic voltammetry and ECL of luminol-H 202 system at different electrodes. Cyclic voltammograms of luminol at different electrodes in 0.1 moUL phosphate buffer (PBS, pH=7.5) were obtained (Fig. 3A). Similarly, an enhancement of redox current from the analyte was observed at the PDDA-chitosan modified GCE, compared with the response at the bare GCE. This is due to the good permselectivity and highly positive charge density of the PDDA-chitosan composite layer. The negatively charged luminol could be easily absorbed on the surface of modified GCE through electrostatic interaction, which was supported by the linear increase of oxidation current vs. scan rates. The ECL response of luminol at bare GCE and modified GCE were obtained (Fig. 3B). It was observed that the ECL intensity of luminol system increased remarkably (Fig.3B-c) when the PDDA-chitosan modified GCE was employed, that indicated the rapidly enrichment ofluminol on the surface of modified GCE. 2000

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200

.

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Fig. 3. (A) Cyclic voltammograms from mixing 2x 10- moUL luminol and 1 xl 0- 7 moUL H 20 2 in 0.1 mol/L PBS buffer solution (pH=7.5) at (a) bare GCE; (b) 5 PDDA-chitosan modified GCE. (B) ECL response from mixing 2 xl 0- moUL 7 luminol and I x 10- moUL H 2 0 2 in 0.1 mollL PBS buffer solution (pH=7.5) at (a) Bare GCE; (b) Chitosan modified GCE; (c) PDDA-chitosan modified GCE. The effect of some experimental parameters on the ECL intensity of luminol-H 2 0 2 system were studied. The optimum conditions for the detection of H 20 2 were scan 5 mode, CV; scan rate ISO mV/s; buffer, 0.1 mollL PBS, pH 7.5; luminol, 2 x 10moUL. Reproducibility and stability of PDDA-chitosan modified GCE. The proposed method was reproducible, and the relative standard deviation (RSD) of ECL from mixing I xIO- 7 moUL H 2 0 2 and 2.0xIO-5 mol/L luminol solution was 2.6% for 10 independently prepared electrodes, and 0.97 % for one electrode. The PDDA-chitosan modified GCE showed good repeatability and long-term stability. The electrochemical and ECL response of the modified electrode remained unchanged even when it had been stored at 4 °c for two weeks.

304

Wu X et al.

Linear range and detection limit for H 20 2 detection. Under the optimization condition, a calibration curve for the determination of H 2 0 2 was obtained in aqueous solution. The ECL intensity (IECL) was linear with the concentration of H 20 2 (CH202 ) in the range of 1.0xlO-9-5xlO-5 moVL [IEcd (a.u.) =3374.5 + 2748.2C H202 1 (moI/L) R2=0.9984]. The detection limit (S/N=3) was 8.5xl0- l omollL. Owing to the size exclusion effect and permselectivity of polyelectrolytes, the PDDA-chitosan modified GCE was free of interference that may co-exist in biological samples, such as K+, Na+, SO/-, N0 3 -, lactose, amylum, sucrose, fructose, maltose, citric acid, uric acid and ascorbic acid. The results indicated a good selectivity to the detection of hydrogen peroxide.

CONCLUSION The PDDA-chitosan modified glassy carbon electrode greatly enhanced the ECL response of luminol-H2 0 2 system and showed good selectivity and repeatabilty for detection of H 20 2 (detection limit ca. 8.5 x 10- 10 mollL) and a five orders of magnitude dynamic working range. The performance of this sensor makes it very attractive for future application, since hydrogen peroxide is not only an essential mediator but also a by-product of several highly selective oxidases in biological processes. ACKNOWLEDGEMENTS This project was supported by the National Nature Sciences Foundation of China (20735002, 20575011), Program for New Century Excellent Talents in University (NECT-06-0572) and Fujian Provincial Natural Science Foundation of China (00510006). REFERENCES 1. Richter M. Electrochemiluminescence (ECL). Chern Rev 2004; 104:3003-36. 2. Marquette C, Leca B, Blum LJ. Electrogenerated chemiluminescence of luminol for oxidase- based fibre-optic biosensors. Luminescence 2001;16:159-65. 3. Yang M, Yang Y, Liu B, Shen G, Yu R. Amperometric glucose biosensor based on chitosan with improved selectivity and stability. Sens Actuat B 2004; I 0 1:269-76. 4. Burchardt M, Wittstock G. Kinetic studies of glucose oxidase in polyelectrolyte multilayer films by means of scanning electrochemical microscopy (SECM). Bioelectrochem 2008;72:66-76.

CAPILLARY ELECTROPHORESIS-ELECTROCHEMILUMINESCENCE DETECTION OF CIPROFLOXACIN IN BIOLOGICAL FLUIDS XIAOMING ZHOU: Ll JIA

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China, Email: [email protected]

INTRODUCTION Ciprofloxacin (CIP) is a potent second generation fluoroquinolone drug (Fig. 1) widely used both in human and veterinary medicine to treat infectious diseases. High-performance liquid chromatography (HPLC) based methods have been em-ployed for its deter~inationY Capill~ry electrophoresis (CE) may have potential for CIP analysis. Advantages of CE for CIP analysis include its speed and cost of analysis, and the possibility of rapid method development. Electrochemiluminescence (ECL) is a type of chemiluminescence produced as a result of electrochemical reactions. ECL detection has many advantages including its simplicity, inexpensive instrumentation, low background noise, high sensitivity, 3 good ~electivity, ~nd wide dynamic linear range. - We have dev~loped a ne~ simple and sensitive CE-ECL method for CIP analysis in biological fluids. The method is based on the CE separation and the detection of secondary amino moieties in CIP with end-column tris(2.2-bipyridyl)ruthenium(II) electrochemiluminescence.

o

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OH N

L Ciprofloxacin Fig. 1. The molecular structure of CIP 305

306

Zhou X & Jia L

EXPERIMENTAL Reagents were of analytical grade. Solutions for CE were stored at 4°C. The solutions were all passed through 0.22 mm filters before being injected into the CE system. The CE-ECL system was described previously.4 A standard stock solution of CIP was dissolved in methanol (1 mg/mL) and stored at 4°C. Working standard solutions were prepared daily by diluting the stock standard solutions in 0.1 % acetic acid. For preconditioning, the capillary was pretreated by rinsing at high pressure with 1 MNaOH for 10 min, pure water for 10 min, and phosphate electrolyte for 15 min. In order to obtain better reproducibility, between runs, the capillary is rinsed at high pressure with 0.1 M NaOH 1 min, pure water for 2 min, and buffer for 3 min. The injection was done electrokinetically and CE was performed at room temperature. Blood samples from a healthy volunteer were collected and immediately centrifuged at 3000 rpm/min for IS min. Serum was spiked with CIP at different concentration levels aliquotted and stored at -20°C. Before use, the serum was diluted 10-fold with 0.1 % acetic acid to decrease the interference of the ionic strength of the sample matrix. Fresh urinary samples from a healthy volunteer were spiked with CIP at different concentration levels, and were filtered through a membrane (0.22 ~m). After that, the samples were diluted 20-fold with 0.1 % acetic acid. The filtrate was injected into the CE-ECL system and analyzed. RESUL TS AND DISCUSSION Optimization of CE-ECL conditions. The detection potential was firstly optimized because ECL is dependent on the potential applied to the electrode. It was found the highest ECL intensity was obtained for a potential of 1.15 Y. The ECL reaction of Ru(bpy)/+ with alkylamine was a pH-dependent process and the maximum ECL emission was observed in pH 8.5 for crp. The concentration of phosphate buffer in the ECL detection reservoir was also carefully examined. The corresponding ECL intensity increased remarkably when the concentration of phosphate buffer was changed from 20 to 100 mM, but the baseline was unstable > 100 mM. Therefore, 100 mM phosphate buffer was selected. The effect of the concentration of Ru(bpy)/+ on ECL intensity was investigated. The experimental results showed that the ECL signals for crp increase almost linearly with the increase of the Ru(bpy)/+ concentration in the range from 2 to 8 mM. While the background noise also increases with the increased of the concentration of Ru(bpy)/+. Considering the signal to noise ratio,S mM Ru(bpy)/+ was selected. Separation voltage was investigated in the range of 8 to 20 kY. When separation voltage increased, ECL intensity increased and reached a maximum at 14 kY. When the separation voltage exceeded 14KY, the ECL intensity decreased. This is due to the strong flow of effluent from the capillary decreased the concentration of Ru(bpy)3 2 + at the working electrode surface, thereby reducing the efficiency of ECL reaction. Thus, 14 kY was selected.

,au"ua,' v

2.

Electrophoresis-Electrochemiluminescence Detection of Ciprofloxacin

pr()or~lm"

307

of serum samples (A), urinary samples (8), and standard CIP samples (C).

Detection limit of CIP. Calibration was linear in the range 0.05-1.5 (y=810 (±26) x + 45 (±18) and R=0.997). Detection limit of 15 with a signal-to-noise of 3 was achieved for CI P. The repeatability of the method was studied by six consecutive injections of standard solution of CIP at 1 Relative standard derivations of the ECL intensity and the migration time were 3.25 and 0.84% for CIP, respectively. to human urine and human serum. The proposed CE-ECL method to the determination of CIP in urinary samples and blood In both urinary samples and blood samples were spiked with different concentrations levels of CI P. Due to the inherent excellent selectivity and of the CE-ECL method, the samples were prepared without extra some simple procedure such as concentration, filtration, and dilution. was the electropherograms of the blank serum sample (a), and 50 CIP standard solution was spiked into serum sample as shown in (b).

308

Zhou X & Jia L

was the electropherograms of the blank urinary sample (a), and 10 ""giL CIP standard solution was spiked into urinary sample as shown in (b). The peak of CIP was verified according to the electropherograms of standard samples as shown in Fig. 2 (C). Reported concentrations ofciprofloxacin varied from 0.1 to 0.65 mg/L in CE-ECL is sensitive enough for the measurement of CIP in biological fluids. We noted that although some unknown peaks were found in the electropherograms of aqueous and from 0.17 to 0.51 mg/L in vitreous after the oral administration of various doses of the antibiotic to humans. s Therefore the current results indicate this both urinary and blood samples, no interferences were found co-migrating with CIP thus showing the proper specificity of the proposed method.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30600128; 30700155), the National High Technology Research and Development Program of China (863 Program) (2007AAI0Z204)), and the Natural Science Foundation of Guangdong Province (7005825). REFERENCES 1. Liang H, Kays MB, Sowinski KM, Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin, trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin determination in human plasma. I Chromatogr B 2002;772:53-63. 2. Zotou A, Miltiadou N, Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids. I Pharm Biomed Anal 2002;28:55-68. 3. Zhu DB, Xing D, Shen XY, Liu IF. A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. Biochem Biophys Res Commun 2004;324:964-9. 4. Gao WD, Liu JF, Yang XR, Wang E. New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection. Electrophoresis 2002;23:3683-91. 5. Lesk MR, Ammann H, Marcil G, Vinet B, Lamer L, Sebag M. The penetration of oral ciprofloxacin into the aqueous, vitreous and subretinal fluid of humans. Am I Ophthalmol. 1993; 115:623-628.

PART 6 BIOMEDICAL APPLICATION OF FLUORESCENT PROTEINS

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A NOVEL MULTICOLOR FLUORESCENT PROTEIN FROM THE SOFT CORAL SCLERONEPHTHYA GRACILLIMA KUEKENTHAL YUKO KATO,I MITSURU JIMBO,2 CHITOSHI SATO,3 T ASTUY A TAKAHASHI, I YUKIMITSU IMAHARA,4 HISAO KAMIY A2 1Department of Environment, Tohwa University, Fukuolw City, 815-8510, Japan 2Department of Marine Biosciences, Kitasato University, iwate, Japan ~ 3Junshin Junior College, Fukuolw City, 815-8510, Japan Walwyama Prefectural Museum of Natural History, Japan Email: [email protected]

INTRODUCTION In recent years marine organisms have been considered as potential sources for useful substances such as medicines and biotechnological reagents. Green fluorescent protein from Aequorea victoria is well known for its usefulness in biotechnology. I In our survey of the fluorescent proteins in marine animals, Scleronephthya gracillima (Kuekenthal) was found to possess multicolored fluorescent proteins. This paper deals with the separation of a new fluorescent protein named "Akane" and also its fluorescent spectroscopic properties. The fluorescence dependency on pH suggests that the fluorescence derives from protonation of the chromophore.") The cDNA cloning of "Akane" was also conducted. METHODS Extraction and separation of fluorescent protein. The soft coral S. gracillima Kuekenthal was first fractionated by a gel filtration, then the fluorescent protein was separated by anion exchange column chromatography on an anion Q-Sepharose High Performance column (Amersham Biosciences) after ultra-filtration with a USY-J membrane (Advantec). All the fractions were analyzed by spectrofluorophotometer (RF-5300 PC Shimadzu). Fluorescent spectroscopic analysis. The fluorescent spectroscopic analysis of the fraction from the anion exchange column was performed using J0 mM-Tris buffer pH 8.5, changing O.lM to 0.15M NaCI. RESULTS AND DISCUSSION The fluorescence intensities at each wavelength (430, 505, 570, and 660 nm) and absorbance (280 nm) of the fraction are depicted in Fig. I. Multiple emissions were observed at one excitation wavelength 298 nm. Fluorescent spectra of Fr (2-10) and Fr (3-6) were examined at different sodium chloride concentrations. Emission peaks were observed at 430 480, 505, 570, 636 and 663 nm. The remarkable result was that the 570 nm emission peak only appeared in 0.15M-NaCI (Fig. 2). pH dependency of the fluorescent spectra were analyzed by changing the pH from 7.0 to 8.5. Results are shown in Fig. 3. 311

312

Kata Yet al. 150

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Fig. 2. Fluorescent spectra of anion exchange column fractions at different sodium chloride concentration levels of Fr (2-10) and Fr (3-6) were excited at 298 nm.

A Novel Multicolor Fluorescent Proteinfrom the Soft Coral S. gracillima Kuekenthal Fluorescence -pH8.S Int.

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450

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700 400

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Fig. 3. Emission spectra of Fr (3-6) (left) and Fr (2-10) (right) in pH range 7.0 to 8.5. left: Fr (3-6) 0.15M-NaCI was excited by 298 nm, changing the pH from 7.0 to 8.5. It was remarkable that the 566 nm emission only appears at pH 8.5, then 477 nm emission appeared at pH 8.0 to 7.0, while the shape of 663 nm and 503 nm emissions did not show significant change. right: Fr (2-10) 0.10 M-NaCI was excited at 298 nm. The 475 nm emission appeared at pH 8.0 to 7.0 not at pH 8.5, while the 436 nm emission only appeared at pH 7.0. LC-ESI-MSIMS analysis and eDNA Cloning. In-gel trypsin digestion of the component followed by peptide fragment sequence analysis by LC-ESI-MS/MS LCQdecaXP gave the amino acid sequence of YPADLPDYFK. The homology search based on MASCOT on the NCBInr Database revealed that the component showed homology to a cyan fluorescent protein Acropora aculeus. Then cDNA was cloned by the RACE method using a gene specific primer designed from amino acid sequences obtained by LCIESI/MS/MS. Four closely related cDNAs were obtained. The ORFs of the cDNAs are composed of 225 residues. Deduced amino acid sequence of "Akane" is contained the putative chromophore sequence ofGFP family at His62-Tyr63-Gly64 for (Table 1). Table 1. Complete gene sequence of "Akane" 1 41 81 121 161 201

MNPIKEDMKV KVYLEGNVNG HAFAIEGEGK GNPLDGTQTM NLTVKEGAPL PFSFDILTTS LHYGNRVFTK YPADIPDYFK QSFPEGFSWE RTMTYEDKGI CTIRSDISLQ GDCFIQKVRF HGINFPSNGP VMQKKTLKWE PSTERMYVRD GVLVGDINNA LLLEGGGHYV CDFKTTYKAK KVVQLPDYHF VDIRIEILSH DRDYNKVKLY EHAVARHSLV PSQAR *

314

Kata Yet al.

CONCLUSION The fluorescent protein was separated from the extract of the soft coral by gel filtration and anion exchange column chromatography. In the fluorescent spectroscopic analysis of the purified protein, multiple emissions from the protein were observed (using one excitation wavelength) and these emissions depended on the pH (7.0 - 8.5) and also on the sodium chloride concentration. The complete gene sequence of "Akane" was gained from cDNA cloning. The partial sequence 081SFPEGFSWER91 is similar to the DsRed sequence' and the 71 80 y pADLPDYFK is a homology to a cyan fluorescent protein Acropora aculeus. Thus, cDNA of "Akane" had strong similarities to green fluorescent protein families especially to Dendronephthya sp." in the sequence of y71_T92MTYEDKGICTIRI04.

ACKNOWLEDGEMENTS We are grateful to Sachiko Matsuhashi, Faculty of Medicine and the Graduate School of Saga University, and Susumu Watanabe, Hitachi High-Tech Manufacturing & Service Corporation Proteome Analysis Laboratory for their involvement in this research.

REFERENCES 1.

2.

3.

4.

5. 6.

George TH, Tim BM, Eun SP, et al. Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 2002;4l:l54 77-88 Brooks R, Ofelia R, Anna VP et al. pH-Dependent fluorescence of a heterologously expressed Aequorea Green Fluorescent Protein mutant: In situ spectral characteristics and applicability to intracellular pH estimation. Biochemistry 1998;37:9894-901. Marc AE, Rebekka MW, George TH, et al. Structural and spectral response of Green Fluorescent Protein variants to changes in pH. Biochemistry 1999;38:5296-301. Ahmed AH, Samuel TH, Watt WW. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chern Phys 2001;274:37-55. Mark A W, Michael S, Rama R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Struct Bioi 2000;7:1133-38. Pakhomov AA, Martynova NY, Gurskaya NG, et al. Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp. Biochem (Mosc) 2004 ;69 :90 1-8.

FLUORESCENCE FROM S2-LEVEL OF COMPLEXES OF TRYPTOPHAN WITH EUROPIUM (III) IN WATER-ETHANOL SOLUTION 10 OSINA, S OSTAHOV, V KAZAKOV Institute a/Organic Chemistry, USc, RAS, Pro Oktybrya, 71, U/a,450022, Russia Email: [email protected]

INTRODUCTION The participation of higher excited singlet states (Sn' n > I) of molecules in photophysical (Sn ~ So fluorescence (FL)) or photochemical (photoinduced electron transfer (PET), isomerization, etc.) processes, which compete with radiation less deactivation, manifests itself in the dependence of the quantum yield (cp) and FL spectra on the wavelength of the exciting light (the violation of the Vavilov law). Such processes were first shown for the FL of azulene solutions due to the transition from the second excited level to the ground state S2 ~ So . I METHODS The FL spectra were recorded on a Hitachi MPF-4 spectrofluorimeter. The absorption spectra were recorded on a Specord M-40 spectrophotometer. The tryptophan (Trp) and EuCI 3 '6Hp were purified by double recrystallJration from twice distilled water and dried in vacuum. The Trp concentration was 10 moUL. RESULTS The FL spectra of Trp in water and dry ethanol are independent of the wavelength of the excitation light (I\.exc). The introduction of europium chloride, which forms complexes with tryptophan,2 (log PI = 4.82 ± 0.07 (HP, 298 K)) quenches FL through PET,1-4 also it has no effect on the FL spectra of Trp in individual solvents. Taking into account the strong Stokes shift of the FL spectra of Trp in H 20 (Amax = 353 nm) relative to the Trp ethanol solutions (Amax = 337 nm), we may assume that an increase in the H 20 content in the water-ethanol solvent will be accompanied by a "red" shift of the FL spectra. However, in 90% C 2H sOH, excitation into the second and shorter-wavelength absorption bands of Trp does not lead to the bathochromic shift, rather, a shortwavelength "shoulder" emerges in the FL spectrum of Trp (Fig. 1, spectrum 1). In the presence of europium chloride, the "shoulder" at 295 nm transforms into a peak (Fig. 1, spectra 2 and 3), which is assigned to the S2 ~ So radiative transition. 315

316

Osina IO et al.

I

I

I

,

I

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270 310 350 390 A,nm Fig. 1. FL spectra ofTrp: (1) without EuCb'6H zO, (2) c(Eu(III» = 1O-5 mo I/L, and (3) c (Eu(III» = 6 x 10-5 mol/L ("-exc = 245 nm, 90% CZH 50H, 298 K). The long-wavelength SI ~ So FL of Trp (Aexc = 245 nm) in 90% CZH 50H IS efficiently quenched by europium chloride, according to the Stern-Volmer equation: loll -1 = K[Q] (Fig. 2, curve 1) with the quenching constant K = 12000 Llmol (298 K). Quenching of the SI ~ So FL ofTrp (Arnax = 337 nm) by Eu(III) chloride makes it possible to clearly observe the short-wavelength Sz ~ So component (Arnax = 295 nm). The latter becomes dominant at a Eu(lII) concentration of 6x 10-5 mollL (Fig. 1, 3). At the same time, we observe the rise of Sz ~ So FL of Trp with an increase in the Eu(III) concentration (Fig. 2, curve 2). The Sz ~ So FL of Trp in aqueous alcohol solutions depends not only on the complexation of the amino acid but also on the excitation energy. Fig. 3 shows the FL spectra and the dependence ofl(295 nm)/I(337 nm) (1(295 nm) and 1(337 nm) are the intensities of the Sz ~ So and SI ~ So transitions, respectively) on the Aexc at a -5

Eu(III) concenration of 6 xl 0 mol/L. In the excitation wavelength range L'1 Aexc = 210 - 260 nm, the intensities of S I ~ So FL and short-wavelength Sz ~ So FL of Trp are redistributed. At 210 nm, the S\ ~ So FL is dominant and the short-wavelength component is observed as a poorly pronounced peak (Fig. 3, spectrum 1).

Fluorescence from S2-Level of Complexes of Tryptophan with Europium (III)

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("'exc =

245 nm, 90% C2HPH, 298 K).

As the "'exc increases, the S2 -7 So FL intensity increases and achieves a maximum value at 245 nm (Fig. 3, curve 4 and spectrum 2). In the wavelength range 245 - 260 nm, the short-wavelength S2 -7 So FL decays (Fig. 3, curve 4), and at "'exc ~260 nm, only ordinary S[ -7 So FL ofTrp is observed (Fig. 3, spectrum 3).

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nm and (4) the plot ofI(295 nm)/I(337 nm) vs. "'exc (c(Eu(III» C2HPH, 298 K).

=

6 xl0

moUL,90%

318

Osina 10 et al.

The /(295 nm)//(337 nm) ratio of the intensities of the Sz -7 So and SI -7 So transitions increases with the concentration of europium chloride upon excitation at a wavelength in the range l1A..xc = 210 - 260 nm. At the same time, at any Eu(III) concentration, the maximal /(295 nm)//(337 nm) value is always observed at 245 nm (Fig. 4, curve 4), which can be naturally attributed to the position of the Sz-Ievel with a minimal vibronic energy. Assuming that E(Sz) = 5.06 eV, we may estimate the -I

energy gap width I1E(Sz - SI)::::: 9400 cm (E(SI) = 3.9 eV \ Despite long-term studies of the photophysics and photochemistry of Trp, our findings are the first observation of its FL due to the transition from the S2 to the Solevel.

ACKNOWLEDGMENTS This work was supported by the Division of Chemistry and Materials Science of the RAS in the framework of program no. 1-0kh and Russian Science Support Foundation. REFERENCES I. Beer M, Longuet-Higgins H. Anomalous light emission of azulene. J Chern Phys 1955;23:1390-1. 2. Batyaev I, Fogileva R. Thermodynamics of tryptophan complexation with rare earth elements. Zh Neorg Khim 1976;21:1199-1201. 3. Ermolaev V, Sveshnikova E, Shakhverdov T. Study of complexation between organic molecules and rare earth elements ions in solutions by method of electronic energy transfer. Usp Khim 1976;45:1753-81. 4. Kazakov V, Ostakhov S, Alyab'ev A, Farrakhova G. Reversible photoinduced electron transfer from tryptophan to Eu(fod)3, Hfod 11 EuCh'6H 20 in the liquid and frozen etanol solutions. High Energy Chemistry 2005;39:97-9. 5. Horrocks W, Bolender J, Smith W, Supkowski R. Photosensitized near infrared luminescence of ytterbium (III) in proteins and complexes occurs via an internal redox process. J Am Chern Soc 1997;119:5972-3.

IDENTIFICA TION OF DEVELOPMENTAL ENHANCERS USING TARGETED REGIONAL ELECTROPORATION (TREP) OF EVOLUTIONARILY CONSERVED REGIONS CU PIRA, SA CALTHARP, K KANA Y A, SK MANU, LF GREER, KC OBERG Department of Pathology and Human Anatomy, Loma Linda University, 24785 Stewart St, Evans Hall Rm B09, Loma Linda, CA 92350, USA Email: [email protected]

INTRODUCTION During development, precise temporal and spatial regulation of critical genes is required to orchestrate body plan morphology. Preservation of a generalized developmental process and body plan across divergent species suggests that regulation has also been conserved. Thus, evolutionarily conserved regions (ECRs) in association with developmentally important genes are likely candidates as regulatory elements. Screening of ECRs has recently been described during early chick development, using in vitro whole embryo electroporation of ECR constructs containing green fluorescent protein (GFP).' The major limitation of this technique is that the chick embryos only survive in vitro for about 48 hrs after electroporation and thus enhancers involved in later development and organogenesis cannot be determined. We previously reported on a method to deliver expression vectors at targeted locations during in ovo development by confined microelectroporation (CMEP).2 We have modified this technique to broaden the targeted region of electroporation and vector delivery, i.e., targeted regional electroporation (TREP). Herein, we demonstrate the ability of this technique to screen for ECR activity at later stages of development. MATERIALS AND METHODS Construction of pTK-EGFP ECR constructs. Evolutionarily conserved regions (ECRs) were identified using the VISTA genome browser. To test ECR activity, we generated expression constructs with pTK-EGFP plasmid (a gift from Dr. Uchikawa, Osaka University), which contains the minimal HSV TK promoter linked to an enhanced GFP reporter gene (Fig.! ).' ECRs were isolated by PCR from genomic mouse (Emx2) or chicken (SHH) DNA. Each was ligated into pTK-EGFP at KpnI and XhoI. Plasmids were isolated and purified using the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA). pCAGGS-RFP plasmid (gift from Dr. Tickle, University of Dundee) was co-e!ectroporated to verify transfection. Electroporation. Whole embryo electroporation in vitro was performed as previously described.' For targeted regional electroporation (TREP) chick embryos were stained (neutral red) and staged according to Hamburger and Hamilton (HH).3 The vitelline membrane overlying the embryo was removed and a small slit was cut on the yolk membrane near the heart. Platinum electrodes (0.3 mm diameter, at 2.5 mm distance) were mounted on a micromanipulator and positioned parallel to the embryo. 319

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Pira CU et al.

11 origin

B

c

L Targeted Regional Electroporation (TREP). ECRs were ligated into pTK-EGFP plasmid. (B) DNA solution (hash was into the embryonic coelom of the presumptive wing region of HH14 chick The cathode (-) was placed underneath the embryo in the yolk while the anode (+) was placed above the embryo. The cathode was slid through the slit and into the yolk. DNA solution (2 0.25 flg/flL pCAGGS-RFP with phenol red and TE was into the embryonic coelom of the lateral plate mesoderm. The anode was above the embryo and 3-5 drops of PBS were used to promote conductivity. was performed using the CUY-2! Electropol'ator San Antonio, TX) at 8 volts, with 3 pulses of 50 msec ONIlOO msec OFF. After each the electrodes were cleaned in bleach, RNase-free water, and PBS to remove residual yolk. Fluorescence was visualized by a fluorescent MZ-8) and digitally recorded (Sony DKC-5000).

2. Identification of Evolutionarily Conserved Regions of the Emx2 locus across divergent species using VISTA Genome Browser revealed mUltiple evolutionarily conserved regions (ECRs; shaded columns).

ldt'ntificcuic'n of Developmental Enhancers Using Targeted Regional Electroporation

321

RESULTS ECR enhancer activity in embryos. Non-coding evolutionarily Fig. 2) may retain regulatory function. Whole could conceptually be used to screen the potential role development, but mUltiple attempts at in ovo EP of early embryos were unsucessful (early embryonic disk and associated membranes were too to survive even slight disruption during electrode placement). electroporated and grown in vitro. The embryo can survive in vitro for but distortion of embryonic growth is evident. We examined ECR associated with Emx2, a transcription factor linked to brain In vitro whole embryo EP demonstrated enhancer of brain development (Fig. 3). With additional manipulations, extended to 60 1m, however, limb buds were still not present. limb development could not be determined by this method.

of an Emx2-related ECR in the after whole embryo EP and 60 hrs of growth in vitro should Hll 18, but under incandescent light (light) it more closely resembles of Efficient transfection is noted by uniform expression) in the forebrain is indicated to the Emx2 expression domain within the brain. To overcome the difficulty of limited survival with whole embryo EP, we developed EP We evaluated TREP using a known limb-specific enhancer ECR (1.8 sonic hedgehog (SHH) expression in the limb's zone of the native limb-specific SHH expression domain done by whole embryo we performed TREP at to limb outgrowth. Adequate transfection was determined by RFP ZPA-related GFP expression 48 hrs after TREP confirmed enhancer of this However, the Emx2-related ECR that showed enhancer in the brain was not detected in the limb.

322

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Fig. 4, SJOT-related ECR Enhancer Activity is Detected in the ZPA after TREP. Broad RFP expression is detected in the limb bud 48 hrs after TREP; in contrast, GFP expression is restricted to the region of the ZPA (arrowheads). No enhancer activity is detected in the limb-related Emx2 expression domain (ISH) by the £mx2-related ECR CONCLUSION Targeted regional electroporation (TREP) extends the capacity of whole embryo electroporation to identify regulatory ECRs that participate at any point in the process of development. Although we targeted the limb, other organs, regions or even later stages could readily be examined. Developmental pathways are frequently re-utilized during development and thus, require tissue/organ specific regulation. We anticipate that this technique will be of great value in the identification of conserved tissuespecific regulatory elements that participate in the process of development and morphogenesis. REFERENCES 1. Uchikawa M, Ishida Y, Takemoto T, Kamachi Y, Kondoh H. Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev Cell 2003;4:509-19. 2. Oberg KC, Pira CU, Revelli J-P, Ratz B, Aguilar-Cordova E, Eichele G. Efficient ectopic gene expression targeting chick mesenchyme. Dev Dyn 2002;224:29i~302. 3. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol 1951;88:49-92.

PART 7 DEVELOPMENT AND BIOMEDICAL APPLICATIONS OF QUANTUM DOTS AND OTHER INORGANIC FLUORESCENT MATERIALS

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QUANTUM DOTS AS FLUORESCENT RESONANCE ENERGY TRANSFER DONORS IN ANTIBODY-ANTIGEN SYSTEM HU SHAN,l YANG HAI,l CAl RUXru,u ZHANG QI,l YANG, XIANGLIANG 1* JCollege 0/ Life Science and Technology, Huazhong University o/Science and Technology, Wuhan, Hubei, China; 2Col/ege o/Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei, China. Email: [email protected]

INTRODUCTION Fluorescence resonance energy transfer (FRET) is a powerful technique to study the structures and interactions of individual biomolecules. Quantum dots (QDs) have gained widespread application as energy donors and acceptors in a variety of FRET based biological studies. They have been used as energy donors to study the FRET process, 1 to sense glucose,2 and to detect oligonucleotide hybridization. 3 Streptavidin-biotin4 as a bridge have been applied in most FRET systems because of its high affinity and small volume. Immunocomplexes of antigen and antibody as a bridge 5 in FRET systems have been studied and FRET processes has been observed. Su's studies 5 proved the size of immunocomplex was small enough to fulfil the need of FRET. Because the combination between antigen and antibody is relatively slow and the reaction is reversible, FRET in an antigen-antibody system differs from the streptavidin-biotin system. Competitive reactions based on reversible combination of antigen and antibody are widely used in ELISA. It also has the potential for a novel quantitative analysis method based on FRET and reversible reaction of antigen-antibody. In this paper, a novel competitive immunoassay to detect the concentration of protein (IgG) based on FRET and is described. EXPERIMENTAL Instrumentation and reagents. Fluorescence experiments were recorded by a Hitachi F-4500 fluorescence spectrophotometer and Perkin Elmer 1420 multilabel counter. QDs were purchased from Invitrogen. ImmunoPure F(ab')2 Preparation Kit that is used to prepare F(ab')2, was obtained from Pierce. Preparation of bioconjugate QD-F(ab')z. Rabbit anti-mouse IgG F(ab'h fragments were prepared following the experimental procedure provided by Pierce. QDs and F(ab')z were conjugated following the experimental procedure provided by Invitrogen. FRET assay between RBITC-IgG and QD-F(ab'h Experiment 1: The fluorescence spectrum of the mixture of diluted QD-F(ab')2 and RBITC-IgG was monitored (excitation 300 nm) every 0.5 h and detected at 2 h. The same procedure was done to the mixture of diluted QD-F(ab'h and PBS. Experiment 2: The fluorescence spectrum of the mixture was detected every 0.5 h. 0.33 mg/mL and Img/mL mouse IgG was added respectively to the mixture above and the fluorescence spectrum was monitored. Experiment 3: PBS and RBITC-IgG were 325

326

Set at.

fluorescence was monitored. Experiment 3: PBS and were added to the diluted QD-F(ab')2 respectively. The fluorescence of the mixtures was detected (excitation 300 nm) every 0.5 h. 1.5 /-lL 1mg/mL mouse was added to both the mixtures and the fluorescence spectrum was detected in the was same way. 3.5 Img/mL mouse IgG and then 10 !!L 1 mg/mL mouse added and the above experiments repeated.

RESULT AND DISCUSSION of the conjugates. Fig. I (a) shows the fluorescence bioconjugate excited at 300 nm and emitted at 562 nm. shows the fluorescence spectrum of the RBlTC-IgG conjugate excited at 562 nm. This indicated that the emission of the QD would excite RBlTC which means that RBITC could be used as the FRET acceptor for the of RBlTC is low and an increase in the fluorescence of RBITC the was not observed. FRET effect of and F(ab')z. Experiment 1: The fluorescence decreased and emission at 562 nm of the mixture of QD-F(ab')2 and became stable after .5 h. The same experiment was done to compare the mixture of and and it was found that the fluorescence was stable but decreased. The reason could be that FRET occurred between QD and RBITC when mouse and rabbit-anti-mouse F(ab')z formed an immunocomplex. 40

(a)

(b) 30

20 10

()-~------~

300

400

500

300

400

450

500

bOO

Emission Wavelength {nm)

'Aroo~,on.~c emission spectrum of diluted QD-F(ab'h conjugate. a of protein was observed at 340 nm because of the strong UV absorbance (b) Emission spectrum ofRBITC-IgG conjugate (emission peak of conjugated RBITC is between 570 nm and 580 nm).

To validate whether FRET occurred between and was added as a competitor. QD fluorescence intensity decreased when was added. Intensity increased when IgG was added showing that FRET occurred. The reason could be that free JgG competed with conjugated to so the number of RBITC-IgG-F(ab')Z-QD complexes was reduced and the of FRET reduced accordingly. Therefore, the fluorescence intensity of recovered. Additionally, the increasing fluorescence with the increase of indicated that the method could be used to determine the concentration of "rA'''''''C

Dots as Fluorescent Resonance

Donors

327

1.5 (hottr)

of vs incubation time order to eliminate the influence of ratio was calculated.

of the fluorescence intensity of the mixture with different concentrations of at different times. influence of fluorometer, the fluorescence ratio was calculated. 100%

2 indicated that the concentration of fluorescence recovery of the mixture of QD-F(ab'h and mixed with the fluorescence intensity of mixed decreased significantly. The fluorescence intensity increased after

328

Hu S et al.

mouse IgG was added to the mixture, while that of QD-F(ab')2 and PBS did not change, which indicated that FRET caused the fluorescence decrease of QDs. To eliminate the influence of the fluorescence change of QDs, QD-F(ab'h plus PBS was taken as blank and compared with QD-F(ab')2 plus RBITC. Fig. 4 shows that fluorescence was reduced over a period of time, which indicated that FRET occurred based on the combination of F(ab')2 and IgG. The experiment also showed that there is a correlation between the extent of fluorescence increase and the concentration of IgG. Therefore, this method is convenient to determine the concentration of proteins.

CONCLUSION This is the first demonstration of the feasibility of determining IgG concentration utilizing FRET in a system comprising dye labeled IgG and QDs labeled F(ab')2. F(ab')2 was prepared to shorten the distance between energy donors and acceptors in favor of FRET occurring. The resu Its showed that the system was suitable for FRET and provided a new way to detect protein simply and quickly. Another aspect of this work was that fluorescence quenching has been used to detect the target, whereas fluorescence enhancement has been widely used in many other FRET systems. A kinetic method may be adopted to study the relationship between the fluorescence and the concentration of protein. Additionally, the fluorescence of QDs was enhanced when IgG was directly added to QD-F(ab')z. The reason may be that FRET occurred between IgG and QDs, the QDs being excitated by the fluorescence oflgG at 340nm. Further research is required to investigate this mechanism. ACKNOWLEDGEMENTS This work was financially supported by the NSF of China (No: 30670552). REFERENCES I.

2. 3.

4.

5.

Clapp AR, Medintz IL, Mauro 1M, Fisher BR, Bawendi MG, Mattoussi H. Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. 1 Am Chern Soc 2004;126:301-10. Duong HD, Rhee n. Use of CdSe/ZnS core-shell quantum dots as energy transfer donors in sensing glucose. Talanta 2007;73:899-905. Algar WR, Krull UJ. Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in FRET. Anal Chim Acta2007;581:193-201. Hildebrandt N, Charbonniere LJ, Beck M, Ziessel RF, Lohmannsroben HG. Quantum dots as efficient energy acceptors in a time-resolved fluoroimmunoassay. Angew Chern Int Ed 2005;44:7612-5. Li Y, Ma Q, Wang X, Su X. Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge. Luminescence 2007;22:60-6.

SYNTHESIS AND PHOTOLUMINESCENCE OF GREEN-EMITTING 3 XdY,GdhSiOs:Tb + PHOSPHOR UNDER VUV EXCITATION ZHANG ZH 1,2 WANG YH 2 LI XX2

of Materials Scien~e and Enginee~ing, Wuhan Institute of Technology, Wuhan 430073, P.R. China; 2Department of Materials Science, Lanzhou University, Lanzhou 730000, P.R. China, Email: [email protected] 1School

INTRODUCTION Recently, attention has been paid to phosphors under vacuum ultraviolet (VUV) excitation due to the demands of plasma display panels (PDPs) and possible generation of mercury-free fluorescent lamps. For green-emitting phosphors for PDPs application, the most widely used is Zn2Si04:Mn2+, but its decay time is so long that it is difficult to exploit the fast response of PDPS. I,2 Therefore, a new green-emitting phosphors with short decay time under VUV excitation must be found. Previously,3 we studied the luminescent properties ofXz-Y19TbolSiOs under VUV excitation, and surveyed its feasibility for PDPs application. The results revealed that XZ-Y19Tbo.ISiOs presented stronger emission intensity and lower decay time than commercial Zn2Si04:Mn2+ phosphor. However, systematic investigations on the VUV photoluminescence of X2 - Y2SiOs doped with different concentration of Tb 3+ have not been reported. In this paper, in order to optimize the performance of Xz- Y2SiOs:Tb3+ and evaluate the effects of Gd 3+ on the photoluminescence ofX 2-Y 2SiOs:Tb3+, X 2-(Y,GdhSiOs:Tb 3+ samples were prepared and their luminescence properties were investigated in VUV regions in detail. EXPERIMENTAL Xz-(Y,Gd)2SiOs:Tb3+ samples X2-Y2_x_yGdyTbxSiOs (0.05 :s; x :s; 0.4,0 :s; Y :s; 0.5) were prepared by a co-precipitation process. 3 Y20 3 (99.99%), Gd 20 3 (99.99%), Tb 40 7 (99.99%) and tetraethyl orthosilicate (TEOS) (AR) were used as starting materials. Crystallinity of the sample was analyzed using Rigaku D/max--2400 X-ray diffractometer with Ni-filtered Cu Ka radiation. Excitation and emission spectra were measured at room temperature by FLS-920T fluorescence spectrophotometer with a VM-504-type vacuum monochromator (deuterium lamp source). Excitation spectrum was corrected with sodium salicylate. The decay time of the samples was examined under 147 nm excitation. The same equipment conditions were adopted for all as-prepared samples and commercial Zn2Si04:Mn2+ RESUL TS AND DISCUSSION All the powder X-ray diffraction patterns of as-prepared X2-Y2_x_yGdyTbxSiOs (0.05 :s; x :s; 0.4, 0 :s; y :s; 0.5) samples could be recognized as a single phase and readily indexed to monoclinic symmetry. Excitation spectra of X2-Y16Gdo.2Tb02SiOs (a) and XrYLSTbo.2SiOs (b) monitored at 542 nm (Fig. 1) consist of a direct Tb3+ 329

330 Zhou L-Yet al.

excitation region for wavelengths> 190 nm and a host lattice excitation region for wavelengths <190 nm. The direct excitation region was assigned to the 4['1-4f75d I transitions absorption of Tb 3+. The excitation band at about 170 nm could be assigned to the absorption ofSi-O groupS.4 Calculating by the J0rgensen's equationS and according to Ref. 6, the absorption position of the charge transfer bands (CTBs) of Tb 3+_0 2- was at about 151 nm. Moreover, the electronic transitions from 02-:2 P6 valence bands to y3+:4 p6 (4d+5s) conduction bands were located at about 155 nm. 7 Therefore, the excitation band in the regions of 130-160 nm which peaked at 141 nm could be attributed to the overlaps of the absorptions of the CTBs of Tb 3+_0 2and the electronic transitions from 02-:2 p6 to y3+:4 P6 (4d+5s). From Fig. 1, it was obvious that the strongest band of X2-Y 1.6Gdo.2Tb o.2SiO s became more intense than that of X2-Y 1.8 Tb o.2SiO s when substituting Gd 3+ for y3+. This was because the CTBs of Gd 3+_0 2- and the f ...... d transitions of Gd3+ were occurred in X2-Y 1.6Gdo.2Tbo. 2SiO s. o The CTBs of Gd3+_0 2 were located in the regions of 130-160 nm,7 and the f ...... d transitions position ofGd3+ was at about 130 nm from the Dorenbos' equation. 8

(a)

Wavelength (nm)

Fig. 1. Excitation spectra of X2-Y 1.6Gdo.2 Tbo. 2SiO s (a) and X2-Y 1.8 Tbo. 2SiO s (b) monitored at 542 nm Under 147 nm excitation, all the emission spectra of X 2- Y2_xoyGdyTbxSiOs (0.05 ~ x ~ 0.4, 0 ~ y ~ 0.5) samples had the most intense emission peak at about 542 nm, 7 which was due to the sD 4- Fs transition of Tb 3+. Fig. 2 exhibits the ralative 7 emission intensity of the sD 4 ...... Fs transition ofTb3+ in X2-Y2oxTbxSiOs (0.05 ~ x ~ 0.4) as a function of x under 147 nm excitation. The results indicated that the most . sD 4...... 7F S transItIOn .. f T b 3+ was observed at x = 0.2, and then the mtense 0 concentration quenching phenomenon was occurred. The inset in Fig. 2 represents the ralative emission intensity dependence of the sD 4- 7Fs transition of Tb3+ in X2-y 1.8_yGdyTbo2SiOs (0 ~ y ~ 0.5) against the concentration of Gd3+ under 147 nm excitation. The results revealed that the optimum emission was obtained at y = 0.2, and then the intensity decreased gradually along with the increasing 3 concentration of Gd +. At first, the CTBs of Gd 3 +_0 2o and the f ...... d transitions of

Synthesis and Photoluminescence of Green-Emitting X2-(Y,GdhSi05:Tb 3 + Phosphor

331

Gd3+ could enhance the emission intensity in the case of 0 < y :::;; 0.2. However, with the further increasing of Gd 3+ concentration, the concentration of y 3 + was decreasing and the role of the electronic transitions from 02.:2 p6 to y 3 +: 4P6 (4d+5s) was weakened, correspondingly, the emission intensity decreased. Therefore, an appropriate ratio between y3+ and Gd 3+ could be achieved with the composition of X 2- y 1.6Gdo.2Tbo.2S i0 5.

. /

.-----.'------.----

.----.

;" t~ /

~

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•

85~~:---:,;:--:;~,.,...--;:r;-I

0.0 0.1

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eon_on of Gd". Y (/role) 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Concentration of1b3., x (rrole)

Fig. 2. Relative emission intensity ofX2-Yz_xTbxSi05 as a function ofx. Inset: relative emission intensity dependence ofX 2-Y L8.yGdyTb 02 Si0 5 versus concentration ofGd3+. C)"ex = 147 nm, Aem = 542 nm)

(a)

450

650

Wavelength (nm)

Fig. 3. Emission spectra ofX r Y 16Gdo.2Tbo2Si05 (a), Xr Y lSTb02SiO s (b), and commercial Zn2Si04:Mn2+ (c). (Aex = 147 nm) Fig. 3 shows the emission spectra of Xz- Y L6Gdo.2 Tbo. 2SiO s (a) and X 2-Y L8 Tbo. 2SiO s (b) under 147 nm excitation, together with that of commercial Zn2S i0 4:Mn 2+ ( c) as a comparison. As seen from Fig. 3, X2-Y\.6Gdo.2Tbo.2Si05 had the most intense emission intensity, which was about 196% of that of commercial Zn2Si04:Mn2+. Based on the resolution of the decay curves (fitted by the single exponential equation), the lie decay time (the time of emission decays to the lie in intensity of the original luminescence) of the 5D4 .... 7Fs transition of Tb3+ under 147 nm

332

Zhou L-Y et at.

excitation was 2.9 ms for X 2-Y 1.6Gdo.2Tbo. 2SiO s and 3.1 ms for X 2-Y 1.8 Tbo. 2SiOs, respectively. Thus, the decay time was reduced by the substitution of Gd 3+ for y3+. It ma~ be that the defect impurities, which were likely to produce a decrease of the Tb + decay time,9 were increaced by the substitution. The lie decay time of commercial Zn2Si04:Mn2+ was about 5.1 ms. Therefore, X2-YI.6Gdo.2Tbo.2SiOs had more proper decay time as well as much stronger emission intensity to meet the requirement ofPDPs.

CONCLUSIONS Single phases of X2-Y2_x_yGdyTbxSiOs (0.05 :0:;; x :0:;; 0.4, 0 :0:;; y :0:;; 0.5) were synthesized and their photoluminescence were systemically investigated under VUV excitation. The excitation bands of X 2-Y 2SiOs:Tb3+ in VUV regions were assigned to the CTBs of Tb 3+_0 2-, the electronic transitions from 02-:2 p6 to y 3+: 4P6 (4d+5s) and the absorption of Si-O bonds. The CTBs of Gd 3+_0 2- and the f--d transitions of Gd 3+ resulted in the enhanced luminescent intensity when Gd 3+ was introduced into X2-Y 2SiOs:Tb3+ and substituted for y3+. The sample of X2-YI.6Gdo.2Tbo.2SiOs had the strongest emission intensity, which was 196% of that of commercial Zn2Si04:Mn2+, and had a much lower lie decay time (2.9 ms) than that of Zn2Si04:Mn2+ (5.1 ms). REFERENCES 1. Kim C H, Kwon I E, Park C H, Hwang Y J, Bae H S, Yu B Y, Pyun C H, Hong G Y. Phosphors for plasma display panels. J Alloys Comp 2000;311 :33-9. 2. JUstel T, Nikol H. Optimization of luminescent materials for plasma display panels. Adv Mater 2000;12:527-30. 3. Zhang Z H, Wang Y H, Hao Y, Liu W J. Synthesis and VUV photoluminescence of green-emitting X 2-Y 2Si05:Tb3+ phosphor for PDP application. J Alloys Comp 2007;433:Ll2-4. 4. Mishra K C, Johnson K H, DeBoer B G, Berkowitz J K, Olsen J, Dale E A. First principles investigation of electronic structure and associated properties ofzinc orthosilicate phosphors. J Lumin 1991;47:197-206. 5. Resfeld R, Jorgensen C K. eds. Lasers and excite states of rare earth. Berlin: Springer, 1977: 45-8. 6. Yang H C, Li C Y, He H, Tao Y, Xu J H, Su Q. VUV-UV excited luminescent properties of LnC~O(B03)3:RE3+ (Ln=Y, La, Gd; Re=Eu, Tb, Dy, Ce). J Lumin 2006;118:61-9. 7. Wang Y H, Guo X, Endo T, Murakami Y, Ushirozawa M. J Solid State Chern 2004;177:2242-8. 8. Dorenbos P. The 5d level positions of the trivalent lanthanides in inorganic compounds. J Lumin 2000;91: 155-76. 9. Kang Y C, Lim M A, Park H D, Han M. Ba2+ co-doped Zn2Si04:Mn phosphor particles prepared by spray pyrolysis process. J Electrochem Soc 2003; 150:H7 -11.

LUMINESCENT PROPERTIES OF Na2Ca4Mg2Si401s:Tb3+ NANO-SIZED PHOSPHOR LI-YA ZHOU, LING-HONG YI, JUN-LI HUANG,JIAN-SHE WEI, FU-ZHONG GONG

School a/Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, People's Republic a/China; [email protected] INTRODUCTION Plasma display panels (PDPs) are a promising candidate for large size flat-panel information-display devices, and much research has been made in exploring phosphors for application in PDPs. ' -J The most widely used green-emitting phosphor for PDP is Zn2Si04:Mn2+ (ZSM). Although it is a good phosphor widely used in plasma display panels, the decay time is too long for color TV application.' Rare earth (RE) ions have a number of efficient and narrow emission lines in the visible region. s Therefore, Re 3+-doped phosphors play an important role for optical applications of PDPs. Some oxide compounds with silicate, borate and aluminate groups have strong absorption in the VUV region."!> Silicates are taking more and more attention as useful luminescent hosts because of the stable crystal structure, and high physical and chemical stability.? Sol-gel method is an efficient technique for preparation of nano-sized phosphors due to good mixing of starting materials and relatively low reaction temperature: In this article, Na2Ca4Mg2Si40Is:Tb3+ nanoparticles were synthesized for the first time by sol-gel method.

MATERIAL AND METHODS Reagents were of analytical grade. Stoichiometric tetraethoxy- silicane [(CH3CH20)4Si, TEOS] and ethanol were mixed with stirring to obtain a TEOS solution. Magnesium nitrate, calcium nitrate, sodium nitrate aqueous solution and Tb(N0 3)3 solution were then added dropwise to the required amount of TEOS solution with vigorously stirring. Then, appropriate amount of HN0 3 was applied as the catalyst for the hydrolysis of TEOS with stirring continued for 4 h. The resultant gel was dried at 120°C for 3 h. The precursor particles were put into a furnace and pre-calcined at 500°C for 2 h, then calcined at 1000 °c for 5 h to obtain phosphor samples. Powder X-ray diffraction (XRD, 40 kV and 35 rnA, Cu Ka = 1.5406 A RigakulDmax - 2200, Japan) was used for crystal phase identification. Field emission scan electron microscopy (LEO-1530 FE-SEM, Germany) was used to observe the morphology and size of the calcined particles. Near UV excitation and emission spectra were measured on a HITACHI F-4500 fluorescence spectrophotometer (Japan).

RESULTS AND DISCUSSION The powder XRD patterns for the particles prepared by the above processes are shown in Fig. 1, and panels a, b, and c show the patterns for the samples calcined 333

334

Zhou L-Y et al.

for 5 h in air at 900 1000 °c and 1100 DC, respectively. When the precursor was calcined at 900°C, the characteristic peaks of Na2Ca4Mg2Si4015 42appear with the existing peaks of CaO (ICDD 28-0775). At 1000 form without impurity phase. When the is increased to 1100 the intensity of the peaks does not change significantly, and no new are observed. So the optimum firing temperature is about 1000 "C. 400-,-------c----------, 350 300 250

50

10

20

30

40

50

60

70

2tJ(degree)

XRD

FE-SEM

of the Na2C34Mg2Si4015 : 4 mol % Tb 3 + phosphor calcined at 900°C, (b): 1000 °c and (c): 1100 for 5 h

ofNa2C34Mg2Si40J5 : 4 mol % Tb3+ phosphor calcined at 1000 °c for 5 h

shows the FE-SEM images of Na2Ca4Mg2Si401S: 4 mol % showed a narrow size-distribution of about 80 ~ 100 nm with There was only minor aggregation ofthe particles. 3 shows the room temperature near UV excitation and emission of the phosphor. When the detection wavelength is monitored at 545 nm, the excitation spectrum consists of four bands, the 4f8 transition at the shorter wavelength (245 nm and 285 nm) with a higher intensity and transition at the longer wavelength (352 nm and 370 nm) with weaker excitation with 245 nm UV light, the characteristic luminescence is due to 4, 3) and 5 D4 -> 7FJ (J 6, 5, 4, 3) line emissions of the

Luminescent Properties ojNa2 Ca4Mg2Si4015: Tb 3 + Nano-Sized Phosphor

335

ions. The strongest emiSSIOn is located at 545 nm corresponding to S D4 --> 7 Fs transition, while thef- ftransition lines from the higher level sD 3 are not observed due to the increased concentration of Tb 3 +., In order to verify the best Tb 3+ doping ratio to Na2Ca4Mg2Si401s, a series of doping experiments with Tb 3 + doping ratios to CaO from 4 mol % to 10 mol % were carried out. The luminescence intensity is enhanced as the increasing of the Tb 3 + doping ratio and reaches a maximum at 8 mol % of Tb 3 +. When the Tb3+ doping ratio is higher above 8 mol %, the 3 luminescence intensity reduces contrarily. As the Tb + concentration increases, the Tb-Tb distance decreases. Tb ions strongly cross-relaxation interact resulting in a decrease of the lifetime. lo Based on a single exponential method, the decays time 3 of the Na2Ca4Mg2Si40ls : 8 mol %Tb + phosphor monitored at 545 nm and excited at 245 nm is 2.96 ms, which was short and suitable for PDP application.

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-~

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-~

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300

500

400

700

600

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0.7

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Fig. 4. Chromaticity diagram for NazCa4MgzSi40ls : Tb3+ phosphors and NTSC green phosphor (NTSC: National Television Standard Committee)

336

Zhou L-Y et al.

The emission color was analyzed with the help of CIE chromaticity coordinates. The coordinates of Na2C'4Mg2Si401s:Tb3+were found to fall in the yellowish green region of the CIE chromaticity diagram and with an decrease ofTb 3+, the phosphor exhibited deeper green emission, as shown in Fig. 4.

CONCLUSIONS 3 Na2Ca4Mg2Si401S :Tb + phosphors with a spherical shape and a size about 80 100 nm were prepared using sol-gel method. Upon excitation with near UV light excitation, the phosphor showed strong green-emission peaked at around 545 nm, corresponding to the SD4 -> 7Fs transition of Tb3+, and the highest PL intensity at 545 nm was found at a content of about 8 mol % Tb 3+. With a decrease ofTb 3+, the phosphor exhibited deeper green emission. All the characteristics indicated that the 3 Na2Ca4Mg2Si4015 :Tb + would be a promising green phosphor for PDP application. ACKNOWLEDGEMENTS Financially supported by grants from the Science Foundation of Guangxi Province (No. 0731014), the Natural Science Foundation of Guangxi University (X051107), the large-scale instrument of Guangxi cooperates and shares network. REFERENCES 1. Liang HB, Tao Y, Su Q, Wang SB. VUV-UV Photoluminescence spectra of strontium orthophosphate doped with rare earth ions. J Solid State Chern 2002; 167:435-40. 2. Rao RP, Devine DJ. Re-activated lanthanide phosphate phosphors for PDP applications. J Lumin 2000;87-89:1260-263 3. Moine B, Bizarri G. Rare-earth doped phosphors: oldies or goldies? Mater Sci Eng B 2003;105:2-7. 4. Ronda CR. Recent achievements in research on phosphors for lamps and displays, 1. Lumin 1997;72-74:49-4. 5. Itoh K, Kamata N, Shimazu T, et al. An improved emission characteristics of 3 Tb +-doped sol-gel glasses by utilizing high solubility of terbium nitrate. J Lumin 2000;87-89:676-8. 6. Kim CH, Kwon E, Park CH, Hwang YJ, Bae HS, Yu BY, et al. Phosphors for plasma display panels, J. Alloys Comp., 2000;311 :33-39 7. Lin YH, Tang ZL, Zhang ZT, Nan CWo Luminescence of Eu2+ and D/+ activated R3MgSi20s-based (R=Ca, Sr, Ba) phosphors. J Alloys Comp 2003;348:76-9. 8. Patra A, Baker GA, Baker SN. Effects of dopant concentration and annealing temperature on the phosphorescence from Zn2Si04: Mn2+ nanocrystals, J Lumin 2005;111:105-11. 9. Kim GC, Park HL, Kim TW. Emission color tuning from blue to green through cross-relaxation in heavily Tb 3+-doped YAI0 3, Mater Res Bull 2001 ;36: 1603-8. 10. Duhamel-Henry N, Adam JL, Jacquier B, Linare C. Photoluminescence of new fluorophosphate glasses containing a high concentration of terbium (III) ions. Opt Mater 1996;5:197-207.

PART 8

BIOLUMINESCENCE, CHEMILUMINESCENCE AND FLUORESCENCE IMAGING

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THE MEASUREMENT OF CYTOSOLIC ATP DURING APOPTOSIS: BIOLUMINESCENCE IMAGING AT THE SINGLE CELL LEVEL RYUTARO AKIYOSHI, HIROBUMI SUZUKI Research & Development Division, Olympus Corporation.2-3Kuboyama-cho Hachioji-shi, Tokyo, 192-8512, Japan Email: ryutaro _ [email protected]

INTRODUCTION Apoptosis is a process of genetically programmed cell death. This distinct form of cell death plays an essential role in embryonic development and metabolic homeostasis. At present, it is known that apoptosis demands energy supplied by ATP for caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, and apoptotic body formation. 1-4 The total amount of ATP during apoptosis has been measured by firefly luciferin-Iuciferase assay system using a luminometer. 5 Beetle luciferases (including those of the firefly and the click beetle) have been used for the highly sensitive detection of ATP because beetle luciferases catalyze the oxidation of D-Iuciferin in the presence of ATP, Mg2+ and molecular oxygen. 6 However, this assay system detects only a total amount of luminescence of the cell population. In order to analyze cellular activity using bioluminescence at single cell level, we developed a luminescence microscope, 7 and attempted to measure cytosolic ATP during apoptosis at the single cell level. MATERIALS AND METHODS The measurement of cytosolic ATP during apoptosis. He La cells were plated on a 35 mm culture dish with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and were transfected transiently with Emerald Luc control vector (Toyobo), click beetle luciferase under the SV40 promoter. For 24 h after transfection, the medium was replaced with Opti-Mem (Invitrogen), and D-Iuciferin was added into the medium at the final concentration of 500 11M. After the 30 min incubation, apoptosis is induced by the addition of the following reagents to the medium at the final concentration; 4 11M staurosporine (STS), 30 11M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 10 Ilg/mL cycloheximide (CHX). The luminescence was continuously monitored using a luminometer (Kronos, ATTO) after addition of apototic inducers by lOs integration of photons with 1 min interval at 37°C. Bioluminescence imaging. Luminescence images were acquired using the Luminoview (LV200, Olympus) luminescence imaging system attached to an electron multiplier charge-coupled device camera (ImagEM, Hamamatsu Photonics). The dish was kept at 37°C in the humidified chamber during observation. Each image was taken by 40 x objective lens [numerical aperture (NA) 1.30] at 10 s exposure, 15 s interval, binning 1 x 1 and EM-gain 255, or 60 x phase contrast objective lens (NA 1.25) at 60 s exposure, 90 s interval, binning lxl and EM-gain 255. Duration time of observation 339

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was 10 h. Luminescence intensity from single cells was measured as an average value in a region of interest (ROI) enclosed for each cell by MetaMorph software (Universal Imaging). RESULTS AND DISCUSSION Cytosolic ATP dynamics of HeLa cell during apoptosis was measured using a luminometer (Fig.I). Total amount of ATP increased within 20 to 120 min and decreased gradually within 10 h via three types of apoptotic induction (STS, FCCP and CHX). As CHX is an inhibitor of protein synthesis, ATP elevation after apoptotic induction is not due to elevation of luciferase synthesis. On the other hand, ATP elevation is suppressed by glucose-free medium and 2-deoxy-D-glucose. It leads to a hypothesis that glycolysis pathway contributes to an elevation of cytosolic ATP. 5 This hypothesis is supported by the result using FCCP, an inhibitor of mitochondrial A TP production.

3.E+06 2.E+06 rJl 4-'

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--+- Staurosporine .... FCCP ....... CHX

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2

3

4

5

6

7

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Time (hour) Cytosolic ATP measurement of HeLa cells during apoptosis by luminometer (Kronos, ATTO).

Fig. 2(a) shows the luminescence image of HeLa cells before apoptotic induction. Cytosolic ATP dynamics during apoptosis induced by STS is analyzed at single cell level [Fig. 2(b)]. ATP elevation within 30 to 45 min was also observed and it paralleled the result obtained using a luminometer (Fig. 1). FCCP and CHX yield similar results (data not shown). Moreover, we observed a phenomenon whereby some of cells emit a flash of light after 4 to 8 h. Fig. 3 shows detailed morphology of the cells emitting flashes of light in bright field and luminescence images. The flash of light was observed just before the cell shrinks [Fig. 3(b) Arrow]. As shown in Fig. 3, most of cells shrink before cell death, but the flash of light occurs at different times in

Measurement ofCytosolic ATP During Apoptosis

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each cell. This stereotypical morphological change occurs with formation of membrane-bound apoptotic bodies that contain cytosolic and nuclear fragments. The apoptotic body is known not to release its contents, but it is considered that intracellular conditions, such as pH, D-luciferin concentration and ATP level are We that the flash of light is caused by some of these But it is uncertain whether the flash of light is due to increased cytosolic ATP or not at the present. Such result cannot be obtained by conventional luminometric assay The bioluminescence imaging assay can detect not only cytosolic ATP level but also of an individual cell. This assay system will make a new window in cell biology.

3.E+07 2.E+07 2.E+07 1.E+07 5.E+06 O.E+OO

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2

3

4

5

6

7

8

9

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Time (hour)

2. Bioluminescence image captured by Luminoview and cytosolic ATP measurement at the single cell level. (a)Bioluminescence image of He La cells Emerald Luc obtained by 40 x objective lense. (b) Time course of cytosolic ATP after addition of STS (4 J.1M) for 7 cells selected in 2(a).

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3. Flash luminescence observed before cell Luminoview. Phase contrast image; (b) Bioluminescence image of HeLa Cell lens. were obtained by 60 x phase contrast apoptosis and emits flash luminescence. but its luminescence intensity does not increase. REFERENCES 1. Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of cellular ATP level. FEBS Lett 1996;378:742-52. a switch in Nicotera P, Leist M, Ferrando-May E. Intracellular between apoptosis and necrosis. Toxicol Lett 1998; I 02-103: 139-42. 3. Hu Benedict MA, Ding L, Nunez G. Role of cytochrome c and in Apaf-l-mediated caspase-9 activation and 8:3586-95. Eriksson JE, Weis M, Orrenuis S, Chow condensation during apoptosis requires ATP. Biochem J 1 Sabirov Maeno E, Ando-Akatsuka Zamaraeva Okada. Y. Cells die with increased cytosolic ATP bioluminescence study with intracellular luciferase. Cell Death Differen 390-7. 6. Lam McElroy WD. Introduction to beetle luciferases J Biolumin Chemilumin 1989;4:289-301. 7. Suzuki H, Dosaka S, Ohashi-Hatta Y, Sugiyama T. Luminescence Hill PJ, Kricka for assay of single live cells. In: Szalay PE. eds. Bioluminescence and Chemiluminescence. 2006:53-6.

BIOLUMINESCENCE IMAGING OF BACTERIA-HOST INTERPLAY: INTERACTION OF E. COLI WITH EPITHELIAL CELLS LY BROYKO,I H WANG,I J ELLIOT,I R DADARWAL,I 0 MINIKH I,2 MW GRIFFITHS I JCanadian Research Institute for Food Safety, University of Guelph Guelph ON N1G 2W1, Canada 2Department of Chemical Enzymology, Lomonosov Moscow State University Moscow 117899, Russia Email: lbrovko@uoguelphca

INTRODUCTION According to the CDC, foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. I The estimated cost for five major food-borne bacterial pathogens (Campylobacter (all serotypes), Salmonella (nontyphoidal), Escherichia coli 0157, E. coli non-0157 STEC, and Listeria monocytogenes) in year 2000 was 6.9 billion dollars. 2,3 The best defense against food-borne illnesses is the early detection and/or identification of contamination events. Currently, the most sensitive and reliable assays for bacterial pathogens are based on specific recognition by antibodies (ELISA) and nucleic acids (PCR, in situ hybridization). However, these methods are relatively time-consuming, may require costly equipment and often involve pre-treatment of samples prior to performing these assays. The time needed to perform the assay and its cost are almost prohibitive for routine, everyday screening of food and environmental samples. Both bacterial and eukaryotic cells are active participants in the infection process. Recently molecular and cellular mechanisms of bacterial pathogenesis were defined for some members of the Enterobacteriaceae family that showed similarity, namely Salmonella, Shigella, enteropathogenic and Escherichia co!i.4-9 The host cell plays an active role in bacterial adhesion, following which bacterial pathogens activate host cell signal transduction pathways. Many pathogens use the same signal molecules (namely Ca2 +, protein kinases, inositol-3-phosphate, etc.) that participate in signal transduction 2 in mammalian cells after the action of different stimuli. Influx of Ca + and/or a subsequent increase in protein phosphorylation were detected in mammalian cells after adhesion of several enteric pathogens. These changes occur within minutes or even seconds after cell-cell interaction. Bioluminescent methods provide researchers with unique opportunities to track growth and movement of cells in real-time and to non-destructively monitor fast metabolic changes with high sensitivity and specificity directly in a live animal or ceIJ. IO- 13 Despite these evident advantages, few results have been reported on the application of bioluminescent methods for monitoring the processes of bacterial infection. I I The goal of our investigation was to apply available bioluminescent techniques for investigation of the early stages of interaction of bacterial pathogens with the host cell 343

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to gain knowledge that could be applied to the detection of infective organisms. As a model system, interaction of Escherichia coli with epithelial HeLa cells was studied. E. coli represents a wide group of related bacteria including harmless, or even beneficial species that produce vitamin K, as well as highly pathogenic strains that cause serious disease. They commonly reside in the gut of warm-blooded animals, including humans, where they colonize intestinal mucosa. Thus, interaction with HeLa cells may provide a good insight into events that occur within the gastrointestinal environment. MATERIAL AND METHODS Cell cultures and their cultivation. All strains of E. coli were obtained from the CRIFS culture collection, University of Guelph. Bacteria were grown at 3rC in LB broth or agar supplemented with 50 Ilg/mL of ampicillin where indicated. Enumeration of bacteria was performed by plating counting and presented in colony forming units (CFU). Construction of the bioluminescent bacterial strains was performed by electroporation of parent strains with plasmid pT7 carrying the full lux operon of Photorhabdus luminesens as described previously.14 CCL-2TM HeLa cells were obtained from ATCC and grown in Eagle's minimal essential media (EMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 • Assessment of the attachment of E. coli to HeLa cells by bioluminescent method. HeLa cells were grown in 24-well microtiter plates until 80-90% con fluency was reached. The monolayer of HeLa cells was then inoculated with the bioluminescent bacteria, incubated for 1 h (37°C 5% CO 2), washed twice and fresh medium was added. The plate was later incubated under the same conditions and bioluminescence was monitored using a Multilabel Plate Reader Victor™ (Perkin Elmer) and/or Night Owl (Berthold EG&G) imaging device. From the time-course of the bioluminescence, lag periods for growth of bacteria attached to epithelial cells were estimated. These were compared with the lag periods of the growth curves obtained under similar conditions for bacterial suspensions in tissue culture media containing a known initial number of cells (range 1 - 106 CFUlmL). On the basis of this comparison, the level of attached bacterial cells was estimated. RESULTS Bioluminescent E. coli. Fifteen strains of E. coli were transformed to give a bioluminescent phenotype and these included 5 non-pathogenic E. coli, 5 enterohemorrhagic E. coli 0157:H7 and 5 enteropathogenic E. coli (EPEC) of different O:H serotypes. Bioluminescence in Relative Light Units (RLU) of bacterial cell dilutions was measured and standard curves (RLU vs CFUlml) were obtained for each strain. Surprisingly, there were significant differences observed between investigated strains. There was no correlation between pathogenicity of the strain and its bioluminescence. The observed difference between the brightest and dimmest was more then one order of magnitude. In further experiments each individual standard curve was used to assess numbers of that particular strain.

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Attachment of E. coli to HeLa cells. Attachment at different levels of inoculation 3 6 (10 _10 CFU/mL) and physiological state of bacteria was investigated as described above. Typical results are presented in Table I. There was no significant difference in attachment of actively growing and starved cells to the monolayer of epithelial cells. The difference in attachment between strains did not correlate with pathogenicity and was not statistically different for all investigated strains. Table 1. Attachment of E. coli bacteria to HeLa cells as assessed by bioluminescence. Attachment, cfu per well Inoculation level 105 cell per Inoculation level 10 6 cell per well well

Strain

IE. coli O157:H7 92-192, 94-37, 666H8, 92-355,

PT67 PT34 PT23 PT23

IEHEC Ol32NM 0103:H2 O153:H25 1N0n-pathogenic E. 'coli ATCC 10798, KI2 ATCC 15597, EC 3000

Growing cells Starved cells Growing cells <10 3 <5x10 3 5xl0 3 (1-5)104 (1-5)10

4

<10 3 10 3 <5x I 0 4 >10 4

10 4 S 5xlO

4 10 4 >5x I 0 (1-5)10 3

5xl0 5 (1-5)10 5 >5x I 0

5xl0 10

5

<10

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>5x104

Starved cells <5x 103 >10 3 5xl0 4 > 10 5 5

10 >5x10 4 >5x104

5xl0 >10 5

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>5x10 4

ACKNOWLEDGEMENTS This work was partially supported by SENTINEL Bioactive paper network, NSERC, Canada; and NCFPD, USA. REFERENCES 1. Mead PS, Shutsker L, Dietz Y, et al. Food-related illness and death in the United States, 1999. Emerging Infect Dis 1999;5 :607-25. 2. Anon. Economics of food-borne diseases: Overview. http://www.ers.usda.gov/Briefing/FoodborneDisease/overview.htm 3. Stinson T. The national economic impacts of a food terrorism event: Preliminary estimates, Proceedings of The Institute of Food Technologists' I sl Annual food protection & defense research conference, November 2005, Atlanta, Georgia, http://www.ift.org/fooddefense/3-Stinson.pdf

346 Brovko LY et al. 4. 5. 6.

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13. 14.

Finlay BB, Cossart P. Exploitation of mammalian host cell functions by bacterial pathogens. Science 1997;276:718-25. Pace J, Hayman MJ, Galan JE. Signal transduction and invasion of epithelial cells by S.typhimurium. Cell, 1993;72:505-14. Oehio C, Prevost MC, Sansonetti PJ. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of contract in by a pp60 c-src -mediated signalling pathway. EMBO J 1995;14:2471-82. Nisan I, Wolff C, Hanski E, Rosenshine I. Interaction of enteropathogenic Escherichia coli with host epithelial cells. Folia Microbiol 1998;43:247-52. Wadsworth, S.1., Goldfine, H. Listeria monocytogenes Phospholipase C dependent Calcium signaling modulates bacterial entry into J774 macrophage-like cells. Infect Immun 1999;67:1770-8 Shin S, Kur GH, Kim YB, Park KJ, Park YM, Lee WS. Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxininduced cytotoxicity. Cell Bioi ToxicoI2000;16:137-44. Rider TH, Petrovick MS, Nargi FE, Harper JO, Schoebel EO, Mathews RH, et al. A B Cell-Based sensor for rapid identification of pathogens. Science 2003 ;30 1:213-5. Hammermueller J, Gyles CL. The development of a rapid bioluminescent Vero cell assay. In: MA Karmali MA and AG Goglio AG, eds. Recent advances in Verotoxin producing Escherichia coli infections. Amsterdam:Elsevier 1994:11316. Allue I, Gandelman 0, Oementieva E, Ugarova N, Cobbold P. Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigorcontracture of metabolically compromised single cardiomyocytes. Biochem J 1996;319:463-9. Brovko L, Young 0, Griffiths MW. Method for assessment of functional activity of antibodies for live bacteria. J Microbiol Methods 2004;58:49-57. Meighen EA, Szittner RB. Multiple Repetitive elements and organization of the lux operon of luminescent terrestrial bacteria. J Bacteriol 1992; 114: 5371-8l.

UL TRASENSITIVE CHEMILUMINESCENT IMMUNOCHEMICAL LOCALISATION OF PROTEIN COMPONENTS IN PAINTING CROSS-SECTIONS LS DOLCI,I G SCIUTTO/ M RIZZOLI/ M GUARDIGLI,I R MAZZEO,2 S PRATI,2 A RODA 1 1Dept of Pharmaceutical Science. University of Bologna. Bologna 40126, Italy 2Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna. Ravenna 48100, Italy Email: [email protected]

INTRODUCTION Identification and localization of binding media and different organic components in multilayer paint samples is crucial in the study of manufactured techniques and for authentication and conservation purposes. To this end, use of immunological techniques has been proposed many years ago based on the unambiguous reaction between antibody and protein target (antigen). These techniques would allow to distinguish between different proteins and also to determine the biological source of a protein (e.g., bovine vs. rabbit collagen). Such techniques are extensively employed in bioanalytical and clinical chemistry,1.2 but only few results are reported in literature concerning the characterization of organic materials in paint crosssections using antibodies labeled with fluorescent markers.'·4 These first applications are characterized by strong interferences due to the presence of painting materials, which can show an intense autofluorescence. We have developed a new method for the localization of ovalbumin (chicken eggwhite albumin, a protein often present in binding media and varnishes) in paint cross-sections based on chemiluminescence (CL) imaging detection. Thanks to the absence of an excitation source, no interference was observed from painting materials. The CL immunochemical method for the localization of ovalbumin relied on the binding to the target protein of a specific primary antibody, which was then detected by a horseradish peroxidase (HRP)-Iabeled secondary antibody and a CL enzyme substrate. The imaging of the CL signal produced by the enzyme-catalyzed reaction allowed the detection and the stratigraphic localization of the target protein. After evaluation of the performance of the method on standard samples, several real samples collected from old paintings were analyzed. MATERIALS AND METHODS Reagents. Anti-chicken egg albumin antibody (whole antiserum, produced in rabbit), HRP-conjugate polyclonal anti-rabbit IgG antibody (produced in goat), albumin from chicken egg white (ovalbumin), and bovine nonfat dried milk were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Primary and secondary antibodies were diluted 1:2000 and 1:4000 (v/v), respectively, in PBS/milk 347

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(phosphate buffer saline, pH 7.4, containing 1.25% dried milk). The luminol-based HRP CL detection reagent Westar Supernova was from Cyanagen (Bologna, Italy). The smalt (ground glass colored with cobalt(II) salts), azurite and malachite (CU3(C03h(OHh), hematite (FeZ03), cinnabar (HgS), and minium (Pb 30 4 ) pigments were obtained from Zecchi (Florence, Italy). Polyester resin for sample embedding (SeriFix Resin and SeriFix Hardener) was purchased from Struers A/S (Ballerup, Denmark). Instruments. Chemiluminescence imaging microscopy experiments were performed using a BX60 epifluorescence microscope (Olympus Optical, Tokyo, Japan) connected to an ultrasensitive CCD camera (LN/CCD Princeton Instruments, Roper Scientific, Trenton, NJ). The microscope was enclosed in a dark box to avoid interference from ambient light. Live color images of the samples were obtained by acquiring separate grayscale images corresponding to the RGB colors by means of a RGB filter (CRI Inc., Woburn, MA) and a white LED light source. Image processing and quantitative analysis were performed using the image analysis software Metamorph v. 4.5 (Universal Imaging Corporation, Downington, PA, USA). Sample preparation. Standard samples were obtained by the application of a thin layer of whole-egg tempera (a mixture of egg white, yolk and water in 1: 1: 1 (v/v) ratio, mixed or not with pigments) on a ground layer of gypsum (purchased from Zecchi) and rabbit glue (purchased from Phase, Bologna, Italy). The following pigment/egg ratios were adopted: 4: 1 for azurite, malachite, smalt and hematite; 3: 1 for cinnabar and minium. Small samples were collected and embedded in polyester resin, following the conventional procedure. The transverse section were obtained by abrasion of the resin and polishing the surface using fine silica abrasive papers (4000- to l2000-grade, purchased from Micro-Surface, Wilton, lA, USA). Experimental procedure. Sample cross-sections were treated for 1 h at room temperature with the blocking solution (5% dried milk in water) then, after washing (3 x) with PBS/milk, incubated overnight at 4°C with the anti-ovalbumin antibody (primary antibody). Afterwards, the samples were washed (5 x) with PBS/milk, incubated for 4 h at 4°C with the HRP-Iabeled anti-rabbit IgG antibody (secondary antibody), and washed again (5 x) with PBS/milk. Then the HRP CL detection reagent was added to cover the cross-section and the CL images were acquired using an integration time of 120 s. RESULTS AND DISCUSSION Due to the porosity of the cross-sections, optimization of blocking and incubation steps was critical to avoid non-specific adsorption of the immunoreagents, which would cause high background signals and decrease the detectability of the target protein. The non-specific binding of the immunoreagents could be reduced with a dry polishing method, thereby decreasing the heterogeneity of the surface, and by lowering the incubation temperature. The specificity of the assay was assessed by performing the immunolocalization of ovalbumin in standard samples with or without the primary antibody. As expected, in the absence of the primary antibody

Ultrasensitive Chemiluminescent lmmunochemical Localisation of Protein Components

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no CL was detected. In addition, no cross reactivity of the primary antibody was observed in standard samples containing other organic binding media (fish intelferences due to metal ions contained in pigments we oil). To standard of whole-egg tempera with different common inorganic pigments malachite, hematite, cinnabar, and minium). The experiments in the absence of the immunoreagents did not show any catalysis of the and for all the pigments we were able to observe CL emission from the I). We plan to include in further experiments other inorganic and of historical interest, also performing investigations on "~''''-'''''' to assess the effect of degradation processes .

. Chemiluminescent immunolocalization of ovalbumin in a standard with smalt and tempera. Left: live image; right: CL image. Bar: 200 Jlm. The CL immunochemical techniques was applied on old in order to evaluate its performance in samples with a natural and riPfrn";
Cross-section of the sample collected from the painting by Rondinelli. Left: live right: CL image showing the signal in the upper-most layer. Bar: 200 !-tm.

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A from a polychrome terracotta altar-piece painted by Mattia della Robbia 1527-1530 (Fig. 3a) was also analyzed. This sample showed a and ovalbumin was detected as organic binding media in different

a 3. (a) Polychrome terracotta altar-piece painted by Mattia della Robbia. Cross-section of the sample collected. Up: live image; down: CL in n. 0 (preparation layer), 2 and 4 (paint layers). Bar: 200 CONCLUSION The CL immunolocalization method allow to detect ovalbumin in sections with a spatial resolution of the order of micrometers. >lfll1f('>I"h could be extended to the detection of other f\rr,tp,n>lc'pm collagen, casein, etc). Different proteins could be also detected in by employing a mixture of analyte-specific nrl1m>llCV one by a secondary antibody labeled with a d iffurent enzyme HRP and alkaline phosphatase) detectable with a specific CL substrate. REFERENCES Roda Pasini P, Musiani M. Girotti S, Baraldini Carrea Suozzi Chemiluminescent low-light imaging of biospecific reactions on macro- and a videocamera-based luminograph. Anal Chem 2.

3.

4.

Marangi M, Casanova S, Grigioni Roda E, Roda A. Chemiluminescence quantitative immunohistochemical determination of MRP2 in liver biopsies. J Histochem Cytochem 2005;53:1451-7. Ramirez Barat de la Vina S. Characterization of proteins in media immunofluorescence. A note on methodological Stud Conserv 2001 A, Millay V, Quick M. The use of immunofluorescence and enzyme-linked immunosorbent assay as for proteins identification in artists' materials. J Am Inst Conserv 05.

DEVELOPMENT OF A NEW DEVICE FOR ULTRASENSITIVE ELECTROCHEMILUMINESCENCE MICROSCOPE IMAGING LS DOLCI,I M RIZZOU,I E MARZOCCHI,2 S ZANARINI,3 LOELLA CIANA,2 A RODA I I Dept of Pharmaceutical Sciences, University of Bologna, Bologna 40126, Italy 2Cyanagen s. r.l. , via Stradelli Guelfi 401c, Bologna 40138, Italy 3 Dept of Chemistry G. Ciamician, University of Bologna, Bologna 40126, Italy Email: [email protected]

INTRODUCTION Electrogenerated chemiluminescence (ECL) is a highly sensItIve analytical technique '" widely used in biosensors',3 and clinical chemistry.IS." Until now no applications have been reported on the use of ECL for ultrasensitive low-light imaging microscopy of immunohistochemical (IHC) and hybridization (ISH) methods on cells or biological tissues. The topographic 20 localization of an analyte down to a few molecules is extremely important in bioanalysis since the evaluation of its distribution in cells is a key factor in physiology, physiopathology and therapeutics. To perform the ECL detection of a biospecific reagent labeled with an ECL-active marker we designed and developed a transparent electrochemical cell using FTOcoated glass compatible with optical microscopy. The cell allows the generation of a luminescence signal which can be activated and switched off during the measurement process. As a model system we used heavy micron-sized beads to simulate biological cells. MATERIALS AND METHODS [Ru ( 4 (4'-methyl-2,2'-bipyridin-4-yl) butan-l-aminium (2,2'-bipyridine)2] (CI0 4 )3 (Ru(bpyh 2+ -NH 2), bis(2,2' -bipyridine)-[ 4-[ 4' -methyl-2,2' -bipyridin-4-yl)butanoic acid] ruthenium bis(hexatluorophosphate) (Ru(bpy)/+ -COOH), 3-sulfo-Nhydroxysuccinimide (s-NHS), and biotin-cadaverine-TFAc were purchased from Cyanagen (Bologna, Italy). Tris(2,2' -bipyridine)dichlororuthenium(lI) (Ru(bpyh 2+), streptavidin from Streptomyces avidinii, MES, tripropylamine, N-(3dimethylaminopropyl)-N' -ethyl-carbodiimide hydrochloride (EDC), and N,N'dicyclohexylcarbodiimid (DCC) were purchased from Sigma-Aldrich Co. (St. Louis, MO). The heavy-core carboxylated beads (diameter 8 flm) were purchased from Spherotech (Libertyville, IL, USA). Fluorine-doped tin oxide-coated glasses (20 Q) were purchased from Flexitec Electronica Organica (Curitiba Parana, Brasil). For microscopy imaging a BX60 epitluorescence microscope (Olympus Optical, Tokyo, Japan) connected to a liquid nitrogen-cooled ultrasensitive CCD camera (LN/CCD Princeton Instruments, Roper Scientific, Trenton, NJ) was used. The microscope was enclosed in a dark box to avoid interference from ambient light and equipped with an OptiScan ES 103 motorized microscope stage (Prior Scientific Instruments Ltd., Fulbourn, England) for sample positioning. 351

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Glass electrochemical cell. A microscope FTO-glass was chemically etched 7 to produce a standard three-electrodes cell. Quasi-reference electrode was prepared flash galvanic deposition directly on the FTO electrode. Electrochemical activity was monitored by cyclic voltammetry using a Fc-MeOH 10-4 mollL solution in PBS 0.1 mollL Electrodes shapes and distances have been optimized to obtain the shown in Fig. t, which produced experimental curves compatible with ext)ected for first oxidation (+0.2-0.3 V vs. Ag).

GALVANIC SlLVER COATING

1. ECL optical imaging system. A: side view; B: top view (R: quasi-reference electrode; W: working electrode; C: counter electrode) profile for a Ru(bpy)/+rrPA solution. Test solution was 10.4 mol/L Ru(bpy)2+3 and 3x1O·2 mol/L TPA in 0.1 M PBS (pH 7.6). voltammetry and ECL measurements were carried out with an AUTOLAB electrochemical station (Ecochemie, Holland) at 0.2 Vis scan speed. The ECL signal a was collected by a photomultiplier tube (Hamamatsu R255, biased at 750 V) few millimeters in front of the working electrode. To register ECL signal, the photomultiplier output signal was sent to an ultralow noise current preamplifier (Acton Research model 181, 10.5 AN). Method I: Direct of Ru(bpy)/+-NH 2 to carboxylate beads. 200 IlL of 8/lm beads suspension were washed three times in 0.1 mollL borate buffer (pH 9.6) and two times in 0.1 mol/L MES (pH 5.5) buffer. The beads were finally resuspended in 250 ofMES buffer. Carboxylic groups were activated adding EDC and s-NHS to a final concentration of 50 mmollL and 2 mmollL, respectively. The reaction mixture was gently mixed for 1 h at RT. After a washing cycle (see above) 500 IlL of a 0.75 mmollL Ru(bPY)32+-NH2 solution in 0.1 mol/L borate buffer (pH 8.6) were added and the suspension incubated overnight at 4°C. The beads were finally washed three times in PBS. Method II: Indirect binding using biotin-streptavidin interaction. of -COOH to streptavidin. Ru(bpy)/"-COOH was dissolved in DMF to obtain 70 IlL of a solution with a concentration of 7.1 xl 0.3 mollL. 1.5 equivalents of DCC and NHS were added and the reaction solution was gently mixed for 4 h at RT. 630 ~L of a 2.0x1O· 5 mollL streptavidin solution in 0.1 mollL borate buffer (pH 9.4) were then added (activated Ru(bpy)/+-COOH:streptavidin 40:1 molar ratio). The solution was incubated overnight and the labeled protein was subsequently purified with dialysis against 5 L of PBS.

New Device for Ultrasensitive Electrochemiluminescence Microscope Imaging

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of biotin to carboxylate beads. Beads were washed and activated using the described procedure. After 1 h of RT incubation 500 J-lL of 9 mmollL biotin-cadaverine in 0.1 mol/L borate buffer (pH 8.6) were added. The mixture was incubated overnight at 4°C and the beads were finally washed three times in PBS. Labeled beads complex. 50 J-lL of biotinylated beads were centrifuged and after buffer removal 50 J-lL of labeled streptavidin were added. The mixture was gently mixed for 2 h at RT then three washing steps "Pl·~"'·nH'rI centrifuging and resuspending with PBS. The characterization of the was carried out using a fluorescence imaging of non-treated and treated beads.

of the new glass cell was evaluated by electrochemical measurements of a Ru(bpy)/+/TPA solution in PBS. A typical light/current/voltage in 2. The cyclic voltammetry shows the of vV''' .... 'VA oxidation potential at about +l.l V vs Ag. Along with the redox reaction concurrent strong light emission was detected confirming the cell was then to the ECL process. The reproducibility of the measurements on the same cell, collecting the ECL solution.

Left: light/current/voltage profile (recorded at 0.2 Vis) for a live image and (B) ECL image of Ru(bpy)/+-conjugated core micron-sized beads was obtained from the second to the tenth cycle of measurement a cathodic cell-cleaning step before each measurement a constant was imposed for 30 s). The satisfactory results obtained by the FTOcell allowed the development of ECL low-light microscope with a highly sensitive nitrogen-cooled CCD. The glass cell was thus to investigate the heavy core micron-sized beads as a model to simulate cells. The Ru-coupled beads (see Method I) were poured onto the cell and a first fluorescence microscope image was acquired exciting the beads with a UV source to cont1rm the Ru complex-beads reaction. Then both the ECL image and the transmitted light (live) image were acquired (Fig. 2). Tn these images each bright

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spot represents a single bead carrying several Ru(bpy)/+ mOIetIes. This result confirms that this technique allows the spatial localization of the ECL signal. The subsequent step focused on a biological assay model in which biotinylated beads were recognized by ruthenium labeled streptavidin (see Method II). Images were acquired from sample solutions containing the purified adduct. Although the observed signal intensity was lower with respect to the previous experiment, the collected images showed a very similar trend. The glass cell described in this work proved to be a successful tool for ECL-based imaging. Once coupled with low-light microscope it was possible to acquire both the live image (thanks to the cell transparency) and the ECL-generated image. These features make the whole system very promlsmg for ultrasensitive immunohistochemical and in situ hybridization techniques in which the comparison of the two images is a critical factor to localize the analyte. As for the near future perspectives, the technique presented has to be further improved and fully investigated using real biological samples. The final goal is to use the glass cell system to detect and precisely localize the binding of labeled antibodies or DNA on a biological sample. Moreover the method can be used in multiplex analysis employing different ECL labels and/or different luminescent principles (BL, CL) to simultaneously localize different targets in the same sample.

REFERENCES 1. Bard AJ. Electrogenerated chemiluminescence. New York: Marcel Dekker, 2004. 2. Knight A. A review of recent trends in analytical applications of electrogenerated chemiluminescence. Trends Anal Chern 1999;18:47-62. 3. Gerardi RD, Barnett NW, Lewis SW. Analytical applications of tris(2,2'bipyridyl)ruthenium(III) as a chemiluminescent reagent. Anal Chim Acta 1999;378: 1-41. 4. Gorman BA, Francis PS, Barnett NW. Tris(2,2-bipyridyl)ruthenium(II) chemiluminescence. Analyst 2006;131 :616-39. 5. Corgier BP, Marquette CA, Blum LJ. Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray. Biosens Bioelectron 2007;22: 1522-6. 6. Marquette CA, Blum LJ. Electrochemiluminescent biosensing. Anal Bioanal Chern 2008;390: 155-68. 7. www.tlexitec.com.br. 8. Zanarini S, Rampazzo E, Bich D, et al. Synthesis and electrochemiluminescence of a Ru(bpY)3-labeled coupling adduct produced on a self-assembled monolayer. J Phys Chern 2008;112:2949-57.

VISUALIZATION OF SEQUENTIAL RESPONSE IN INTRA CELLULAR SIGNAL TRANSDUCTION CASCADE BY FLUORESCENCE AND LUMINESCENCE IMAGING IN THE SAME LIVING CELL

Y. HATTA-OHASHI, T. TAKAHASHI, H. SUZUKI Research & Development Division. Olympus Corporation. Hachioji. Tokyo 192-8512. Japan, Email: [email protected]

INTRODUCTION Cells recognize changes in their environment through the cell surface receptors, resulting in initiation of the intra-cellular signal transduction followed by gene expression of the downstream transcription factor. We have observed protein-protein interaction in cell signaling by fluorescence imaging, and have detected gene expression by reporter assay with luminescence detection. Until now, it had been impossible to observe both of the processes in the same living cell. To observe the two processes sequentially, we developed a luminescence imaging system that is also applicable to fluorescence imaging and we applied it to (I) observation of translocation ofPKC (Protein Kinase C) e from cytoplasm to the cell membrane and following gene expression of the downstream transcription factor, NF-KB, in HeLa cells and (2) Raf-l activation and following gene expression of API in PCI2 cells. MATERIALS AND METHODS Visualization of PKC activation and monitoring NF-K BI gene expression. The pPKCe-EGFP vector contains a fusion of PKCe and EGFP (enhanced Green Fluorescence Protein) under the control of the SV40 promoter. The pGL4-NF-KBI contains the GL4 luciferase under the control of the NF-KB I promoter. HeLa cells were plated on a 35 mm culture dish with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and were co-transfected transiently with the pPKC6-EGFP and the pGL4-NF-KBI vectors. At 48 h incubation after transfection, the medium was replaced with DMEM containing 1% FCS and 10 mM HEPES (pH 7.2). Cells were treated with PMA (phorbol myristate acetate 5ng/mL), and then 500 !!M O-Iuciferin (Promega) was added before the imaging experiment. Visualization of Ras activation and monitoring API gene expression. The pCI-EGFP-RBO+RSV-HA-Ras vector contains the human RBO (Ras-binding domain: 1-149 amino acid) of the Raf-I controlling expression of EGFP and Ras coding sequence. I The pGL4-APl contains the GL4 luciferase under the control of the API promoter. PCI2 cells were plated on a 35 mm culture dish with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FCS and were cotransfected transiently with the pCI-EGFP-RBO+RSV-HA-Ras and the pGL4-API vectors. At 48 h incubation after transfection, the medium was replaced with RPMIl640 containing 1% FCS and 10mM HEPES (pH 7.2). Cells were

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treated with EGF (epidermal growth factor 50 ng/mL), and then 500 f-tM D-luciferin was added before the imaging experiment. ' . .Hit"."" _.. ___ ......... '" of fluorescence and luminescence. Images were acquired the luminescence imaging system Luminoview (LV200, Olympus) attached with cooled charge-coupled device (CCD) camera, ImagEM (Hamamatsu Photonics) under dark conditions. The operating temperature of the CCO camera was set to -65°C. In this system, the optical parameters such as numerical aperture (NA) of and tube lens, total magnification were optimized for luminescence of a single cell. 2 The dish was kept at 37°C in the humidified chamber of f'rr,c("·r>np> during observation. For fluorescence imaging, the excitation and emission filters were used (conditions of observation described in figure legends).

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358

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Luminescence intensity from single cells was measured as an average value in a region of interest (ROI) encircled for each cell by using MetaMorph software (Universal imaging). RESULTS AND DISCUSSION We successfully observed the translocation of PKCe by fluorescence imaging from the cytoplasm to the cell membrane after PMA stimulation, and following gene expression of the downstream transcription factor, NF-KB, by luminescence imaging in the same HeLa cells (Figs. I A and I B). We monitored the chronological response of the NF-KBI promoter activity in each individual cell, and found that the response was different among cells (Fig. IC). Accordingly, the two processes of protein localization and transcription in signal transduction could be sequentially measured in the same cell. We next examined the Ras activation after EGF stimulation in PCI2 cells. As shown in Fig. 2A, the translocation of Raf-l could be visualized by fluorescence imaging. In Fig. 2B, the line analysis clearly indicated that Raf-I was translocated from the cytoplasm to the plasma membrane with EGF stimulation. The luminescence images of the API gene expression were shown in Fig. 3A. In the time course analysis, the API promoter activity revealed the heterogenetic response in each cell (Fig. 3B). Although the protein-protein interaction in cell signaling and the following gene expression have been observed separately so far, the present method enables us to measure the different stages of the signaling pathway in real-time in living cells. Recently, the single-cell analysis of the gene expression with bioluminescence imaging was reported in several studies 3,4 and they provide insight into mechanisms of gene regulation that could not be obtained by averaging effects in cell population. In addition, with the visualization combined with the fluorescence detection, bioluminescence imaging can be applied to a wide range of kinetic imaging applications. Thus, our imaging system will be helpful in understanding gene regulation in signal transduction,

REFERENCES I.

2.

3. 4.

Kao S, laiswal RK, Kolch W, Landreth GE. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PCI2 cells. 1 BioI Chem 2001;276:18169-77. Suzuki H et al. Luminescence microscope for reporter assay of single living cells, Proceedings of the 14th International Symposium on Bioluminescence and Chemiluminescence. 2006;53-56. Welsh DK et al. Bioluminescence imaging in living organisms. Curr Opin Biotechnol. 2005;16:73-8. Ukai H Kobayashi TJ, Nagano M, et al. Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell BioI. 2007;9:1327-34.

BIOLUMINESCENCE IMAGING OF INTRACELLULAR CALCIUM DYNAMICS BY THE PHOTO PROTEIN OBELIN MA Y MAW THET, T SUGIYAMA, H SUZUKI Research & Development Division, Olympus Corporation, Hachioji, Tokyo 192-8512, Japan Email: [email protected]

INTRODUCTION The bioluminescent system (luciferase reporter assay system) is widely used for the study of gene expression, signal transduction and other cellu lar activities. The luciferase assay is conventionally performed by the photon-counting luminometer method. In this system, light emitting from cells is measured as integrated value through all cells. Recently, we developed a luminescence microscope to monitor expression activity of genes of interest in each cell spatially and temporally as images, 1.2 and demonstrated heterogeneous response of c-fos gene promoter activity in each cell by ATP stimulation. ATP stimulation leads to calcium release from intracellular membranes, mitochondria and endoplasmic reticulum. The c-fos promoter contains a calcium response element region and responds to numerous environmental changes from outside the cell. We tried to visualize the process from calcium signalling to gene expression of c-fos at the single cell level using the calcium-regulated photo protein, obelin, and firefly luciferase as a c-fos reporter. MATERIALS AND METHODS Reporter gene construction. Apoobelin gene 3 was inserted into mammalian expression vector, pcDNA3.1 (Invitrogen). The complete region of c-fos gene promoter 4 was inserted into the firefly luciferase vector (pGL3 basic promoter vector, Promega). HeLa cell was co-transfected the apoobelin and c-fos promoter constructs by FuGene HD (Roche). Ca2+ imaging. HeLa cell co-transfected apoobelin and c-fos promoter constructs were incubated in Dulbecco's modified Eagle's medium containing coelenterazine (Renilla luciferase assay system, Promega) for 4 h to reconstruct obelin. The cell was stimulated by 500 ~MATP, and the luminescence image captured on a luminescence microscope (Luminoview, LV 200, Olympus) equipped with iXon EM-CCD camera (Andor). Binning of the CCD was 2x2, and the exposure time was 25 s with 30 s interval. The cell was re-stimulated by I 0 ~M ionomysin at 20 min after A TP stimulation. c-fos imaging promoter assay. After Ca2+ imaging without ionomysin stimulation, 1 mM luciferin was added into the medium, and luminescence image was captured by 5 min exposure time at 12 min interval. The c-fos signal light was separated from Ca2+ signal light by an optical long pass filter (610 nm), and the promoter activity was monitored sequentially after ci+ response of the same cells. 359

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RESULTS AND DISCUSSION Luminescence image of obelin regulated by and its field in HeLa cells are shown in Fig. I, and intracellular dynamics in some chronologically (see Fig. 2). responses are observed selected cells are

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362

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c-fos imaging promoter assay. The cjos promoter activity after Ca2+ response is analyzed sequentially at the same cells. Fig. 3 shows time course analyses both of ci+ and c-fos responses after ATP stimulation of the three cells. In Fig. 3A and 3C, Ca 2+ response appears at the early stage in the two cells, but c-fos activity appears at 2.S and 7.S hours for each cell after stimulation (Fig. 3B and 3D). In Fig. 3E, Ca2+ response appears at the late stage, and cjos activity appears at 1.S hours after stimulation (Fig. 3F). Thus heterogeneous response of c-fos promoter activity does not correspond to the early and late response of Ca2+ signalling which is upstream trigger for the c-fos activation. The heterogeneous response of promoter activity at single cell level has been already reported in some genes, 5,6 but the role and mechanism of the phenomena are still unknown. Therefore, luminescence imaging system will be a suitable tool for promoter assay at single cell level and will contribute for further understanding of signal transduction, gene expression and other cellular activities.

REFERENCES l.

2.

3.

4.

S.

6.

Suzuki H, Dosaka S, Ohashi-Hatta Y, Sugiyama T. Luminescence microscope for reporter assay of single live cells. In: Szalay AA, Hill PJ, Kricka LJ, Stanley PE. eds. Bioluminescence and Chemiluminescence. Singapore:World Scientific.2007:S3-6. Hatta-Ohashi Y, Hayasaka N, Takahashi T, Shigeyoshi Y, Suzuki H. Application of a luminescence microscope with novel optical system for detection of the gene expression pattern of individual cells. In: Szalay AA. Hill PI, Kricka LJ, Stanley PE. eds. Bioluminescence and Chemiluminescence 2007; Singapore:World Scientific. 2007:4S-8. Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee 1. Obelin from the bioluminescenct marine hydroid Obelia geniculata: Cloning, expression, and comparison of some properties with those of other Ca2 + regulated photoproteins. Biochem 2002;41 :2227-36. Chen C, Clarkson RWE, Xie Y, Hume DA, Waters MJ. Growth hormone and colony-stimulating factor 1 share multiple response elements in the c-fos promoter. Endocrinol199S;136:4S0S-16. Castano JP, Kineman RD, Frawley LS. Dynamic monitoring and quantification of gene expression in single, living cells: A molecular basis for secretory cell heterogeneity. Mol Endocrinol 1996;10:S99-60S. Takasuka N, White MRH, Wood CD, Robertson WR, Davis IRE. Dynamic changes in prolactin promoter activation in individual living lactotrophic cells. EndocrinoI1998;139:l361-8.

APPLICATIONS OF DELAYED FLUORESCENCE AND LASER CONFOCAL SCANNING MICROSCOPE TECHNIQUES IN MONITORING ARTIFICIAL ACID RAIN STRESS ON PLANTS HAIXIN ZHANG, FENG WEN, XIAOMING ZHOU MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China [email protected] INTRODUCTION

Acid rain can retard plant growth and development, perturb photosynthesis, and finally decrease crop production. 1 Delayed fluorescence (DF) is the phenomenon of photon emission by a living system. 2 It has been demonstrated to be a sensitive indicator of energy utilization efficiencies and many stress factors. 2-8 The production of H20 2 and other reactive oxygen species (ROS) is a common feature of plant responses to adverse stress. 9 Using ROS-specific probe and laser confocal scanning microscope (LCSM) techniques, the site of ROS production could be clearly localized and monitored in situ. The aim of the present study was to explore the effects of artificial acid rain (AAR) on intact seedlings and single living cells. MATERIALS AND METHODS

Materials. Arabidopsis seedlings were cultured according to the procedures described previously.9 The components of artificial acid rain are H2S04, HN0 3 and HCI in a ratio of 1:1:1.1 Catalase (CAT, bovine liver) and 2', T-dichlorodihydrofluorescein diacetate (H 2DCFDA) were from Sigma (St. Louis, MO, USA). Methods. DF was recorded with custom-built detection system. Photosynthesis rate (Pn) was determined with a photosynthesis system (Model: LI-COR 6400 LI-COR, Lincoln, NE, USA) using a method described previously.7 All DF and Pn measurements were performed according to the method described previously.7 Detection of ROS was performed using a LCSM (LSM510/ConfoCor2, Zeiss, lena, Germany) according to a method described previously. to The pigment extraction was performed as described described previously. 1 RESULTS AND DISCUSSION

Effect of artificial acid rain on DF and Pn. Clearly, DF intensity declined in a way consistent with Pn with decreasing pH of artificial acid rain. Only 15 min after exposure to AAR at pH 3, the decreases in DF intensity and Pn both became 363

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Fluorescence and Laser Confocal Scanning Microscope Techniques

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ROS pmduction and effects of antioxidant on D F. in many cells at 5 min after the exposure to AAR at 3 DF fluorescence could be detected at 15 min the cells with CAT effectively prevented the increase DCF fluorescence induced by AAR (Fig. 3A, B). Moreover, the decreases in DF could be inhibited by pre-incubation with CAT (Fig. 3C). In summary, DF could be used an marker for monitoring AAR stress.

ACKNOWLEDG EMENTS This research is by the National Natural Science Foundation of China and the National High Technology Research and Development (2007 AA 1OZ204). REFERENCES 1.

CL. Effects of simulated acid rain on seed germination and of three crops. 1 Trop Subtrop Bot 2003; 4:400-4. Amesz 1, Van Gorkom HI. Delayed fluorescence in photosynthesis. Annu Rev Plant 1978;29:47-66.

366 Zhang H et ai.

3. Badretdinov DZ, Baranova EA, Kukushkin AK. Study of temperature influence on electron transport in higher plants via delayed luminescence method: experiment, theory. Bioelectrochem 2004;63:67-71. 4. Zhang LR, Xing D, Wang JS, Li LL. Rapid and non-invasive detection of plant senescence using a delayed fluorescence technique. Photochem Photobiol Sci 2007;6:635-41. 5. Wang JS, Xing D, Zhang LR, Jia L. A new principle photosynthesis capacity biosensor based on quantitative measurement of delayed fluorescence in vivo. Biosens. Bioelectron 2007;22:2861-8. 6. Kurzbaum E, Eckert W, Yacobi YZ. Delayed fluorescence as a direct indicator of diurnal variation in quantum and radiant energy utilization efficiencies of phytoplankton. Photosynthetica 2007;45 :562-7. 7. Zhang LR, Xing D. Rapid determination of the damage to photosynthesis caused by salt and osmotic stress using delayed fluorescence of chloroplast. Photochem Photobiol Sci 2008;7:352-60. 8. Chen WL, Xing D, Tan SC, Tang YH, He YH. Imaging of ultra-weak bio-chemiluminescence and singlet oxygen generation in germination soybean in response to wounding. Luminescence 2003; 18: 19-24. 9. Gao CJ, Xing D, Li LL, Zhang LR. Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant cell death induced by ultraviolet-C overexposure. Planta 2008;227:755-67.

DELAYED FLUORESCENCE AND OPTICAL MOLECULE IMAGING TECHNIQUES FOR DETECTING THE STRESS RESPONSE OF PLANTS TO HIGH TEMPERATURE LINGRUI ZHANG, FENG WEN MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China Email: [email protected]

INTRODUCTION Temperature is one of major environmental factors that affects plant growth. Heat stress could completely inhibit photosynthesis before other stress symptoms are detected.! Delayed fluorescence (DF), emitted from chloroplasts, has been demonstrated to be a sensitive indicator of photosynthetic efficiencies and stress factors. 2-9 ROS production is a common feature in responses to environmental stress.!O It has been demonstrated that heat-shock transcription factor, HsfA2, plays a crucial role in maintaining redox balance. I Using a ROS-specific probe and laser scanning IO confocal microscopy (LCSM), ROS production could be clearly monitored in situ. Here, we used a DF technique combined with LCSM imaging to detect the responses of HsfA2 mutants and wild type (WT) plants to heat stress. MATERIALS AND METHODS Materials. Arabidopsis seedlings were cultured according to the procedures as described previously.8 Catalase (CAT, bovine liver) and 2', 7'-dichlorodihydrofluorescein diacetate (H 2DCFDA) were from Sigma (St. Louis). Methods. DF was recorded with a custom-built DF detection system. Photosynthesis rate (Pn) was determined with a portable photosynthesis system (Model: LI-COR 6400 LI-COR, Lincoln, NE, USA). DF and Pn measurements were performed according to the methods as described previously? In situ detection of ROS was performed using a commercial laser scanning microscope (LSM510/ConfoCor2) combination system (Zeiss, Jena, Germany) according to the method described previously.!O RESULTS AND DISCUSSION Effect of heat stress on DF in WT and HsfA2 mutants. DF intensity declined more markedly in HsfA2 mutants than in WT plants after a 2 h heat stress at 40°C (Fig. I). There was no significant difference in DF intensity in heat-stressed relative to unheated-stressed leaves for WT; however, a significant difference in DF 367

368

Zhang L & Wen F.

intensity at P < 0.01 could be found for HsfA2 mutants (Fig. 1). 140 ~

,0

~

.0

'[ij

=
.S .... 0

120

_ Before heat stress [=:::J After heat stress

100 80 60 40 20 0

KoHsfA2

WT Fig.1.

Effect of heat stress on DF intensity in WT and HsfA2 mutants.

** indicates a difference at P < 0.01. A

14()

~

120

~

.El '" ~

B .S

....Ci

HX)

Values are the mean ± SE of seven replicates.

_'W'T 'KoH~!A::

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llO

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fi-

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_WI' ~KoHs/12

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5

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20 ()

24 21

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B ~

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15 12

;=;

9 6

Control

4

6

8

10

Recovery time (11)

12

P-

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I

8

lU

L

I

0... '--i---'-~-"---'--'-~---'----"",\--L-""'I-...L

~

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o

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-l

(,

12

Recovery time (h)

Fig. 2. Recovery dynamics of DF (A) and Pn (8) in WT and HsfA2 mutants after heat stress. Data are the mean ± SE of seven replicates,

Recovery dynamics of OF and Pn after heat stress. As shown in Fig. 2, after heat stress, DF intensity and Pn consistently recovered the level of control (at 25°C) after cooling the leaves from high temperature for 12 h in WT but not in KoHsfA2 mutants (Fig. 2A, 8). This indicated that heat stress caused an irreversible damage to HsfA2 mutants but a reversibe to WT.

In vivo monitoring ROS production and effects of antioxidant on DF. Fig. 3 showed that H2 0 2 produced a more marked in the HsfA2 mutants than in WT after a 2 h HS at 40°C, as shown by bright green fluorescence resulting from staining with

Delayed Fluorescence and Optical Molecule Imaging Techniques

369

In the presence of 100 units/mL CAT, H 20 2 burst could be inhibited in both WT and HsfA2 mutants (Fig. 3A). The declines in DF could also be arrested by infiltrating the leaves with CAT (Fig. 3B). Taken

plays an important role in ROS burst. DF is an excellent detecting the response to oxidative stress caused by HS.

Chloroplast DCF

Overlay

B

140 120

heat stress

. . CAT heat stress

100

80 60 >'-<

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o ROS production (A) and effect of antioxidant on DF intensity (B) after 2-h heat stress. Data are the mean ± SE of seven replicates. Scale bars 10 ).im.

ACKNOWLEDGEMENTS Thanks to Dr. Yee-yung Charng (National Taiwan University, Taiwan) for mutant seeds. This research is supported by the National Natural Science Foundation of China (30470494) and the National High Technology Research and Development Program of China (863 Program) (2007 AA 1

REFERENCES 1. Liu Liu NY, Chi WT, Wang CN, Chang heat-inducible transcription factor, HsfA2, is required for extension thermotolerance in Arabidopsis. Plant Physiol 2007; 143 :251-62.

2.

D. Rapid determination of the damage to photosynthesis caused salt and osmotic stress using delayed fluorescence of chloroplast. Photochem Photobiol Sci 2008;7:352-60.

3. Kurzbaum E, Eckert W, Yacobi YZ. Delayed fluorescence as a direct indicator of diurnal variation in quantum and radiant energy utilization efficiencies of phytoplankton. Photosynthetica 2007;45:562-7. 4. Zhang Jia L. A new principle photosynthesis biosensor based on quantitative measurement of delayed fluorescence in vivo.

370

Zhang L & Wen F.

Biosens Bioelectron 2007;22:2861-8. 5. Badretdinov DZ, Baranova EA, Kukushkin AK. Study of temperature influence on electron transport in higher plants via delayed luminescence method: experiment, theory. Bioelectrochem 2004;63:67-71. 6. Wang CL, Xing D, Chen Q. A novel method for measuring photosynthesis using chloroplasts delayed fluorescence, Biosens Bioelectron 2004; 20:454-459. 7. Wang CL, Xing D, Zeng LZ. Effect of artificial acid rain and SOz on characteristics of delayed light emission. Luminescence 2005;20:51-6. 8. Amesz J, Van Gorkom HJ. Delayed fluorescence in photosynthesis. Annu Rev Plant Physiol 1978;29:47-66. 9. Chen WL, Xing D, Tan SC, Tang YH, He YH. Imaging of ultra-weak bio-chemiluminescence and singlet oxygen generation in germination soybean in response to wounding. Luminescence 2003;18:19-24. 10. Gao CJ, Xing D, Li LL, Zhang LR. Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant cell death induced by ultraviolet-C overexposure. Planta 2008;227:755-67.

PART 9 ASPECTS OF FLUORESCENCE AND PHOSPHORESCENCE

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THE INTERACTION OF Tb3+-PROTOCATECHUIC ACID COMPLEX WITH NUCLEIC ACIDS AND ITS APPLICATION IN DETERMINATION OF NUCLEIC ACIDS BASED ON FLUORESCENCE QUENCHING CHEN Y ANJING, 1,2 YANG YUNHONG, 1 YANG JINGHE 2 JSchool a/Chemistry and Chemical Engineering, University 0/ Jinan, Jinan 250022, PR China, email: [email protected] 2Sc haal a/Chemistry and Chemical Engineering, Shandang University, Jinan 250100, PR China INTRODUCTION Quantitative determination of nucleic acids based on fluorescence and absorption properties is limited by low sensitivity and interferences,I,2 Methods based on quenching or enhancement of light properties of a probe after interacting with nucleic acids have been developed 3 ,4 based on luminescence rare earth ions that have narrow spectral width, long luminescence lifetime, large Stokes shift and strong binding with biological molecules, o-Phenanthroline and pyridine are commonly used as rare-earth ligands. When the complex is used as probe, the sensitivity of determination of nucleic acids is improved greatly.5,6 We used protocatechuic acid (PCA) as a ligand for Tb 3+ and Tb 3+-PCA as a probe for determination of nucleic acids. Under the optimum conditions, binding of PCA to Tb 3 + leads to a marked enhancement in the fluorescence emission intensity of Tb3+, but with the addition of nucleic acids, the fluorescence emission intensity of the complex is quenched in proportion to the concentration of nucleic acid with a detection limits of23 ng/mL for ctDNA and 9,9 ng/mL for yRNA. EXPERIMENTAL Reagents. Stock standard solutions (0.01 mollL) of Tb3+ were prepared by dissolving 0.1868 g of terbium oxide (99.99%, Shanghai Yuelong Chemical Co,) in hydrochloric acid, and diluting to 100 mL with deionized water. Stock solution (0.01 mollL) of PCA was prepared by dissolving PCA (0.0770 g) (Shanghai Chemical Reagent Co, of the Medical Department of China) in 50 mL water. Stock solutions of nucleic acid (100 Ilg/mL) were prepared by dissolving commercial ctDNA and yRNA (Beijing Baitai) in 100 mL water and stored at 0-4°c, All the chemicals used were of analytical reagent grade and doubly deionized distilled water was used throughout. Apparatus. The spectrum and intensity of fluorescence were measured with a RF-540 spectrofluorometer (Shimadzu, Japan). An UV-260 (Shimadzu, Japan) spectrophotometer was employed in all absorption spectra recordings, The circular dichroism spectrometer JASCO J-81 0 (Hitachi. Japan) was used for measurement 373

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circular dichroism. The viscosity was measured with a Ubbelodhe viscosimeter. All pH measurements were made with a pHs-3C acidity meter (Leici, Shanghai).

(.)

60

40 LL

300

320

340 ~nm)

1IiII

1IiII

o~~~~~~~~~ 480

500

520

540

560

580

600

A(nm)

Fig. 1. Excitation and emission spectra. (a) excitation O"em = 546 nm) (b) emission spectra (Aex =320 nm): 1. Tb3+; 2. Tb-DNA; 3. Tb-RNA; 4. Tb 3 +-PCA-RNA; 5. Tb 3 +-PCA-DNA; 6. Tb3+- PCA . Conditions: Tb 3 + 1.00xlO-6 mollL: PCA 2.5xlO-5 mollL; ctDNA 1.00xlO-6 g/mL; yRNA: 1.00xlO-6 g/mL; pH 8.10. Procedure. To a 10 mL test tube, solutions are added in the following order: Tb 3+, PCA, nucleic acids and Tris-HCI buffer. The mixture is diluted to 10 mL with deionized water. The fluorescence intensity was measured in a 1 cm quartz cell (excitation and emission slits 10 nm). The quenched fluorescence intensity of Tb 3+-PCA by nucleic acids (emission peak 546 nm) is represented as ~F = Fa-F (F and Fa are the intensities of the systems with and without nucleic acid, respectively). The excitation wavelength was 320 nm.

RESULTS AND DISCUSSION Features of fluorescence spectra. The excitation and emISSIOn fluorescence spectra of Tb 3 +, Tb-DNA, Tb-RNA, Tb3+-PCA, Tb 3 +-PCA-RNA, Tb 3 +-PCA-DNA are shown in Fig. J(a) and (b). No characteristic fluorescence ofTb 3 + was observed in Tb 3+, Tb-DNA and Tb-RNA systems, but Tb3+-PCA system emits three strong 3 characteristic fluorescence peaks of Tb + located at 489 nm, 546 nm and 587 nm, 7 which correspond to the sD 4- F6 , SD4-7FS and sDc7F4 transitions of Tb 3+, respectively. The maximum excitation wavelength was 320 nm. The fluorescence intensity of the Tb 3+-PCA system was strongly quenched with the addition of nucleic acids. However, the quenching fluorescence intensity of RNA is much stronger than that of DNA. We chose a peak of excitation wavelength 320 nm, and

The Interaction ojTb3+ -Protocatechuic Acid Complex with Nucleic Acids

375

emission wavelength of 546 nm in further studies. Selection of optimized analytical parameters. In order to select an optimized analytical system, experimental parameters - pH, buffers, concentration of Tb 3+ and PCA - were studied at [DNA] = 1.00x 10,6 g/mL. The experimental results indicate that tlF has the largest value in Tris-HCL buffer at pH8.10, so the Tris-HCL buffer of pH 8.10 was chosen for the assay and the optimum volume of buffer was 1.0 mL. The study of effect of concentration of Tb 3+ and PCA showed that the optimized concentration is 1.00xlO-6 mo1lL for Tb 3 +, 2.5 x lO- 5 mollL for PCA, respectively. Effect of foreign substances. Under the optimum conditions, interference of foreign substances, such as metal ions, amino acids, BSA and lactose, were tested at a ctDNA concentration 1.00x 10'6 g/mL and the results were satisfactory. ANALYTICAL APPLICATION Calibration curve and detection limit. A linear relationship was obtained between the tlF and the concentration of nucleic acids over the range 3 .Ox 10'8 -1.0xlO-6 g/mL and 1.0xlO'6 _5.0xlO'6 g/mL for ctDNA, 1.8xlO'8-1.0xlO'6 g/mL and 1.0 x lO- 6-8.0xl0,6 g/mL for yRNA, the detection limits (S/N=3) were 23 ng/mL for ctDNA, and 9.9 ng/mL for yRNA, respectively. Table 1. Sample

Analytic results for nucleic acids in synthetic samples. Added (x 10,6 g/mL)

Mean founded (xl0'6 g/mL)

Recovery (%)

RSD (%)

ctDNA

2.00 3.00

2.07 2.89

103.5 96.3

3.6 2.7

yRNA

2.00 3.00

2.04 3.12

102.0 103.0

3.1 1.9

Determination of DNA in synthetic samples. The concentration of fsDNA and ctDNA in synthetic samples that were prepared by mixing 2x 10'7 mollL NaCl, 2x 10'7 mollL CUS04 and 5 x 10-7 mollL alanine with nucleic acids, were determined. Results show satisfactory recovery and precision of this method are (Table 1). REFERENCES 1. Tuite E, Kelly JM. The interaction of methylene blue, azure B, and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems. Biopolymers 1995;35: 419-33. 2. Nordmeier E. Absorption spectroscopy and dynamic and static light-scattering studies of ethidium bromide binding to calf thymus DNA: implications for outside-binding and intercalation. J Phys Chern 1992;96:6045-55

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4.

5. 6.

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Rossetto FE, Nieboer E. The interaction of metal ions with synthetic DNA: induction of conformational and structural transitions. Inorg Biochem 1994;54:167-86. Patty KL, Claudia TF. Energy transfer from nucleic acids to Tb(III):selective emission enhancement by single DNA mismatches. J Am Chern Soc 1999;121:1-7. Liu RT, Yang JB, Wu X. Study of the interaction between nucleic acid and oxytetracycline-Eu 3+ and its analytical application. J Lumin 2002;96: 201-9. Wu X, Sun SN, Yang JB, Wang YB, Li YX, Su BY. Study of the reaction between the nucleic acid and Y-BPMPBD-CTMAB complex and its analytical application. J Fluor 2004; 14: 1l3-8.

FLUORESCENCE ENHANCEMENT OF KI FOR THE MORIN-fsDNA SYSTEM AND ITS ANALYTICAL APPLICATION HONG HONG DING, XIA WU, JINGHE YANG, FEI WANG Key Laboratory a/Colloid and Inter/ace Chemistry (Shandong University), Ministry a/Education, School o/Chemistry and Chemical Engineering, Shandong University, Jinan 250100, Shandong, China; E-mail: [email protected];[email protected]

The study on selective recognition of DNA by small molecules has drawn considerable attention because it is important in the design of new and more efficient drugs targeted to DNA." 2 Studies on the binding mode between probe and DNA have been performed with various methods.'-? It is a generally accepted concept that there are three models for binding of small molecules to the DNA double helix: intercalative binding, groove (or surface) binding and electrostatic binding.' Potassium iodide (KI) is commonly used as fluorescent quenching agent because of the heavy atom effect. Therefore, it has wide applications in the study of the interaction mechanism between fluorescence probes and biomacromolecules.' But in the study on the interaction mechanism of morin-fish serum nucleic acid system,'O it was found that KI can enhance the fluorescence intensity and lifetime ofa system. We studied the effect of the different concentration of KI on morin-hsDNA system. Our results indicated that there was no heavy atom effect in the system at low concentrations of KI « 1.0 x 10-3moI/L) and the fluorescence spectra are presented in Fig. 1. Fig. 1 showed that the fluorescence intensity of morin at 510 nm was enhanced by hsDNA or smDNA with a remarkabe blue shift in the emission spectra. Furthermore, when KI was added to morin-hsDNA or morin-smDNA, the fluorescence intensities of the systems were still further enhanced; however, the peak of morin-hs(sm) DNA showed little change. ctDNA did not exhibit this phenomenon. There were two possible explanations for the fluorescence enhancement of morin-fsDNA upon the addition ofK!. The first reason for the enhancement effect was the formation of the more favorable structure for luminescence. The studies on the effect of different halogen anion compounds (KCl, KBr and KI) on the morin-hsDNA systems were conducted. The results showed that at the same concentrations and conditions «l.Ox 10-3 mollL), KCl weakened the fluorescent intensity of the system, while both KBr and KI enhanced it, and the enhancement order was KI > KBr. It is proposed that the lone electron pair of electronegative r (or BO, bonds to the conjugated system of morin, and that the combined system then interacted with fsDNA. With the increase of atomic radius from B( to r, the attraction between nucleus and outer electrons of 377

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Ding H et al.

wlJvelengtJI

wavelength

Fig.1.

Fluorescence spectra: (a) Excitation spectrum; (b) Emission spectrum (1): morin; (2): morin-KI; (3): KI-morin-ctDNA; (4): morin-hsDNA; (5): morin-smDNA; (6): KI-morin-hsDNA; (7): KI-morin-smDNA. Conditions: morin: 4.0x 10-6 mollL; KI: 6.0x 10-4 mollL; hsDNA: 1.0x 10- 5 g/mL; ctDNA: 1.0x 10-5 g/mL; smDNA: 1.0xl0-5 g/mL. the halogen diminished gradually. Therefore the conjugation effect between morin and outer electrons increased accordingly, and then the fluorescent intensity was enhanced. For KCI, which is not an anionic quencher of the f1uorophores, its influence on fluorescence intensity arises only from the ionic strength."" The second reason was that the addition of KI changes the microenvironment of the morin-fsDNA system. This change could be monitored by both the microviscosity estimated using the fluorescence polarization (P) of fluorescence probe," and polarity reflected by the ratio of the first to the third fluorescence bands of pyrene monomer (I/h)." The value of P in morin-fsDNA was 0.183 (mPa.s), then changed into 0.184 (mPa.s) after adding KI. Meanwhile, the values OfIl/I3 changed to 1.54 from 1.56. The above data signified that there was little change in the microenvironment of morin-hsDNA with and without KI. Therefore, it seemed that the first reason was the most likely expalanation for the enhancement effect by KI on the morin-hsDNA system. KI reinforced the conjugate effect of morin-hsDNA, formed the more favorable structure for luminescence, so the fluorescence of morin-hsDNA was enhanced. We also studied the binding properties of KI-morin-hsDNA system by absorption, circular dichroism and the ionic strength effect experiments. The studies indicated that the groove binding mode was dominant in the KI-morin-DNA system. Fluorescence titration result revealed that KI-morin showed a good linear response to fsDNA. The calibration curves for the determination ofhsDNA and smDNA were constructed under the optimal conditions. All the analytical parameters are presented in Table 1.

Fluorescence Enhancement of Klfor the Morin-fsDNA System

379

Table 1. Analytical parameters of this method DNA

Linear range

Correlation coefficient

(g/mL)

hsDNA smDNA

8.0x I 0- 9-2.0x 10- 5 9

5.0xlO- _l.OxI0-

5

Limit of detection (ng/mL)

0.997

4.5

0.996

3.5

In order to explore the method's selectivity, the highest permissible concentrations of other substances (concentrations that cause a ± 5% relative error in the 7 fluorescence intensity) were tested at I.OxlO- mollL of hsDNA. Results indicated that most of amino acids and metal ions except Fe 3 +and Fe 2 + had no or little effect on the determination ofhsDNA.

ACKNOWLEDGMENTS This work is supported by Natural Science Foundations of China (20575035) and Shandong Province (Y2003B02). REFERENCES l. Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB. Regulation of gene expression by small molecules. Nature 1997;387:202-5. 2. Dervan PB, Poulin-Kerstien AT, Fechter EJ, Edelson BS. Regulation of gene expression by synthetic DNA-binding ligands. Top Curr Chern 2005;253:1-3l. 3. Alonso A, Almendral MJ, Curto Y, Criado JJ, Rodriguez E, Manzano IL. New fluorescent antitumour cisplatin analogue complexes. study of the characteristics of their binding to DNA by flow injection analysis. J Fluoresc 2007;17:390-400. 4. Arya DP, Coffee RL, Willis B, Abramovitch AI. Aminoglycoside-nucleic acid interactions: Remarkable stabilization of DNA and RNA triple helices by neomycin. I Am Chern Soc 2001;123:5385-95. 5. Wang XP, Pan IH, Yang XD, Niu CD, Zhang Y, Shuang SM. Porphyrin binding to DNA investigated by cyclodextrin supramolecular system. Anal Bioanal Chern 2002;374:445-50. 6. Cao Y, He X W. Studies of interaction between Safranin T and double helix DNA by spectral methods. Spectrochim Acta A 1998;54:883-92. 7. Long YF, Huang CZ. Spectral studies on the interaction of Amido black lOB with DNA in the presence of cetyltrimethylammonium bromide. Talanta 2007;71 :1939-43. 8. Sovenyhazy KM, Bordelon lA, Petty IT. Spectroscopic studies of the multiple binding modes of a trimethine-bridged cyanine dye with DNA. Nucleic Acids Res 2003;31:2561-9. 9. Zhu QZ, Li F, Guo XQ, Xu IG, Li WY. Application of a novel fluorescence

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probe in the determination of nucleic acids. Analyst 1997;122:937-40. 10. Wang F. Doctoral thesis, Jinan: Shandong University, 2006. 11. Prativel G, Bermadou J, Meunier B. Carbon-hydrogen bonds of DNA sugar units as targets for chemical nucleases and drugs. Angew Chern Inti Ed 1995;34:746-69. 12. Jiang YC, Wu SK. A study on the micellar viscosity in surfactant solution by fluorescence polarization. Photagr Sci Photochem 1995;13:180-5. 13. Nakajima A. A study of the system of pyrene and p-cyclodextrin in aqueous solution utilizing the intensity enhancement phenomenon, Spectrochim Acta A 1983;39:913-5.

MICROEMULSION SENSITIZED DETERMINATION OF BSA WITH 3-(4'-METHYLPHENYL)-5-(2'-SULFOPHENYLAZO) RHODANINE BY RESONANCE RAYLEIGH SCATTERING METHOD GE SHENGUANG, DAI PING, YU JINGHUA, LI BO, TAN YUN School a/Chemistry and Chemical Engineering, University 0/Jinan, Jinan 250022, Email: [email protected]@vip.sina.com

INTRODUCTION Protein malnutrition leads to the condition known as kwashiorkor, hence it is important to develop a method for monitoring protein levels. Several protein methods have been developed including nephelometric detection, Kjeldahl determination, I absorption spectrophotometry,2 spectrofluorimetryJ and resonance Rayleigh light scattering.' The accuracy and precision are low in nephelometric detection and Kjeldahl determination. Absorption spectrophotometry has poor sensitivity and is subject to interference. Spectrofluorimetry has been developed as a powerful tool for high sensitivity protein determination but requires a fluorescent moiety. Resonance Rayleigh scattering (RRS) is a new analytical technology with remarkable characteristics of high sensitivity, simple operation and good selectivity. We found that intense RRS was produced based on the aggregation of biomacromolecules and dye chromophore. 3-(4'-Methyl phenyl)-5-(2'-sulfophenylazo) rhodanine (4MRASP) is an azo compound based on rhodanine. The sulfonic acid group is strongly acidic, therefore the dye molecules can exist as anions owing to the dissociation of sulfonic in both acidic and weakly acidic mediums. We have now found that in an acidic medium, the RRS intensity of 4MRASP itself was weak but it was sharply enhanced when the dye molecule associated with protein. Furthermore, the RRS intensity was found to have a linear relationship with concentration of protein under specified conditions. Based on this, a new method for determination of protein was established, and the method was applied to determine protein in milk with satisfactory results. MATERIALS AND METHODS A Perkin-Elmer LS55 (America) fluorescence spectrophotometer was used with a quartz cell (1 cm; Perkin-Elmer, USA). UV absorption spectra were measured on a UV-31 0 1PC spectrophotometer (Shimadzu, Japan). pH measurements were made by using a home-made PHS-3C digital pH-meter (Shang Hai Lei Ci Device Works, Shanghai, China) with a combined glass-calomel electrode. The stock solution of BSA (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) was 1.0 mg/mL. A 4MRASP stock solution (2.0 x 10-4 mol/L) was 381

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prepared by dissolving 81.4 mg of 4MRASP in anhydrous alcohol and diluting to 1 L with anhydrous alcohol. The B-R buffer solution was pH 2.84. A sodium dodecylsulphate (SDS) micromulsion was prepared by mixing n-butanol, n-heptane and water to give a final total volume of 200 mL. All chemicals were of analytical reagent grade or better. Doubly distilled demineralized water used throughout and was obtained by SYZ-550 quartz sub-boil high-purified water distiller (Jiang Su Jin Tan, Jiang Su, China). Procedure. 2x 10-5 moUL of 4MRASP (1.0 mL) was added to a 10 mL color comparison tube with a cover, 10 Ilg /mL of BSA standard solution (1.0 mL), 2.0 mL of B-R buffer solution (pH= 2.84), and 0.2 mL of SDS micromulsion were added in turn, and the mixture diluted to the standard volume. The scattering intensity of the blank solution (Io) and sample or BSA standard solution (II) were measured at 391 nm (slit = 5.0/2.5). The concentration of BSA was quantified via the peak height (relative scattering intensity), which was obtained by subtracting the blank solution scattering intensity from that of the sample or BSA standard solution. RESULTS AND DISCUSSION RRS spectrum. RRS spectrum of the different components is shown in Fig. 1. The scattering intensity of BSA or 4MRASP was weak, and weak in the acidic medium with SDS. However, when BSA was injected into 4MRASP in the presence of SDS, the scattering intensity enhanced sharp Iy (curve 5). Optimum conditions. It was found that the BSA with 4MRASP solution scattering intensity was most enhanced in the B-R buffer solution (compared to disodium hydrogen phosphate-citric acid and tartaric acid-sodium tartaric acid buffer). B-R buffer (2.0 mL) at pH 2.84, and 1.0 mL of2 x 10-5 moVL4MRASP was selected for further study. SDS micromulsion (2.0 mL) gave the highest sensitivity, hence it was chosen to be sensitization agent in our experiments. The interaction between BSA and 4MRASP was also influenced by ionic strength. The scattering intensity was unchanged with an increase of NaCI concentration until the concentration of NaCI reached 0.03 mollL. Hence, 0.03 mollL of NaCI was chosen to control the ionic strength of the solution. Effect of coexisting species. The to lerab Ie concentration ratios of foreign species on the determination of 10.0 Ilg of BSA in 10 mL of BSA-4MRASP system were as follows (the relative error less than ±5 %; fold): L-histidine, DL-aminoisovaleric acid, D-phenylalanine (60); L-Ieucine, DL-alanine (40); L-tryptophan, L-cysteine, L-tyrosine, DL-methionine (50); glycine (60); L-arginine (45); L-glutamic acid, L-lysine, L-methionine (70); DL-phenylalanine (100); ascorbic acid (200) Zn 2+, A1 3 +(20); K+, Fe 2 +(30); Fe3 +(5); Ca2+(20).

Microemulsion Sensitized Detennination of BSA

383

900 750 600

-.. 450 300

150 340

360

380

400

420

440

i.inm Fig. 1. Spectrum of resonance Rayleigh light scattering. BSA (1.0 Ilg/mL); 2. 4MRASP(2.0 x 10-6 mollL); 4. 4MRASP(2.0 x 10-6 mollL) + SOS; 5. 4MRASP (2.0 x 10-6 mollL) + BSA (0.5 Ilg/mL) + SOS; 7. 4MRASP (2.0 x 10-6 mollL) + BSA (1.4 Ilg/mL) + SDS. It can be seen from the data that the common ions and amino acid do not interfere

in the determination of BSA except for Fe 3+. To eliminate the interfere of Fe 3+, ascorbic acid was added to convert Fe3 + to Fe2 +. We conclude that the new rhodanine-based reagent is an effective probe with good analytical performance, and the BSA-4MRASP system has good selectivity. Analytical characteristics. The proposed RRS method was studied for linearity, precision, and sensitivity. Under the optimum conditions, a linear relationship between BSA concentration and enhanced scattering intensity was obtained over the range of 0-1.4 Ilg/mL, with a regression equation of t:J= -39.17 + 2.661 (Ilg IL) and correlation coefficient (r) of 0.9966. The detection limit of BSA was found to be 2.04 x 10-9g/mL according to 11 parallel determinations of the blank solution. Applications. A milk sample was diluted to 100 mL with water, and then determined by the proposed method. The recovery rates of the method were 96.2 % and 102.8 % RSD was 3.08 and 0.93 that indicated the results were satisfactory.

ACKNOWLEDGMENTS This work is financially supported by Science Research Foundation of Shandong Province, China (Y2007B07) and Key Subject (Laboratory) Research Foundation of Shandong Province, China (XTD0705). REFERENCES 1. Qin W, Dan W, Hui Z, et al. The trend of development of analytical

384

2. 3.

4.

Ge S et al.

techniques for protein. J Jinan Univ 2003;4:312-9. Changrong; Z Baosheng, Hongyi Z, et al. Spectrophotometric determination of albumin with acid brown NR. Anal Lab 2005; 1:63-5. Yongnian N, Shaojing S, Serge K. Spectrofluorimetric studies on the binding of salicylic acid to bovine serum albumin using warfarin and ibuprofen as site markers with the aid of parallel factor analysis. Anal Chim Acta. 2006;2:206-15. Dejing G, Na H, Yuan T, et al. Determination of bovine serum albumin using resonance light scattering technique with sodium dodecylbenzene sulphonate-cetyltrimethylammonium bromide probe. Spectrochim Acta A Mol Biomol Spectrosc 2007;3:573-7.

FLUORIMETRIC DETERMINATION OF RUTIN USING RUTIN-Fe(III) MM KARIM,! CW JEON,z SH LEE,! SM WABAIDUR! JDepartmento/Chemistry, KyungpookNational University, Taegu 702-701, Korea 2Korea 1nstitute o/Geoscience & Mineral Resources, Daejon, 305-350, Korea Email: [email protected]

INTRODUCTION Rutin is a flavonol type flavone derivative, consisting of quercetin and disaccharide rutinose (rhamnose and glucose).! It is found in black tea, buckwheat, and apple. This has been widely used in food and plant-based beverages. 2- 3 Rutin exhibits distinct biological and pharmacological activities including antibacterial, antispasmodic,4-5 and anticarcinogenic properties. 6 Rutin is able to prevent hemorrhages and ruptures in the capillaries and connective tissues, and is used to treat epitaxis, hemorrhages and venous insufficiency.7-8 This bioflavonoid also acts as a potent radical scavenger.8 Therefore, there is an increasing demand for simple, sensitive, inexpensive and fast analytical techniques for the determination of rutin for medical and biological use. In this paper, we report the development of a novel, sensitive, simple and practical method for the determination of rutin with Fe(III) as a probe by fluorimetric determination. Several analytical techniques have been described for determination of rutin. For example, direct spectrophotometric detection,9 indirect spectrophotometric methods,1O-12 liquid chromatography,13 HPLC with photodiode array detection (DAD).!o.!4 In this paper we report the development of a fluorimetric method for rutin that is sensitive and simple. EXPERIMENTAL Chemicals. Rutin was obtained from Aldrich (Milwaukee, WI, USA). Stock solution of rutin was prepared by dissolution of these compounds in methanol, followed by dilution with water of equal volume (methanol-water, 50:50, v/v). All other chemicals and solvents are used of analytical grade and doubly deionized distilled water was used throughout. Apparatus. Fluorimetric measurements were recorded on SPEX Fluorolog-2 spectrofluorimeter equipped with a xenon lamp of 450W. The fluorescence intensities of solutions were obtained using 1 cm quartz cells. The excitation and emission monochromators were fixed with 0.25 mm slits. Fluorescence was collected and detected by photomultiplier tube (Hamamatsu Model R 928) powered at 950V. All spectral data were obtained by SPEX DM 3000F 385

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spectroscopy computer. A pH meter (Model Orion 520A USA) was used for pH adjustment. General procedure. The excitation and emission spectra of fluorescence were measured at room temperature and optimum excitation and emission wavelengths were selected from these spectra. Three mL of rutin and I mL of 1 x 10-4 mo ilL Fe(III) were taken into a vial, mixed well and stood for 5 min. The content of the vial was made up to 10 mL with distilled water. The fluorescence intensity of the system was then measured in a 1 em quartz cell. The fluorescence intensity of the solution was measured at 506 nm with excitation at 491 nm. All fluorescence measurements were made using 1 nm increment, 1 s integration time, S acquisition mode and YES auto zero. RESUL TS AND DISCUSSION Fluorescence excitation and emission spectra. The fluorescence excitation and emission spectra of rutin (a, b) and rutin-Fe(III) systems (a', b') are shown in Figure 1. From the figure it can be shown that the fluorescence intensity of rutin was increased several fold with respect to the signal obtained from rutin when Fe(III) was added to the system (emission wavelength 506 nm with excitation at 491 nm). Considering the non-interference effect, stability and lower value of blank signal an excitation wavelength of 491 nm was selected for recording the emission spectra (Aem = 506 nm) in the subsequent experiments.

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Fluorimetric Determination of Rutin Using Rutin-Fe(Ill)

387

Effect of Fe(III) on fluorescence intensity. Addition of Fe (III) to the final solution of rutin caused the fluorescence intensity to increase significantly. Varying concentrations of Fe(III) (from 1 x 10-7 to 0.1 moUL) were added to the standard solution of rutin (1 x 10- 2 moUL). The maximum signal for rutin was 4 observed at iron concentration 1 x 10- mollL. Further increasing concentration decreased the emission intensity. Therefore, a concentration of 1 x10-4 moUL of Fe (III) was selected for subsequent experiments. Analytical parameters. Calibration curve for rutin run under the optimum conditions (i.e., [Fe(III)] = 1 x 10-4 moUL; Aex = 491 nm) was obtained by using a series of 6 standard solutions. The calibration curve shows linearity in the range 4 6 IxI0· mollL-1 x 10. mol/L with a detection limit of2.5xlO· 7 mollL. The R.S.D. was 1.35% (n=5). CONCLUSION This is a convenient and efficient method for determination of rutin in dosage forms. Sample preparation was simple and samples stable at 4.0 'c for 3 days. The method has a linear range of 1 xl 0-4 mollL-1 x I 0- 6 moUL with detection limit of 2.5xlO- 7 mollL. The instrumentation and methodology are simple, economical and easily adaptable to clinical analysis compared to the previously reported methods. ACKNOWLEDGEMENT The support of this research by Korea Research Foundation Grant (KRF-2004005-C00009) is gratefully acknowledged. REFERENCES I. Harborne JB. The Flavonoids. Advances in Research Since 1986, London:Chapman and Hall, 1993. 2. Herrmann K. Flavonols and flavones in food plants: a review. J Food Technol 1976; 11 :433-48. 3. Hetrog MLG, Hollman PCH, Katan MB. Content of potentially anti carcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J Agric Food Chern 1992;40:2379-83. 4. Deschner EE, Ruperto J, Wong G, Newmark HL. Quercetin and rutin as inhibitors of azoxymethanol-induced colonic neoplasia. Carcinogenesis 1991;12:1193-6. 5. Mata R, Rojas A, Acevedo L, et al. Smooth muscle relaxing flavonoids and terpenoids from Conyzajilaginoides. Planta Med 1997;63:31-5. 6. Webster RP, Gawde MD, Bhattacharya R K. Modulation of carcinogen-

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induced DNA damage and repair enzyme activity by dietary riboflavin. Cancer Lett 1996;109:129-35. 7. http://www.pdrhealth.com/drug info/nmdrugprofiles/nutsupdrugs/rut, 23 8. Ne'gre-Salvayre A, Affany A, Hariton C, Salvayre R. Additional antilipoperoxidant activities of alpha-tocopherol and ascorbic acid on membranelike systems are potentiated by rutin. Pharmacology 1991;42:262-72. 9. Pharmacopoea Helvetica VII, Department Federal de I'Interieur, Berne, 1995. 10. Kuntic V, Kosanic M, Malesev D, Radovic Z. Spectrophotometric investigation of Pd(II)-rutin complexes and its application to rutin determination in tablets. Pharmazie 1998;53:724-6. 11. Kuntic V, Malesev D, Radovic Z, Kosanic M, Mioc U, Vukojevic V. Spectrophotometric investigation of uranil(II)-rutin complex in 70% ethanol. J Agric Food Chern 1998;46:5139-42. 12. Kuntic V, Malesev D, Radovic Z, Vukojevic V, Vladana. Spectrophotometric investigation of the complexing reaction between rutin and titanyloxalate anion in 50% ethanol. Monatsh Chern 2000; 131: 769-77. 13. Daigle DJ, Conkerton EJ. Analysis of flavonoids by HPLC: an update. J Liq Chromatogr Relat Technol 1988; 11 :309-25. 14. Paganga G, Rice-Evans CA. The identification of flavonoids as glycosides in human plasma. FEBS Lett 1997;401 :78-82.

MICELLE ENHANCED FLUORIMETRIC DETERMINATION OF BENSERAZIDE IN PHARMACEUTICAL FORMULATIONS 1

SH .LEE,' WH KIM,' K MEEA,2 MA KHAN' Dep~rtment ofChemlst~, KyungpookNational University, Taegu, 702-701, Korea Department of Environmental Engineering, Andong National University, Andong, 760-749, Korea; e-mail: [email protected]

INTRODUCTION Benserazide (BZ), 2-amino-3-hydroxy-N-[(2,3,4-trihydroxyphenyl) methyl] propane hydrazide is an irreversible inhibitor of peripheral L-aromatic amino acid decarboxylase (AADC). The decarboxylase inhibitor drugs, e.g., carbidopa and benserazide, inhibit dopamine production outside the brain and permit direct delivery of dopamine (LD metabolite) to the brain. This synergistic therapy also minimizes the side effects such as nausea and vomiting induced by levodopa.'·2 Benserazide at the recommended therapeutic dose does not cross the blood-brain barrier to any significant degree. Synergistic effect of levodopa and benserazide reduces the required dose of levodopa for the optimal and earlier therapeutic response. 3 Few methods have been reported for the determination of BZ as a single analyte. 4•s The literature also contains few reports of methods specifically for the simultaneous determination of LD and BZ, like spectrophotometry,6.7 capillary electrophoresis (CE)2.s.9 and high performance liquid chromatography'O in pharmaceutical preparations and biological fluids. Most of the reported spectrophotometric methods for BZ are based on a derivatization technique, and these have poor selectivity, sensitivity and require additional software. Although electrophoresis and liquid chromatographic methods have high sensitivities, they are expensive, involving the use of complex procedures with several sample manipulations and involve long analysis time. Hence, the development of a new effective and efficient method for the determination of BZ in pharmaceutical preparation and biological fluids is an important task. EXPERIMENTAL Materials. Benserazide (Aldrich, USA). Triton X-100 (Sigma, USA) Phosphoric acid (Osaka, Japan) Sodium dihydrogen phosphate (Merck, Germany). Apparatus. Spectrofluorometer SPEX Fluorolog-2 (Edison, NJ, USA). Xenon lamp 450-W (OSRAM, Germany) and photomultiplier tube (R 928 Hamamatsu Co.) powered at 950 V as the detector. Excitation and emission monochromator slits, wavelength increment, and integration time were set at 1 mm, 1 nm and 1 second respectively. A pH meter (Model Orion 520A, USA) was used for pH adjustment. 4 Basic procedure. Benserazide solution 2 mL (1.0 x 10-6 - 1.0 x 10- mol/L) was 4 added to 2 ml of Triton X- I 00 solution (4.0 x 10- moJlL) and 2 mL pH 4.0 389

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phosphate buffer. Final volume was made up to 10 mL with distilled water. The fluorescence intensity of the mixture solution was measured at excitation and emission wavelengths of 279 nm and 318 nm, respectively. The fluorescence intensity of a reagent blank solution was similarly measured under the same conditions. RESUL TS AND DISCUSSION Spectral characteristics. Increase in the fluorescence intensity of the benserazide solution in the presence of Triton X-I00 was investigated using spectrofluorometer at excitation and emission wavelengths of 279 nm and 318 nm respectively (Fig. 1). As can be seen from Fig. 1 the poor fluorescence response of benserazide solution was enhanced by using the Triton X-I00 solution. This micelle-enhanced phenomenon was exploited for the spectrofluorimetric determination ofbenserazide Parameter optimization. Various parameters such as pH and Triton X-I00 concentration were optimized for the spectrofluorimetric determination of benserazide. Phosphate buffer pH 5 was found to be the optimum for the investigated method. Similarly, addition of Triton X-IOO up to 1.0 X 10-4 mol/L increased the emission intensity. Above this concentration the response was linear, 4 therefore 1.0 x 10- mol/L of Triton X-I 00 was chosen as optimum concentration. Calibration curve for benserazide. In order to evaluate analytical characteristics of the method, the linear fluorescence response for various benserazide concentrations was investigated using the optimum conditions. The results obtained are shown in the Fig. 2. It was found that linear fluorescence response was observed for benserazide concentration in the range of 1.0 x 10-7 to 1.0 x 10-4 mol/L. The limit of detection was found to be 3.5 x 10-8 mol/L. The relative standard deviation (R.S.D) for 6 repeated measurements of 1.0 x I 0-4 mollL benserazide was 1.75%. Analytical application. To study the reliability and suitability, the proposed method was applied to the determination of benserazide in Madopar tablets. The results are given in Table 1. As can be seen from Table I, the benserazide concentrations measured using our new method was in close agreement with the label claims.

Micelle Enhanced Fluorimetric Determination of Benserazide

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392

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Sample 1

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2

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CONCLUSION A simple fluorimetric method has been developed for the determination of benserazide. The anionic surfactant of SDS showed a strong sensitizing effect for the fluorescence of benserazide in a pH 5.0 buffer. The proposed method can be applied to the assay of benserazide in real sample with good results.

REFERENCES 1.

Wolters EC. Deep brain stimulation and continuous dopaminergic stimulation in advanced Parkinson's disease. Parkinsonism Relat D 2007;13:S18-3. 2. Fanali S, Pucci V, Sabbioni C, Raggi MA. Quality control of benserazide-Ievodopa and carbidopa-Ievodopa tablets by capillary zone electrophoresis. Electrophoresis 2000;21 :2432-7. Treseder SA, Rose S, Summo L, Jenner P. 2003. Commonly used L-amino 3. acid decarboxylase inhibitors block monoamine oxidase activity in the rat. J Neural Transm 2003;110:229-38. 4. Tang Y, Cao L, Qian X, Zhang R. Quantitative analysis of technical samples of benserazide by reversed-phase high-performance liquid chromatography. Sepu 1985;2:56-8. 5. Di P, Yin F, Mao H. Determination of dl-benserazide by the 2.5th order differential anodic stripping voltammetry. Fenxi Huaxue 1992;20:1416-8. 6. Dinc E, Kaya S, Doganay T, Baleanu D. Continuous wavelet and derivative transforms for the simultaneous quantitative analysis and dissolution test of levodopa-benserazide tablets. J Pharmaceut Biomed 2007;44:991-5. 7. Pistonesi M, Centurion ME, Band BSF, Damiani PC, Olivieri AC. Simultaneous determination of levodopa and benserazide by stopped-flow injection analysis and three-way multivariate calibration of kinetic-spectrophotometric data. J Pharmaceut Biomed 2004;36:541-7. 8. He WW, Zhou XW, Lu JQ. Capillary electrophoresis-chemiluminescence detection of levodopa and benserazide in Madopar tablet. Chin Chern Lett 2007;18:91-3. 9. He WW, Zhou XW, Lu JQ. Simultaneous determination of benserazide and levodopa by capillary electrophoresis-chemiluminescence using an improved interface. J Chromatogr A 2006; 1131 :289-2. 10. Wang C, Huang P, Liu Y. Simultaneous determination of levodopa and benserazide in madopar tablets by HPLC-ECD. Zhongguo Yiyuan Yaoxue Zazhi 2003;23:517-9.

IMPROVEMENT IN CARBARYL ASSA Y BY FLUORESCENCE IN A MICELLAR MEDIUM SH LEE,! CW JEON,2 WH KIM,! HY CHUNG,! SM WABAIDUR,! HW PARK,! YS SUH,! MA KHAN! I Department o/Chemistry, Kyungpook National University, Taegu, 702-701, Korea 2Korea Institute o/Geoscience & Mineral Resources, Dafjon, 305-350, Korea e-mail: [email protected]

INTRODUCTION Carbaryl (I-naphthyl methylcarbamate) is a chemical in the carbamate family used chiefly as an insecticide. It is a colorless white crystalline solid. Carbaryl disrupts the nervous system by adding a carbamyl moiety to the active site of the acetylcholinesterase enzyme, which prevents it from interacting with acetylcholine.! It is classified as a likely human carcinogen by the EPA. The pesticide is used indiscriminately, so the toxicity has raised public concern about the ecosystem and human health. Carbaryl is lethal to many non-target insects such as the honeybee. Accumulation of the pesticide occurs in many aquatic organisms such as catfish and algae. 2 Due to public health and ecosystem concerns a number of analytical procedures have been used to determine carbaryl concentrations. Most of analytical methods employed for quantification of pesticides are based on spectrophotometry,3.4 separation by chromatographic techniques such as thin-layer chromatography (TLC),5 gas chromatography (GC)6 and high pressure liquid chromatography (HPLC).7 Chromatographic methods have high sensitivity and accuracy but have disadvantages such as high cost, high volumes oftoxic solvents and complex operation and this limits their application. Fluorescence spectroscopy can be used for determination of carbamate pesticides residues, and the micellar medium fluorescence enhancement method has been exploited in recent years because of its inherent sensitivity. EXPERIMENTAL Apparatus. SPEX Fluorolog-2 spectrofluorometer (Edison, NJ, USA)., 450-W xenon lamp, R 928 photomultiplier tube powered at 950V (Hamamatsu Co.), SPEX DM 3000F spectroscopy computer. A pH meter (Model Orion 520A, USA) was used for pH adjustment. Reagents. Carbaryl (Fluka, USA), Sodium dodecyl sulfate (SDS) (Fluka, USA), Ethyl alcohol (Duksan, Korea), Sodium hydrogen phosphate (Na2HP04) (Merck, Germany), Sodium dihydrogen phosphate (NaH 2P0 4) (Merck, Germany). Basic procedure. Carbaryl standard stock solution was prepared in distilled water through ultrasonication for 6 h at 40 to 50°C. Carbaryl solution (0.1 flmollL - 0.1 mmollL) was diluted with distilled water in a 10 mL calibrated flask containing 2 mL phosphate buffer solution (pH 7), 2 mL ethanol (20%) and 2 mL SDS solution (0.1 mmollL). The fluorescence measurements were performed at "'em = 349 nm and "'ex = 281 nm. 393

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Lee SH et at.

RESULTS AND DISCUSSION Spectral characteristics and selection of surfactant. Increase in the fluorescence intensity of the carbaryl solution was observed using SDS as surfactant at excitation and emission wavelengths of 281 nm and 349 nm respectively (Fig. 1). As can be seen from Fig. 1, Triton X-100 and dodecyl pyridinium chloride (DPC) decreased the fluorescence. This phenomenon clearly indicates that cationic surfactant (DPC) and neutral surfactant (Triton X-100) have negative effects on the fluorescence intensity for carbaryl while anionic surfactant (SDS) enhances the fluorescence intensity of carbaryl. This surfactant-enhanced phenomenon by SDS was used for the spectrofluorimetric determination of carbaryl. Parameter optimization. For maximum fluorescence various parameters, e.g. alcohol, pH and SDS concentration were optimized for the spectrofluorimetric determination of carbaryl. By addition of ethanol an increase in fluorescence was observed. Further it was found that increasing the ethanol percentage up to 20 % increased fluorescence. Beyond 20 % ethanol a decrease in fluorescence was found. Therefore, 20 % ethanol was used in further experiments. Phosphate buffer pH 7 was found to be the optimum buffer. The optimum concentration for SDS was found to be 1.0 x 10-4 mol/L. Above this concentration of SDS, a decrease in response was observed.

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Improvement in Carbaryl Assay by Fluorescence in a Micellar Medium 2.0xlO·

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Fig. 2. Calibration curve for carbaryl obtained by peak height as function of carbaryl concentrations. pH 7.0,20% ethanol, SDS, 1.0 x 10.4 moUL. Calibration curve for carbaryl. The linear calibration curve was investigated using the optimum condition for the spectroflurimetric determination of carbaryl. The results obtained are shown in the Fig. 2. Under the optimum conditions, the fluorescence intensity responds linearly to the CA concentration in the range of 5 x 10. 7 to I.Ox 10.4 moUL with a detection limit of2.3 x 10.3 !!g/mL. Determination of carbaryl in commercial available pesticide. To check the application of the investigated method, carbaryl was determined in commercially available pesticide samples. Carbaryl was determined in Dongbu nac (Dongbuhitek South Korea) and the results are given in Table 1. As can be seen in Table 1, the concentration of carbary I found with the new method were in close agreement with the label claims. The results confirm that the new method is reliable and may be applied for the determination of carbaryl in pesticide samples. Table 1. Analytical results in commercial pesticide sample Sample 1 2 3

Carbaryl present (mollL) 1 1.5 2

Found (moI/L) 1.02 1.56 1.93

CONCLUSION A simple, rapid and highly sensitive fluorimetric method for the determination of carbaryl was developed. The method was based on the formation of the micelle with SDS. Under the optimum conditions, the fluorescence intensity responds linearly to

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the carbaryl concentration in the range of 5 x 10- 7 to 1.0 x 10-4 mollL with a detection 3 limit of 2.3 x 10- Ilg/mL. The method was successfully applied to the determ ination of carbaryl in real samples.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

Caetano J, Machado SAS. Determination of carbaryl in tomato "in natura" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity. Sensor Actuat B-Chem 2008;BI29:40-6. Zhu SH, Wu HL, Xia AL, Han QJ, Zhang Y, Yu RQ. Quantitative analysis of hydrolysis of carbaryl in tap water and river by excitation-emission matrix fluorescence coupled with second-order calibration. Talanta 2008;74:1579-5. Bhaskara BL, Nagaraja P. Highly sensetive reaction for the estimation of carbaryl using 4,4 - diaminodiphenyl sulfone in environmental samples. E-J Chern 2006;3 :250-6. Agrawal 0, Gupta VK, Sub-parts-per-million spectrophotometric determination of phenol and related pesticides using diazotized p-aminoacetophenone. Microchem J 1999;62:147-3. Tang F, Ge S, Yue Y, Hua R, Zhang R. High-performance thin-layer chromatographic determination of carbamate residues in vegetables. J Planar Chromatogr-Mod TLC 2005;18:28-33. Lal A, Tan G, Chai M. Multiresidue analysis of pesticides in fruits and vegetables using solid-phase extraction and gas chromatographic methods. Anal Sci 2008;24:231-6. Mukherjee I, Gupta S, Kulshrestha A, Pant S, Singh A, Kumar A, Gajbhiye V T, Dikshit AK, Kulshrestha Gita. Simultaneous estimation of aldicarb, carbofuran and carbaryl in water by HPLC. Pesticide Res J 2007; 19: 128-30.

STUDY OF THE INTERACTION BETWEEN HUMAN SERUM ALBUMIN AND 7-ETHYL-IO-HYDROXYCAMPTOTHECIN LI GUIZHI, LIU YONGMING Chemistry College of Yantai University, Yantai, 264005, P. R. China E-mail: [email protected]

INTRODUCTION Studies on the interaction between drugs and proteins are important for understanding of the absorption, transport and receptor binding of drugs at the molecular level. Human serum albumins (HSA) are often chosen as an object of study because they are fluorescent due to tryptophan residues, and they easily accept drug molecules as donors. As a result, albumin plays a role in storing and carrying drugs in blood and the formation of the drug-HSA complex quenches the fluorescence ofHSA.1 Camptothecin is one of the bioactive components isolated from the fruits and leaves of Camptotheca acuminata Decne in China. Also camptothecin has anti-cancer effects? 7-Ethyl-10-hydroxycamptothecin (EHC), a camptothecin derivative, has stronger anti-cancer effects and weaker side-effects than camptothecin,3 however, its interaction mechanism with protein is poorly understood. In this paper, we studied the interaction of HSA and EHC. The fluorescence quenching mechanism of HSA by EHC was investigated and the binding constant and the number of binding sites were calculated. The shortest binding distance (r) and energy transfer efficiencies (E) between donor (HSA) and acceptor (EHC) were obtained (Forster's nonradiative energy transfer mechanism). In addition, HI NMR spectral studies provided an understanding of the interaction ofHSA and EHC at the molecular level. EXPERIMENTAL Apparatus. The excitation and emiSSIOn spectra were measured on a Varian Cary-Elips fluorescence spectrophotometer. UV absorption measurements were taken in a Hitachi U-3400 UV-VIS spectrophotometer. The HI NMR spectra were measured on Bruker AV400 NMR spectrophotometer. Reagents. A 1.00 X 10.4 mol!L stock solution of EHC was prepared in alcohol. HSA (Sigma) was directly dissolved in water to prepare a stock solution of 1.00 gIL, and stored at 0-4·C. The buffer solution was adjusted to 7.4 with 0.10 mollL Tris and 0.10 mol!L HCI. AlI reagents were of analytical reagent grade and used without further purification. Doubly distilled water was used throughout. Procedure. Into a 10 mL volumetric calibrated tube, 2 mL ofHSA(100 mglL) and 2 mL of buffer solution were transferred. Then an appropriate amount of EHC stock solution was added and diluted to 10 mL with water. The fluorescence intensity was determined using an excitation wavelength of 225 397

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nm and an emission wavelength of 340 nm (excitation and emission slits, 8 nm in width) in a 1 cm quartz cuvette at 2S"C and 37"C. Into a 10 mL volumetric calibrated tube, 1 mL of EHC (1.00 X 10-4 mollL) and O.S mL of buffer solution were transferred and diluted to 10 mL with water, and then the absorbance determined. Into a NMR tube, appropriated amounts of EHC were transferred and diluted with DMSO-d 6 . The HI NMR spectra were recorded, then was added an appropriate amount of HSA, mixed and reacted for 30 min and 24 h at 2S"C, respectively. The HI NMR spectra were then recorded again. RESULTS AND DISCUSSION Interaction of HSA and EHC. The emission spectra of HSA and HSA in the presence of EHC were obtained in pH=7.4 buffer solutions and are shown in Fig.l (a is at 2S"C and b is at 37"C). As shown in Fig. 1, the emission spectra of HSA at 340 nm (excitation 22S nm) in pH=7.4 buffer displayed a remarkable decrease upon addition of EHC. The result showed that EHC could quench the fluorescence of HSA. The quenching efficiency increased greatly with an increase in the concentrations of EHC, and this suggested that this was to due to an interaction between HSA and EHC. Analysis using the Stern-Volmer relationship6 indicated that the probable quenching mechanism of the fluorescence ofHSA by EHC is a static quenching process.

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Interaction Between Human Serum Albumin and 7-Ethyl-IO-Hydroxycamptothecin

399

The binding constant, number of the binding sites and the binding distance between HSA and EHC. When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the equation: 4 Log [(Fo-F)/F] = IgKo+nlg[Q] Where Ko is the binding constant to a site, and n is the number of binding sites per HSA. For the reactions of EHC and HSA, Ko=6.20X 10 4 Llmol, n = 1.0179 (25°C) and Ko=6.67 x l0 4 Llmol, n = 1.0095 (37°C) were obtained. From the overlapping absorption spectra of EHC and fluorescence spectra of HSA, the shortest binding distance (r) and energy transfer efficiencies (E) between donor (HSA) and acceptor (EHC) were obtained by Forster's nonradiative energy transfer mechanisms as follows, r=2.94nm; E=0.35. Thermodynamic parameters and nature of the binding force. The interaction forces between a drug and a biomolecule may include hydrophobic forces, electrostatic interactions, van der Waals interactions, and hydrogen bonds. From the thermodynamic parameter calculation, the acting force can be identified. 6 For the interaction of HSA and EHC, at 25°C and 37 °C, 6. H=4.68 kJ/mol and 6. S=107 J/mollK. While 6. G = -27.3 kJ/mol (25°C), -28.6 kJ/mol (37°C). It can be seen the positive value of i::.S indicate that the hydrophobic forces played a major role in the reaction. At the same time, hydroxyl and carbonyl groups in the EHC presumably interacted with polar groups of HSA, so the main sort of binding force between them probably includes both hydrophobic and dipole forces. HI NMR spectral of interaction between HSA and EHC. The 'H NMR spectra of EHC and the complex of EHC and HSA in DMSO-d6 were determined. The results indicated that the 10-0H and 20-0H of EHC binds with the amino-acid residues of HSA. The interaction between 10-OH of EHC and HSA was stronger than that for 20-0H of EHC and HSA.

1.

2. 3. 4. 5.

REFERENCES Yang MM, Yang P, Zhang LW. Study on interaction of caffeic acid series medicine and albumin by fluorescence method. Chin Sci Bull 1994;39:31-5. Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase. J Bioi Chern 1985,260:14873-8. Fang YX, Xiong XJ, Lv QJ. The development of synthesis of campothecin derivatives. Chin J Synth Chern 2003,11: 119-23. Feng XZ, Jin RX, et al. Chemical. Studies on the Ions' Effect on the Binding Interaction Between HP and BSA. Chern J Chin. Univ 1996,17:866-9. Chen GZ. In: Fluorescence Analytical Methods, Beijing: Science Press, 1990:122.

400

6.

Li G & Liu Y

Nemethy G, Scheraga HA. The structure of water and hydrophobic bonding in proteins. III. The thermodynamic properties of hydrophobic bonds in proteins. Phys Chern 1962:66:1773-89.

RESONANCE RAYLEIGH SCATTERING METHOD FOR DETERMINATION OF ALGINIC SODIUM DIESTER WITH METHYLENE BLUE LIU YONGMING, LI GUIZHI

Chemistry College ofYantai University, Yantai, 264005, P. R. China E-mail: [email protected] INTRODUCTION Alginic sodium diester (ASD) is made from sodium alginate extracted from seaweed. It is an important medicine in China, and is widely used to cure ischemic cardiovascular and cerebrovascular diseases and hyperlipemia, based on its functions of reducing viscosity, resisting coagulation and improving circulation of blood. Indirect titration and gravimetric methods based on oxidation of ASD to produce sol- have been developed in recent years.! But in the titration method it is difficult to distinguish the end point; and the gravimetric method procedure is complicated. Resonance Rayleigh scattering (abbreviated RRS) is a new analytical technique that has received much attention because of its high sensitivity, simplicity and good selectivity. It has been widely applied to the determination of biomacromolecules such as nucleic acids/ proteins,3 and heparin. It also can be used for the determination of some inorganic ions, organic compounds, and pharmaceuticals. 4 However, there is no report on the determination of ASD with the RRS method. We have investigated the interaction between the cationic dye, methylene blue (MB) and ASD in acid solution. We found that when MB and ASD form an ion-association complex by virtue of electrostatic and hydrophobic interaction forces, the intensity of RRS enhances greatly and a new RRS spectrum appears. The intensity of RRS at 545 nm and the concentration of ASD in the range of 0.5-5 ~g/mL obey a linear relationship. This simple and rapid method for determination of ASD has high sensitivity and good selectivity. The detection limit for ASD is 3 ng/mL. EXPERIMENTAL Apparatus. A Cary-Elipse spectrofluorophotometer (Varian, USA) was used for recording RRS spectra and measuring the RRS intensity. A UV-vis spectrophotometer (Beijing P GENERAL Instruments Co., Ltd, China) was used for recording absorption spectra. Reagents. Alginic sodium diester (ASD) standard solution: 500 ~g/mL. Methylene blue (MB) solution: 1 x 10-3 mollL. All other reagents were of analytical reagent grade and doubly distilled water was used throughout. 401

402

Liu Y & Li G

General procedure. Place I mL of 50 Ilg/mL ASD standard solution into a 10 mL calibrated flask, add 1 mL of NaAc-HCI buffer and 3 mL of lxl0· 3 mollL MB solution, then dilute to the mark with water, mix and set the solution aside for 15 min. Record the RRS spectrum of the ion-association complex with synchronous scanning at Aex=Aem and measure the RRS intensity, I, for the ion-association complex. Analysis of tablets. Following removal of the outside of 10 tablets of ASD they were ground into a powder, then the powdere dissolved in water and filtered. The filtrate was transferred into a I L flask and diluted to I L with water. Then 1.0 mL of this solution was transferred into a 100 mL calibrated flask and dilute to 100 mL to prepare the sample solution.

400 ~--------------------------------------~

300

~ 200

100

250

350

450

550

650

)." Inm Fig. 1. Resonance Rayleigh scattering spectra of MB-ASD system CASD From 1 to 6: 4, 3, 2, 1,0.5,0 (llg/mL ),CMB =3 Xl O' 5 moIlL, pH=2.3

Resonance Rayleigh Scattering Methodfor Alginic Sodium Diester Determination

403

RESULTS AND DISCUSSION Spectral characteristics. Under the experimental conditions, the RRS intensities of ASD or MB, are very weak. When trace amounts of MB coexist with ASD in solution, an ion-association complex can form and an obvious enhancement of RRS intensity for MB-ASD system can be observed. In the MB-ASD system, the RRS peaks of the reaction products for MB and ASD are located at 360 nm and 545 nm. The RRS intensity of the ion-association complex at 545 nm is higher than that at 360 nm and increases with an increase in the concentration of ASD (Fig. 1). Effects of pH and temperature. The effects of pH on RRS intensity were tested 5 at MB concentrations of 3 x 10- mollL and 4 /!g/mL for ASD. The results show that the optimum pH range of the system was pH l.4-3.l. As a result, pH 2.3 NaAc-HCl buffer solution was used as the reaction medium in our experiments. The effects of temperature on the RRS intensities of MB-ASD system at 10, 18, 20, 25 and 30 'c were tested. The results show that the RRS intensities of the system decrease with temperature increase. In our experiment, the temperature was controlled in the range 18-20'C. Effects of MB concentration. When the concentration of MB was low, the RRS intensity of the system was low, due to the incomplete reaction of MB with ASD. With an increase in MB concentration, the RRS intensity was gradually enhanced. However, when the concentration of MB was over a certain range, the RRS intensity also decreased. In our experiment, the optimum concentration of MB was 3 x 10-4 moUL. Stability of I RRs . Under the optimum conditions, the I RRs of MB-ASD system increased gradually during 0-15 min and became stable within 2 h. Relationship between the RRS intensities and the concentration of ASD. Different amounts of ASD were used to form the ion-association complex with MB under the optimum conditions. The calibration graphs were plotted with IRRs against the concentration of ASD. In the ASD concentration range of 0.5-5 /!g/mL, the linear regression equation of the calibration graph was as follows: IRRs =88.I43C AsD (/!g/mL) + 22.26 r= 0.999l. The detection limit was 3 ng/mL (calculated from 3 0 IS). Selectivity of the method. A lxIO- 3 mollLconcentration ofNa+, Ca2+, Mg2+, Mn2+, Zn 2+, NH/, cr, F, N0 3-, C20/, SO/-, glycine, tyrosine, urea, glucose, did not 3 interfere with the determination of ASD. Only 0.5/!g/mL of A1 3+ and Fe + interfered and gave relative errors of the determination of 9% and 17%, respectively. Determination of amounts of ASD in tablets and recovery. Results of the tablet analysis are listed in Table 1. The results compare with that of the improved complex formation titration method for determination of ASD. 4 The recovery for ASD was 10l.3% (n=3); RSD is l.84%. Consequently, the RRS method can be applied for determination ASD in tablets.

404

Liu Y & Li G

REFERENCES 1. 2.

3.

4.

Wang HM, Zhang YM. Determination of alginic sodium diester amounts in tablets by gravimetric method. Strait Pharm 1 2003;15:43. Wu X, Yang lH, Sun SN, et al. Determination of nucleic acids based on the quenching effect on resonance light scattering of the Y(III)-1 ,6-bi(l '-phenyl-3'-methyl-5'-pyrazolone-4'-)hexanedione system. Luminescence 2006;21 :129-34. Wu X, Sun SN, Guo CY, Yang lH, Sun CX, Zhou CR, Wu T. Resonance light scattering technique for the determination of proteins with Congo red and Triton X-I00 . Luminescence 2006;21:56-61. Chen S, Liu SP, Luo HQ. Resonance Rayleigh scattering spectra of ethyl violet 2. Anionic surfactant systems and their analytical application. Chin 1 Anal Chern 2004;32:19-23.

EFFECTS OF METAL IONS ON PEROXYNITRITE NITRIFYING PROTEIN YUNJING LUO: SHUANG CUI, LONG ZHANG, RUGANG ZHONG College 0/ Life Science and Bioengineering, Beijing University o/Technology, Beijing, 100022, China, [email protected]

INTRODUCTION Peroxynitrite (ONOO -) can induce the nitration and oxidation of amino acid residues in proteins in vivo, leading to serious biological consequences. Nitrated tyrosine (3-nitrotyrosine, 3-NT) is related to various diseases including Parkinson's disease, stroke and cardiovascular disorders. l Oxidized fibrinogen (Fg) has been shown to inhibit thrombus formation, 2 and platelet aggregation. 3 Many metal ions participate in oxidation and nitration of protein induced by ONOO-. Some small molecular metal complexes, such as FeEDTA and CUS04, have been shown to catalyze the nitration of a wide range of phenolic compounds. 4 Previous workS 6 showed that cobalt complexes enhanced the yield of nitrated phenolics. Alissa reported that copper, zinc, and selenium were associated with the risk of atherosclerosis. We have screened the effects of several common metal ions on Fg and bovine serum albumin (BSA) nitration, via the yield of 3-NT by use of a spectroscopic assay. Then at physiological conditions in vitro, we further analyzed their abilities to facilitate protein nitration by 3D fluoresence spectra. The study will assist in elucidating the roles of metal ions on peroxynitrite nitrifying protein. MATERIAL AND METHODS Peroxynitrite was prepared as previous described, and was stored at -20 'C. 12 Its concentration was determined spectrally in 0.1 moUL NaOH (f:302 nm = 1670 L'morl'cm- I ) prior to use. 7 CoCI 2 '6H 20, MgCI 2, MnCIz, CaCIz, ZnS04, CUS04, NiCIz, Fg and BSA were obtained from Sigma-Aldrich corporation. Other reagents were of analytical purity and were used without further purification. Damage assays. ONOO- was added as a sequential addition with an interval of 30 s to stirred Fg (2 mg/mL) or BSA (5 mg/mL) solutions in 0.1 moUL pH 7.4 potassium phosphate buffer at 3TC in the absence or presence of metal ions, such as Cu(IT) and Co (IT). The yields of 3-NT for tyrosine residues were measured at 428 nm with U-30 10 UV- Vis spectrophotometer (Hitachi, Japan) and calculated from f: = 4400 L·mor l ·cm- I . 8 3D-Fluorescence spectroscopy. Protein solutions (BSA/Fg) were mixed with 0.3 mM ONOO- and a series of CUS04 and Co(Ach solutions with different concentrations, preparing for 3D fluorescence experiments. The fluorescence intensities were recorded with a F-4500 fluorescence spectrophotometer (Hitachi, Japan). Results are reported for the mean of at least three separate experiments. 405

406

Luo Y et al.

RESULTS AND DISCUSSION Selection of metal ions. We examined the effects of metal ions (MgCh, MnCI 2, CaCI 2, ZnS04, CUS04, CoCh, and N iCh) on peroxynitrite damaging tyrosine through comparing the yields of 3-NT in the presence and absence of metal ions. The yields of nitrated products showed different results with metal ions: 3-NT enhanced from 0.03 mM (control) to 0.05 mM for 0.15 mM CUS04, but decreased to 0.016 mM for 0.15 mM Mn (Il). Other ions were without major effect (Fig.1A).

~r-< o

~

0.06

0.14

~ r-< 0.12

0.05

o

3

3 0.04

0.1 :;: O. 08

~ 0.03 ~ 0.02

"" eO.06

~ 0.04

J, O. 01

J, O. 02

'-'

'-'

Fig. 1. Change of 3-NTyr(Fg/BSA) with different metal ions Co(II) and Cu(II) had positive effects on nitration (Fig. 1B). The concentration of 3-NT with CO(AC)2 increased from 0.077 mM to 0.124 mM, Cu(II) also contributed to the enhancement of 3-NT. Mn (II) or Ni (II) reduced the nitrification of tyrosine, and yields of 3-NT reduced to 0.055 mM and 0.025 mM, respectively. Ca(II) and Zn(II) did not show obvious effects on the output of 3-NT. 10000

7000

2000

Fig. 2. 3D Fluorescence ofFg reaction with ONOO-

Effects of Metal Ions on Peroxynitrite Nitrifying Protein

407

The changes of 3D fluorescence spectra reflect the conformation modification of 9 protein. Two obvious peaks could be observed in peroxynitrite modified Fg (Fig. 2A), which were peak I (230 nm, 344 nm) and peak 2 (280 nm, 342 nm). In the presence of 20 f!M CuCI 2 (Fig. 2B), the intensities of the two peaks were attenuated, peak 1 decreased 18.9%, and peak 2 decreased 32.1 %. Copper ion promoted Fg nitration more at higher concentrations, in the presence of 80 f!M copper ion (Fig. 2C), the fluorescence intensity for peak 1 decreased 34.1 %, peak 2 reduced 63.3%. Similarly, Co(TI) ion also can promote BSA nitration, major peak (280 nm, 340 nm) reduced from 9686 to 110 I, in the presence of 160 f!M Co(TI) (data not shown).

CONCLUSIONS In this study, UV-Vis and 3D fluorescence spectra were used to investigate the catalysis and inhibition of metallic ions on the production of 3-NT and protein in vitro. Our results showed that Co(TI) and Cu(TI) enhance the production of 3-NT in proteins, but Mn(TI) and Ni(TI) inhibits its production. 3D-fluorescence showed that Co(TI) could promote Fg nitration in a concentration dependent manner, while Cu(TI) facilitated BSA nitration. Consequently, essential trace metal element for biological systems, such as copper and cobalt salts, have a synergistic action with peroxynitrite on protein nitration. ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (20605002) and Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality. REFERENCES 1. Peluffo G, Radi R. Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc Res 2007;75:291-302. 2. Stief TW, Kurz J, Doss MO, Fareed 1. Singlet oxygen inactivates fibrinogen, factor V, factor VIII, factor X, and platelet aggregation of human blood. Thromb Res 2000;97:473-80. 3. Belisario MA, Di Domenico C, Pelagalli A, Della MR, Staiano N. Metal-ion catalyzed oxidation affects fibrinogen activity on platelet aggregation and adhesion. Biochimie 1997;79:449-55. 4. Ramezanian MS, Padmaja S, Koppenol WHo Nitration and hydroxylation of phenolic compounds by peroxynitrite. Chern Res Toxicol 1996;9:232-40. 5. Zhang W, Luo Y, Wang Y, Zhong R. Influence of metallic ions on tyrosine modification by peroxynitrite. Chinese J Inorg Chern. 2006; 22: 1113-7. 6. Eman M, Alissa, Suhad M, Bahjria, Waqar H. Ahmeda. Trace element status in Saudi patients with established atherosclerosis. J Trace Elem Med BioI 2006;20: 105-14.

408

7. 8.

9.

Luo Yet al.

Uppu RM, Pryor WA. Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite and hydrogen peroxide. Anal Biochem 1996;236:242-9. Van der Vliet, Eiserich JP, O'Neill A, Halliwell B. Cross C E. Tyrosine modification by reactive. nitrogen species: a closer look. Arch Biochem Biophys 1995 ;319:341-9. Lu J, Jin F, Sun T, Zhou X. Multi-spectroscopic study on interaction of bovine serum albumin with lomefloxacin-copper(II) complex. IntI J BioI Macromol 2007;40:297-304.

MECHANISM AND PROPERTIES OF BIO-PHOTON EMISSION AND ABSORPTION OF PROTEIN MOLECULES IN LIVING SYSTEMS PANG XIAO-FENG Institute of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, P.R. China and International Centre for Materials Physics, Chinese Academy ofSciences, Shenyang 110015, PR China Many experiments show that bio-tissues in human beings, animals and plants can emit bio-photons with different frequencies, which can be thought to be a basic 7

features of bio- tissues. 1- However, the mechanism of bio-photon emission is not clear up to now. We believe that bio-photon emissions are caused by energy levelstransitions of excitons excited in protein molecules after absorbing bio-energy (0.43eV) released from hydrolysis of adenosine triphosphate (ATP) molecules. The excitons are primarily vibrational quanta of stretch and contraction of the

c=o bond

of amino acid residues. Such an excitation results necessarily in the deformation of amino acid residues. The excitation and the deformation balance each other to make the excitons form a soliton by the nonlinear interaction between the excitons and deformation of amino acids through a self-trapping mechanism. 4 The soliton propagates along the protein molecular chain, collective excitation of the protein molecules occurs and a great number of bio-photons are emitted from the protein molecules in virtue of the transition of energy level of the excitons. According to this mechanism of excitation of excitons and bio-energy transport in protein molecules, we can gain insight into the properties of bio-photon emission through calculation of the energy-spectra of protein molecules by the dynamic equation in the theory of bio-energy transport established on the basis of protein molecular structure. The Hamiltonian and wave function of the a-helix protein molecules with three channels in the improved bio-energy transport model proposed by Pang 4

Davydov model are represented as: 409

8 12 -

based on the

410

Pang X-F

H = 2:Co(B;aB"" +1/2) na

JI (B;, Bn+la +BnuB;+IJ+ I[~a 12M +f 12(unu - un_IJl na

na

I

+ {[XI (un+la - un_la)B;,Bnfl +X2(B;" Bn+lfl +BnuB:+lfl )(un+lu -unJ +Lnu (B", B~u+1 + B~u Bna +I )} (1) nfl

and

I

I[l 2

+ CPna (t)B: +~ CPna(t)B:jll 0) ex X exp{-~ [qna (t)Pan -nna (t)u na ]} 10) ph na 'na na

1(2

2

(2)

Where

:8 ~ (:8 n)

is boson creation (annihilation) operator of quantum (exciton)

with energy fa =0,205eV at site n(C=O stretch mode or amide-I),

Un

and Pn are the

molecular displacement and momentum operators for the amino acid residue at site n, M and

13

are molecular mass and force constant of molecular chains,

respectively, J=7,8cm,1 is the intersite transfer energy produced by dipole-dipole interactions. The nonlinear coupling constant Xl arises from modulation of the one site-energy by the molecular displacement, X 2 is another coupled constant which represents the modulations of resonant (or dipole -dipole) interaction energy of excitons caused by the molecular displacement, A is a normalization constant, 10>ph and 10>ex are the vacuum states of phonon and exciton, respectively, L interaction energy among three chains, the subscript three channels,

a

a

is

=1,2,3 denote number of

In Pang's model, the soliton formed from the exciton is thermally

stable, has a sufficiently long lifetime of 120-300 ps at 300 oK, Thus it is very useful in the biological processes at 300 oK, From Eqs. (1 )-(2) and Schr6dinger equation:

IfI I >= ilia I
Mechanism and Properties ofBio-Photon Emission and Absorption of Protein Molecules

we can find

here

15-22

CPna (t) is the wave function of state of exciton at nth site and a th chain, IS

f 0

411

= I1wo (11 = 1)

nonlinear

interaction

constant,

is the energy of exciton, s=v/vo, v is velocity of exciton, Vo is the

sound velocity in amino acid lattice. The effective Hamiltonian for the excitons corresponding to Eq. (3) can be represented b/- 12

In the system the particle number is denoted by,

N

=

2: na ICPna 12

In second

quantum representation the above effective Hamiltonian operator and number operator are as follows

here

N=

2: CQ.; Q./ + 1 / 2), i

t;t;

and ~i are creation and annihilation operators

of the exciton. The common eigenfunction of the Hamiltonian and particle number operators is now taken as I \jJ m>=Cdm,1>+C 2 Im,2>+---+C d (m)lm, d(m», From the eigen function equation

He 11.fJ m >= Em 11.fJ m >

we can obtain

(6)

412

Pang X-F

(7)

where

em = Col .(Cl, C

2,

"',

Cd(m)), Hm is a d(m) X d(m), real, symmetrical

matrix with the diagonal elements. Applying the above formulae, we calculate numerically the quantum energy levels of

a

-helix protein molecules by using

generally accepted experimental data of physical parameters, which are'-" W=(39~ 58.5) N/m; M=(3.51 ~ 5.73) X 10-25 kg is mass of amino acid,

XI =(56 ~

o

62)PN,X2 = (10 -15)PN, J=7.8 em-I, ro=4.5 A, L,=14.63 em-I, L2=12.45 cm-',

G2 =49.73 em-I for Pang's model. The calculated results of quantum vibrational energy levels from the Davydov's model 4 and Pang's model 7-'2 together with the experimental data are presented in Table 1. We see from the Table 1 that the energy-spectrum of the protein is quite complicated, but its distribution has many particularities: (1) The vibrational energy spectra consist of a series of manifolds or energy-bands, i.e., there are several energy-levels corresponding each vibrational quantum-number m.

For example, the first excitation state (m=l) (from

1610-1678cm-') contains 8, there are 44 and 164 in m=2 (from 3179-3358cm-'), respectively. Hence, as m increases, the energy discrepancy, 6. E, between energy-levels decreases gradually. For instance, the discrepancy, 6. E, changes from 6cm-' to 23cm-' at m=l, but 6. E is 0-14cm-' at m=2 , respectively. We can suppose that there is individual energy-bands at large m. (2) The vibrational spectra have strong local mode pattern, or the discrepancy between the energy-levels depends strongly on nonlinear interaction, Y, as we see from Eqs. (12) and (14). This is due to the fact that

Y

is much greater than J,

fl,

f2

and

f3.

(3) The local-mode

degeneracies of energy levels appear at higher-lying vibrational states which begins to occur at m?=2, e.g., degeneracy at 3242cm-' and 3259cm-' at m=2, etc.). Therefore, the degeneracy increases with increasing m. We can see from the energy-spectra shown in Table 1 that the protein molecules can absorb or radiate the

Mechanism and Properties of Bio-Photon Emission and Absorption of Protein Molecules

413

Table 1. Vibrational energy-spectra of protein with three channels in cm- 1

M

exp

cal

M

I

1610.42

I

1612.01

I

1627.64

I

1630.11

I

exp

cal

I

1650

1653.81

I

1666

1667.65

I

1678.73

2

3150

3179.40

2

3203.19

2

3205

3204.71

2

3211.85

3212.95

2

3213.21

3216

3216.84

2

3218.19

3242.48

2

3242.45

2 2 2 2

3250

1662

1661.98

3249.68

2

3258.78

2

3259.87

2

3261.77

2

3260.95

2

3262.97

2

3263.67

2

3269.43

2

3278.89

2

2

3282.84

2

3283.97

2

3285.44

2

3286.49

2

3287.44

2

3290.49

2

3298.96

2

3300.09

2

3301.15

2

3302.13

2 2

3279

3267

3267.39 3277.71

3280

3280.21

2

3309.47

2

3310.21

2

3312.91

2

3313.37

2

3321.54

2

3322.49

2

3323.56 3329.16

2 2

3327.96 3333.91

2

*Where "exp" is experimental values, cal is calculated values in Pang's theory . bio-photons with a wavelength of 5-7

~m

and < 3

~m,

which are infrared emissions.

Very clearly, the bio-photon emissions of proteins are caused by the transitions of energy levels of the quanta or excitons after absorbing the bio-energy released from

414

Pang X-F

ATP hydrolysis.

ACKNOWLEDGEMENTS The authors would like to acknowledge the National "973" project of China for financial support (grant No:2007CB9361 03).

REFERENCES 1. Popp FA, Li KH, Gu Q, eds. Recent advances in biophoton research and its application. Singapore:Worid Scientific, 1992:47-154. 2. Gu Q, Popp FA. Biophoton physics: Potential measure of organizational order. In: Ernst G, Jung M, Holick F, eds. Biological effects of light. Berlin:Walter de Gruyter, 1994:425-44. 3. Pang X-F. The theory of non-linear quantum mechanics, Chongqing: Chinese Chongqing Press, 1994, 567-634. 4. Davydov AS. Space-periodical excitations in nonlinear systems. In: Solitons in molecular systems. DordrechtReideel, 2nd edn, 1991: 132-87. 5. Pang X-F. Phys Rev E Improvement of the Davydov theory of bioenergy transport in protein molecular systems Phys Rev E 2000;62:6989-98. 6. Pang X-F. The lifetime of the soliton in the improved Davydov model at the biological temperature 300 K for protein molecules Eur Phys J B 2001 ;19:297-316. 7. Pang X-F, Feng V-Po Quantum mechanics III nonlinear systems. Singapore:Worid Scientific 2005,:471-576. 8. Pang X-F Soliton physics. Chengdu: Sichuan Technology Press, 2003:673-723. 9. Pang X-F, Yu J-F, Luo V-H. Influences of quantum and disorder Effects on soliton excited in protein in improved model. Commun Theor Phys 2005;43:367-76. 10. Pang X-F. Vibrational energy-spectra of protein molecules and non-thermally biological effect of infrared light. J Int Infr Mill Waves 2001 ;22:291-306. 11. Pang X-F, Zhang HW, Luo YH. The influence of the heat bath and structural disorder in protein molecules on soliton transported bio-energy in an improved model J Phys Cond Mat 2006;18:613-27. 12. Pang X-F. Quantum vibrational energy spectra of molecular chains in crystalline acetanilide. J Phys Chern Solids 2001 ;62:793-6.

THE MECHANISM OF PHOTON EMISSION OF BIO- TISSUES AND ITS PROPERTIES

PANG XIAO-FENGY CAO XIAN-YU 1 1Institute

of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, PR.China and 2Internationai Centre for Materials Physics, Chinese Academy ofSciences, Shenyang I 10016, China Bio-photon emission from tissues in human beings, animals and plants is a general biological phenomenon, every bio-tissue can radiate bio-photons with a certain strength and frequency.I-6 It can provide an insight into the state of activity in living systems. However, the mechanism of bio-photon emission is not clear. It is known that bio-photon emission is a result of energy transition of electrons or atoms, therefore, it is necessary to relate this to various biological activities, such as, the changes of structures and conformations and states of bio-tissues, including cells and bio-macromolecules. We think that the bio-photon emission arises from the growth and development of biological tissues after absorbing the energy, material and information from the environment through bio-organs. In these processes the states of bio-molecules, atoms and bonded electrons in these bio-tissues are changed and these changes and transitions of states result in the emission of bio-photons. In these biological processes, the interactions of light-carried information plays an important role in the conversion and transfer of bio-energy and biomaterial as well as the processing of bio-information. We know from experiments that 90% of the bio-information needed for living organisms comes from this process. Although this process is complicated we can study only the interaction of the absorbed photons with the small biomolecules and their final variations of state resulting from the interactions In general, the small biomolecules vibrate or move around their equilibrium positions, we here refer to them as "localizors". Hence living systems are composed of a large number of localizors, but the latter consist also of many atoms and electrons including bonded electrons that can interact with the light. Then the states of the localizors will be changed and can transit to other quantum states. In the meantime, the bio-entropy of the systems will also be changed. We can calculate the change of bio-entropy of the living systems by 415

416

Pang X-F & Cao X-Y

using quantum statistical-theory of the non-equilibrium state. 9- 13 In such a case, the changes of number and states of particles arising from the energy, material and information absorbed from the environments can be represented by: 3-6

where n i is numbers of localizer with the energy E i ' mv is number of photon with the energy E v ,the n i and mvare the functions related to time, I denotes the type of bound. The gl in Eq. (1) is a function related to time and represents the conversion of material, energy and information, and the features of non-equilibrium state of the living system, thus it embodies the open characteristics of living systems due to the interaction of the system with the incoming photons, energy and material. In the combined system, the numbers of microscopic states of this great system can be represented by:

where N is totality of the localizors, energy-level

Ei

'

W, is degeneration numbers of the

Wv is the degeneration numbers of energy-level E v. We can

determine the probability distribution of the microscopic states by the Lagrange

~ a, g, I = 0 where al is Lagrange uncertainty factor. Thus we ~rther obtain: uncertain factor method of

Ln(m. + W. -\)/ m.)+

<5 ( LnQ +

:? a, ( ag, / am. - ! (ag, / amv )) ~

0

(3)

The entropy of the whole system is S = KBLnQ . The change of total biological entropy resulting from the transitions for the localizors from ith

Mechanism of Photon Emission of Bio-Tissues and Its Properties

417

energy-level to jth energy-level, when the living system absorbs the photon

hv = EO j

-

EO, ,

is denoted as in such a case as:

(4) From Eq. (4) we see that if the number of particles (photons or localizors) in the non-equilibrium system is increased, then the entropy of this system will increase. Otherwise, when the localizors transit to other quantum states through radiation energy or absorbing energy, the total entropy of this living system will change. When this system is in steady state, namely, n localizors in the ith level with low energy all transit to the jth level with high energy after absorbing n photons, i.e.,

13.n = -n'13.n. = n' 13.mv = -n . then change of entropy of J I

system is:

13.S

=

-nKs (Ln (n/V, / n,W] ((mv + Wv -1)/ mv

Due to the fact that (( mv + W v-1)/ mv»

)+ (n] - n, )/ n]n,)

(5)

1 in Eq. (12), then the change of

entropy of the living system is negative, when n)n, and (n] / W]) > (n, / W, ), i.e., the numbers of localizors with higher energy is more than in the lower energy-levels or the numbers of localizors in each microscopic state in the higher energy-levels is more than in the lower energy-levels. This shows that the bio-entropy of the living system decreases, if the energy, material and information acquired from the environments are not to increase kinetic energy of thermal motion of the localizors, but to make these localizors transit to higher energy-levels from the lower energy levels, or to make the particle numbers with higher energy be more than one in the lower energy levels which forms a reversed distribution of particle numbers. This represents a degree of self-organization, it has the features of high energy and low entropy, and is formed naturally in the living system after absorbing energy, material and information from the environment. Substituting now Eq. (3) into Eq. (5), we obtain:

418

Pang X-F & Cao X-Y

This shows that the change of bio-entropy is negative, only if an appropriate energy, material and information are absorbed from the environments, e.g., the condition:

a a a dad a d a [ - - - + - - - ( - ) + - ( - ) - - ( - ) ] g ,>0 anj ani amv dt a· dt a· dt a . .

~

~

~

is satisfied. Thus the bio-self- organization can be spontaneously formed. 7 or enhanced, and it is also a kind of dissipation structure. 7 because the above condition of n quanta in ith state converting to n quanta in the jth state after absorbing n photons is just the condition of form of dissipation structure formed in a non-equilibrium state. Meanwhile, the bio-self-organization has the features of higher energy and lower entropy. From Eq. (6) we know that if Ll S <0, then it is necessary to have: (7)

The boundary function, g" represents the changes of energy, material and information incoming into the living systems through the bio-boundary, hence the first equation in Eq. (7) shows that the energy, material, and information needed for changing the states of one localizor in the higher energy-levels are more than one in the lower energy-levels. This means that changing the states of localizors with the higher energy are more difficult than one in the lower energy-levels. Therefore the localizor in excitation states with the higher energy are more stable than ones in lower energy states. Second equation in Eq. (7) represents the speed of change of localizors in the lower energy-levels in unit time, and is larger than one in the higher energy states, i.e., the numbers of localizors transited from the lower energy states to the higher energy states in the unit time are more than one for the reverse transitions. In such a case, the number of localizors in the higher energy states is always more than in the lower

Mechanism of Photon Emission of Bio-Tissues and Its Properties 419

energy states. Thus, the localizors exhibit a reversed distribution in living systems. The distributions of energy are consistent with the experimental data of Popp et al. I -2 From this distribution curve we know that bio-organisms can spontaneously emit bio-photons because the localizors in the higher energy states could always transit to the lower state. We can also find out the distribution function of localizors in accordance with the energy levels in the living things. In fact, since the number of localizors is very large (n i ) ) 1) in living things, then we may neglect the small term, lInj in Eq. (3) then we obtain from Eqs. (3)

ni /Wi where

= /; =

exp(B(ni ,iZi ,EJ -1),

d

.

B(n;,npE) = ~ G, / an; --(ag, / an)). ~ dt

This is just the distribution function of the localizors in accordance with the energy levels in the living systems. From Eq. (7) we know that the numbers of localizors occupying each energy level depends on the numbers and its speed of change as well as the energy of the localizors. However, when B(n;,

n;, E; )

constant, i.e., the bio-energy, material and information including in the bound function gl in Eq.(1) are linear functions of variables t and n. Thus the above distribution function is represented by /;

=

exp(1- E' - 1') -1)

=

constant

where: 1'="1;001 L,i '

This shows that the probability of localizors occupying each energy state are the same and independent on the excitation energy in the living systems. This is the same as the experimental result of Popp et al l -2 obtained from measuring the bio-photon emission in the same conditions as shown in Eq. (7). This shows that the above distribution function of localizors is correct for living system and from this study we know that the intensity of bio-photon emission is related closely to the degree of bio-self-organization.

420

Pang X-F & eao X-y

ACKNOWLEDGEMENT The authors would like to acknowledge the National "973"project of China: for financial support (grant No: 2007CB936100).

REFERENCES 1.. Popp F.A, Li. K.H, Gu Q. Recent advances in biophoton research and its application. Singapore: World Scientific. 1992:47-154 2.

Trzebiotowska BJ, Kochel B, Slawinke J, Strek W. Photon emission from biological systems. Singapore: World Scientific. 1987:98-187

3.

Pang X-F. Molecular theory of bio-photon emission of living systems.

Chin J

4.

Pang X-F. Dynamical theory for spontaneous emission of bio-photons in living

Atom Mol Phys 1995; 11 :411-26. systems.

Proc 12th IUPABP, Amsterdam. 1996:234-7.

5. Pang X-F. A statistical theory ofbio-photon emission of the living systems. Proc. 19th IUPAP-LCSP, Xiamen. 1995:220-5. 6.

Pang X-F. The theory of non-linear quantum mechanics. Chongqing:Chinese Chongqing Press. 1994:567-634.

7.

Nicolis G, Prigogine 1. Self-organization in nonequilibrium systems. New York:Wiley.1977:9-76.

SYNTHESIS OF A NOVEL FLUORESCENCE PROBE OF 6-CD AND CUPROUS IODIDE PYRIDINE AND ITS APPLICATION HE QIAO,1' RUlFENG DONG,' DUXIN LI,' CHUAN DONG,' SHAOMIN SHUANG" I Environmental Science and Engineering Research Cente r, Shanxi University, Taiyuan 030006, PR China; 2 Shanxi Medical University, Taiyuan 030001, PR China; [email protected]

INTRODUCTION f3-CD consists of seven D-glucopyranoside units, which are linked by a-I, 4-glycosidic linkages. The shape of CD can be represented as a truncated cone. Its interior is hydrophobic and the exterior is hydrophilic (hydroxyl groups), which results in CD's ability to include different compounds. The primary hydroxyl groups are on one side and secondary hydroxyl groups are the other side of the molecule. The I, 4 secondary groups of C-2 and C-3 are located at the wider open end of the torus and point outwards. The primary hydroxyl groups of C-7 are located at the smaller open end. A guest molecule is located or partly located into the main body of the CD.l.2 CD inclusion compounds are formed for the modification of physical or chemical properties of the guest molecule, so f3-CD is widely used in the field of food, environment protection, biology and medicine. 3 . 5 The solubility of hydrophobic drugs in water can be increased by inclusion in CD. A CD can be used for both increasing or decreasing the availability of an active substance and protecting it from degradation. 6 The cuprous iodide-pyridine complex is a novel fluorescence probe but it is not stable in the air. We have synthesized the inclusion complex of f3-CD and cuprous iodide-pyridine. The structure of the inclusion compound was confirmed by IR and lH NMR. Its fluorescence performance and the relationship between structure and fluorescence were investigated. The results indicated that the inclusion complex was soluble and exhibited steady fluorescence in air. This novel fluorescence probe has not been reported previously. As the fluorescence of the complex can be quenched by methane, it was a candidate probe for detection of methane. EXPERIMENTAL SECTION Apparatus. Fluorescence measurements were performed with Cary Eclipse Mode spectrofluorometer (Varian, Australia). The FT-IR measurements were performed with a Perkin Elmer l700FT-IR (Perkin Elmer, USA). All pH values were measured with a pHS-2 acidometer (The 2nd Instrument Factory of Shanghai, China). lH NMR spectra were recorded in D20 on a Bruker - DKX - 300MHz spectrometer (Switzerland). 421

422

Qiao Jet al.

RESULTS AND DISCUSSION We synthesized the inclusion complex of f3-CD and cuprous iodide-pyridine. The structure of the compounds was confirmed by JR, ! H NMR, and fluorescence spectra (Fig.!). It was found that inclusion comp lex of f3-CD and cuprous iodide-pyridine can emit intense yellow fluorescence.

,..

(+)

0

I Cul-

0

+

.-

CuI

N I

Cul-

CuI

a N

N

0

+

N

N

1 Cul-

d

_

~ N

Cul-

Fig. 1. Inclusion complex of f3-CD and cuprous iodide-pyridine.

Curr .. n t

D .. t a

>'",,,,,,metD"''' bb10~O~

N ...... '"

£:X"'.O

6'" 1.

",ROCNO

_ "'"c"e"";'''9

p.~"'''''''' :32,,"6&

:aD'-I'J.,l300l;;i ...

.~::g It' h

,

F

7

Fig. 2.

'"

. o

,

ppon

IH NMR spectra of f3-CD and fJ-CD and cuprous iodide-pyridine.

Synthesis of a Novel Fluorescence Probe of (3-CD and Cuprous Iodide Pyridine

423

To study the structure of the inclusion complex, IH NMR experiment was performed (Fig. 2). According to the shift of the IH (below), it was shown that an inclusion complex of f3-CD and cuprous iodide pyridine was formed. Fig. 2 illustrates the alteration of the fluorescence spectrum of the inclusion complex of f3-CD and cuprous iodide-pyridine upon addition of different concentrations of methane. The fluorescence maximum excitation and emission wavelengths of the inclusion complex of f3-CD and cuprous iodide-pyridine were at 282 nm and 365 nm, respectively. Addition of different concentrations of methane caused a noticeable decrease in fluorescence intensity. The maximum emission wavelength produced a small red shift from 365 nm to 367 nm and the corresponding excitation wavelength was slightly red shifted from 282 nm to 286 nm. The marked fluorescence quenching and the bathochromic displacement proved that there was interaction between the methane and this inclusion complex.

260

280

300

320

340

360

380

400

420

440

460

v.eveiength Inm

Fig. 3.

Fluorescence spectral changes of inclusion complex f3-CD and cuprous iodide-pyridine bubbling in various concentrations of methane.

ACKNOWLEDGEMENTS We appreciated the support of the key Funding of the National Natural Science of China (No: 50534100) and Shanxi Province graduate student innovative plan (No: 07010700). REFERENCES 1. Wenz G. Cyclodextrins as building blocks for supramolecular structures and functional units. Angew Chern Int Ed 1994;33:803-22. 2. Harada A. Cylodextrin based molecular machines. Acc Chern Res 2001;34: 456-64. 3. Liu Y, Li L, Zhang H-Y, Zhao Y-L, Wu X .. Bis(pseudopolyrotaxane) s possessing copper ions formed by different polymer chains and bis(~-cyclodextrin)s bridged with 2,2' -bipyridine-4,4' -dicarboxyether. J

424

4. 5.

6.

Qiao Jet al.

MacromoI2002;35:9934-8. Ma shikun, Wang Jinlin, Li Aixiu, et al. Synthesis and crystal structure of P-CD and paradioxy benzene. J.Chinese Science Bulletin 2000;45:1383-1386. Liu Y, Zhao YL, Zhang HY, et al. Polymeric rotaxane constructed from the inclusion complex of p-cyclodextrin and 4,4-dipyridine by coordination with nickel ions. Angew Chern Int Ed 2003;42:3260-3. Song L, Meng QJ, You X. Cyclodextrin and inclusion compounds. J Inorg Chern 1997;13:368-74.

PHOSPHORESCENCE PROPERTIES OF 2-BROMOQUINOLINE-3BORONIC ACID IN SODIUM DEOXYCHOLATE AND ITS POTENTIAL APPLICA TION IN RECOGNITION OF CARBOHYDRATES QJ SHEN,I WS ZOU,I WJ JIN,1,2· Y WANG 1• 1School a/Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P.R. China 2College a/Chemistry, Beijing Normal University, Beijing 100875, P.R.China *Email: [email protected]

INTRODUCTION Recently, a considerable effort has been devoted towards aromatic boronic acids because of their remarkable luminescence properties which can be altered when they bind to carbohydrates. I,2 The signal measured in such research was usually fluorescence rather than phosphorescence, because the latter is easily quenched by oxygen. However, phosphorimetry has its own advantages, such as large Stokes shift, high signal-to-noise ratio and long lifetime, which make these signals easy to separate from background signal and thus become a potential tool for analysis of biological samples. Here we report a new approach using 3-bromoquinoline as a phosphorescent sensing molecule for the detection of carbohydrates in sodium deoxycholate (NaDC) solution. NaDC can form aggregates in aqueous solution and provide a rigid hydrophobic environment that can protect a phosphor from quenching by external molecules such as oxygen. Although 5-quinolineboronic acid and 8quinolineboronic acid have been reported to be used as fluorescent agents for the detection of carbohydrates3,4 there is still little development of phosphorescencebased sensors for carbohydrates. MATERIALS AND METHODS Materials and apparatus. The 2-bromoquinoline-3-boronic acid (BrQBA) (97%), D-glucose (99%) and D-mannose (99%) were purchased from Alfa Aesar. NaDC (+99%), D-fructose (99%) and D-galactose (99%) are products of Acros Organics. A Cary Eclipse luminescence spectrometer (Varian Company) was employed to obtain room temperature phosphorescence (RTP) spectra and measure lifetime. Intensity and lifetime measurement of BrQBA. IOO!J.L BrQBA ethanol stock solution of 5.0 mmoIlL and an appropriate volume of NaDC solution were transferred into a 10 mL comparison tube, and mixed and finally diluted to 10 mL. The excitation and emission slits were both set at 20 nm. The delay time and gate time were set at 0.10 and 5.0 ms for intensity measurement, and both at 0.1 ms for lifetime measurement. 425

426

Shen QJ et al.

RESUL TS AND DISCUSSION Room temperature phosphorescence (RTP) spectrum and lifetime of BrQBA in NaDC. BrQBA gave a weak fluorescence and no phosphorescence in aqueous solution because of the quenching by dissolved oxygen. However, a strong RTP signal was observed in a NaDC solution without any deoxygenation, with emission at 500 nm. ~~--------------------~ '0;;

~

~

0 ~asured value - - Fitted decay curve

~

~

0·~0---::1;0~~20~~3~0~~40~~~5'0 Lifetime/ms

10

20

30

40

50

Lifetime/ms

Fig. 1. RTP decay curve (left) and analysis of residual (right), [BrQBA] =5.0xI0- 5 mollL, [NaDC] =4.0 mmollL. This is because NaDC in aqueous solution formed micelle complexes that provide a near rigid and hydrophobic structure,5 that are favorable not only for the stabilization of the triplet states of BrQBA, but also preventing from the quenching of oxygen. As 6 shown in Fig. I, phosphorescence decay showed as diexponential decay model, with a long lifetime of 6.99 ms and a short lifetime of 1.39 ms. The fractional contribution of long-lived component was> 73.5%, suggesting that the majority of BrQBA molecules were well protected by NaDC aggregates. Effect of NaDC concentration on RTP intensity. The RTP intensities of BrQBA increased at first upon increase of NaDC concentration and reached a maximum at 4.0 mmollL. Then, RTP intensity fell sharply to zero when the NaDC concentration was greater than 6.0 mmollL. This result suggests that it is essential to maintain the NaDC concentration so as to obtain optimal RTP emission. The reason is that NaDC in aqueous solution exists as a variety of clusters with different sizes and is prone to form a dimer in the lower concentration range, 5,7,8 which can tightly capture individual phosphors as a "sandwiched" structure that isolates the phosphor from quenchers such as molecular oxygen. RTP change with incubation time. When the NaDC concentration was fixed at 4.0 mmollL, the RTP intensity of BrQBA increased with incubation time, accompanying by an increase in the fractional contribution of the long-lived species up to 95%. This is because the equilibrium between BrQBA and NaDC is reached slowly at room temperature. However, we found that heating accelerated this process and also produced the stronger RTP intensity.

Phosphorescence Properties of2-Bromoquinoline-3-Boronic Acid 427 1.7r-T"<"""7=ct'""o.,-se--------, 1.6 • D-galactose 1.5 • D-g1ucose e • D-marmose 1":4

1.3

0.4 Wavelengthfnm

Fig. 2. RTP excitation and emission spectra of BrQBA (5.0xlO- 5 moliL) upon addition of D-fructose. ([NaDC] =4.0mmoIlL)

0.6

0.8

1.0

[Carbohydrate]/rrnmllL

Fig. 3. Changes of RTP intensity of BrQBA (5.0xlO- 5 mollL) in 4.0mmollL NaDC solution in the presence of carbohydrates.

Recognition of carbohydrates based on binding induced RTP enhancement. Fig. 2 shows the RTP spectra of 5.0xlO-5 mol/L BrQBA in 4.0 mmollL NaDC solution upon adding different concentrations of D-Fructose. It can be seen that the RTP of BrQBA was enhanced gradually with increasing [D-Fructose], suggesting that the new BrQBA-fructose complex is formed that is favorable for RTP emission. Other carbohydrates of interest were also investigated and were found to enhance the BrQBA RTP emission_ Fig. 3 shows the RTP enhancement CIllo) as a function of the concentration of carbohydrate (fitted by the equation below): 9

1= (lo+hmKIC])1 (1+KIC]) Where 1 is the RTP intensity for a particular concentration of carbohydrate; 10 is the initial intensity without carbohydrate while hm is the limiting intensity; K is the stability constant of the receptor with guest; [C] is the concentration of carbohydrate. The stability constants of BrQBA-carbohydrate complex were 3 obtained as 2.6x 10 3 mollL for fructose, 1.8x 10 3 mollL for galactose, 1.6x 10 mollL 3 for glucose and 1.3xl0 mollL for mannose, respectively.

CONCLUSION The BrQBA emits strong phosphorescence in NaDC solution without deoxygenation. This phenomenon will have potential applications in recognition of biological important carbohydrates. ACKNOWLEDGEMENT Authors thank the support by the NFSC for this project (No. 20675009).

428

Shen QJ et at.

REFERENCES 1.

2.

3. 4.

5.

6. 7.

8.

9.

Yoon J, Czarnik A. Fluorescent chemosensors of carbohydrates. A means of chemically communicating the binding of polyols in water based on chelationenhanced quenching. J Am Chern Soc 1992;114:5874-5. Van J, Fang H, Wang B. Boronolectins and fluorescent boronolectins: An examination of the detailed chemistry issues important for the design. Med Res Rev 2005;25:490-520. Yang W, Lin L, Wang B. A new type of boronic acid fluorescent reporter compound for sugar recognition. Tetrahedron Lett 2005;46:7981-4. Yang W, Yan J, Springsteen G, Deeter S, Wang B. A novel type of fluorescent boronic acid that shows large fluorescence intensity changes upon binding with a carbohydrate in aqueous solution at physiological pH. Bioorg Med Chern Lett 2003;113:1019-22. Li GR, Wu ]J, lin WJ, Xie JW. Anti-oxygen-quenching room temperature phosphorescence stabilized by deoxycholate aggregate. Talanta 2003;60:55562. Lakowicz JR. Principles of Fluorescence Spectroscopy, 3rd Ed. New York: Springer, 2006:141-2. Zhang HM, Wang Y, Jin WJ. Study on the kinetic properties of phosphor in deoxycholate aggregates by phosphorescent quenching methodology. J Photochem Photobiol B 2007;88:36-42. Li G, MeG own L. A new approach to polydispersity studies of sodium taurocholate and sodium taurodeoxycholate aggregates using dynamic fluorescence anisotropy. J Phys Chern 1993;97:6745-52. Valeur B, Pouget J, Bourson J, Kaschke M, Erusting N. Tuning of photoinduced energy transfer in a bichromophoric coumain supermolecule by a cation binding. J Phys Chern 1992;96:6545-9.

STUDY ON THE INTERACTION BETWEEN METHYL BLUE AND HSA IN THE PRESENCE OF ~-CDIHP-~-CD BY MOLECULAR SPECTROSCOPY SHENG MEl SONGY XIAOLI HOU/ SHAOMIN SHUANG,1 CHUAN DONG 1JResearch Center 0/ Environmental Science and Engineering, Shanxi University, Taiyuan, 030006, P.R China 2Department a/Chemistry, Shangqiu Normal University, Shangqiu, 476000, P.R China, E-mail: [email protected]

INTRODUCTION Studies of supramolecular interactions of organic dyes and biological molecules are significant to the understanding of the structure and function of biological macromolecules' which can be used to simulate certain biophysical processes. In this paper, a fluorescence method has been used to study the interactions between methyl blue (MB) and human serum albumin (HSA) in the presence or absence of CDs. MATERIALS AND METHODS All the fluorescence measurements were carried out on an F-4S00 spectrofluorometer (Hitachi, Japan). TU-1901 dual-beam UV-vis spectrophotometer (Beijing Purkinje General Instrument Co. Ltd) was used for scanning the absorption spectrum. All pH measurements were made with a pHS-3C digital pH-meter (Shanghai REX Instrument Factory, China). 1x 10-4 mo 1/L HSA, 1 x 10-3 moUL MB and j3-CDI hydroxypropyl- {J -cyciodextrin (HP-j3-CD) (DS=9.0) stock standard solutions were diluted with Britton-Robison buffer solution when used. The water used in experiments was doubly distilled water and all other reagents were of analytical purity. RESULTS AND DISCUSSION The interaction between HSA and MB. The fluorescence quenching spectra of HSA with the addition concentrations of MB was shown in Fig. 1. The fluorescence intensity of HSA was decreased regularly with the increasing of MB concentration, and the emission wavelength significantly red shifted. It was inferred from Fig. 1 that a complex was formed between MB and HSA which was responsible for the fluorescence quenching of HSA. The UV-vis spectra of MB with different concentration ofHSA confirmed the formation of this complex. 429

430 Song Set al. 1800 lGOO

1400 1200 1000 IL.

800 GOO

400 200 0

280

38~ (run) 430

330

480

Fig. 1. Fluorescence quenching of HSA by MB, Ae,=283 nm, pH=4.1. (MB]: (l) 0; (2) 0.25; (3) 5; (4) 0.75; (5) 1.25; (6)1.5; (7) 1.75; (8) 2.25 x lO-'mollL Usually the fluorescence quenching of proteins by small molecules is divided into static and dynamic quenching. The quenching constant decreases with increasing temperature in static quenching, but it is the inverse in dynamic quenching. 2 The possible quenching mechanisms were inferred from the Stern-Volmer equation 3 (1) of fluorescence quenching of HSA by MB at 290 oK, 300 oK and 310 oK. Results indicated that the Stern-Volmer curve was linear and the slope decreased with the increasing of temperature. This showed that the quenching mechanism of HSA by MB was static quenching. Log[(Fo

F)/F]= 10gK+ nlog(Q]

(1)

The linear regression equation was made according to log [(Fo-F)/F]-log[Q], and the linear equation: y=1.l28x+5.411(r=0.9988) was obtained. The binding site nand binding constant K of MB and HSA was obtained from the linear equations (n=1.128:::::1, K=2.58 x 10'moIlL). The interaction of MB with fJ-CDIHP-fJ-CD. The effect of pH on the interactions ofMB and CDs was investigated at pH=4.1, 7.0 and 9.2. Fluorescence method was used to determine the inclusion ratio and binding constant of MB with ~-CDIHP-~ CD, according to: 4 --=

F-Fo

J (Kk[P]o[CD]o)

1

+--

kQ[Pl o

(2)

The results showed that the lI(F-Fo) vs. lI[CD]o double reciprocal plots of MB and CDs were in good linear relationships at different pH and 1:1 inclusions were formed between MB and ~-CDIHP-~-CD. The inclusion constants of MB-CDs in

Interaction Between Methyl Blue and HSA in the Presence of f3-CDIHP-f3-CD

431

different pH values are shown in Table 1. It can be seen that the inclusion constants of MB-CDs in neutral and alkaline media were larger than in acidic media. HP-f)CD had better inclusion ability than f)-CD. This can be explained based on the fact that HP-f)-CD has reduced the fluorescence quenching of HSA by MB in water because it expanded the cavity and enhanced the hydrophobicity, and so HP-f)-CD provided a better protective environment. Table 1. Binding constants between MB and CDs at different pH values Cyclodextrins

pH 4.1

pH 7.0

pH 9.2

f)-CD

3286

8000

9000

HP-f)-CD

6000

13500

14650

The interaction of MB with HSA in the presence of CDs. Fluorescence spectra of MB-CDs in the presence of different concentrations of HSA were scanned and the results showed that the emission wavelength of MB-f)-CD inclusion complex was at 443 nm and blue shifted with increasing concentration ofHSA, and at the same time the emission intensity increased, which showed that a complex was formed through the reaction of the three compounds. Benesi-Hildebrand variable equation was used to calculate: 4

[Go]

-=

DF

1 1 +KkQ[HSA]" kQ

(3)

Table 2. Binding constants of MB combining to HSA in the presence of CDs linear regression equation Cyclodextrins n=1

Binding constant (Limo))

n=2

y=7x I 0-9 X +7x 10- 3

y=1 x IO-"x +7 x 10-"

(r=0.9954)

(r =0.9887)

y=7 x 10-'x +6x 10- 2

y= 1 x IO-"x + 1.3 x 10-5

(r =0.9979)

(r=0.9712)

1 xl 0"

f)-CD

8.6xI0"

HP-f)-CD

432

Song S et al.

The linear regression curve was obtained by 11 6F vs 1I[HSA]O and n for different values. The best linear n value was used in the equation, and K can be obtained from the intercept divided by the slope. When n=l, the linear regression equation was better than n=2, and this showed that MB-j3-CDIHP-j3-CD-HSA was a complex of 1: 1: 1. The binding constant could be obtained by substituting n values into the formula (3). The results are shown in Table 2. Experimental results showed that the existence of j3-CDIHP-j3-CD did not change the interaction trend of MB with HSA, and the addition of j3-CD made the interaction of MB and HSA more facile. Similarly HP-j3-CD further promoted their reaction. Both CDs can increase the binding constant of MB and HSA.

CONCLUSIONS The interaction between MB and HSA in the presence or absence of j3-CD)IHP-j3CD were studied using UV -vis and fluorescence methods. The results showed that the fluorescence of MB can be enhanced by CDs because MB can enter the hydrophobic cavity of CDs and the radiant transition from S, to So was protected. Meanwhile, CDs can strengthen the binding between MB and HSA by changing the microenvironment of MB and forming a MB-CDs-HSA ternary supra-molecular system. The inclusion ratio and inclusion constants of MB-HSA, MB-j3-CD/HP-j3CD and MB-j3-CDIHP-I3-CD-HSA were calculated. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (No. 20575037). All the authors express their deep thanks. REFERENCES 1. Li Y, He WY, Liu HX, Yao XJ, Hu ZD. Daidzein interaction with human serum albumin studied using optical spectroscopy and molecular modeling methods. J Mol Struct 2007;831: 144-50. 2. Iwata K, Takashima T, Kawabata K. Japan, Kokai Kokkyo Koho 7962, 019(CI.C09Dl1l16).1979;5-18. 3. Lakowicz JR, Principle of Fluorescence Spectroscopy, New York:Plenum Press, 1986. 4. Catena GC, Bright FV. Thermodynamic study on the effects of p-cyclodextrin inclusion with anilinonaphthalenesulfonates. Anal Chern 1989;61 :905-9.

STUDY OF INTERACTION OF KAEMPFEROL WITH HUMAN SERUM ALBUMIN BY SPECTROSCOPY AND MOLECULAR MODELLING JIANNIAO TIAN,t JIAQIN LIU, 3 ZHIDE HU,2 XINGGUO CHEN 2 ICollege of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China, 2Department of Chemistry, Lanzhou University, Lanzhou 730000, China, 3 Mianyang Teacher's College, Mianyang 621000, China

INTRODUCTION Albumin is the most abundant protein in mammalian systems, and plays an important role in the transport and deposition of endogenous and exogenous substances in blood such as hydrophobic organic anions of medium size, long-chain fatty acids, haem and bilirubin.'" Molecular interactions are often monitored using optical techniques because they are sensitive and relatively easy to use. With HSA, many compounds have been used successfully as probes, e.g., drugs. 3 However, the binding of the components of natural plant medicine to proteins has seldom been investigated.' The flavonoids are a large group of polyphenolic natural products that are widely distributed in higher plants. Such compounds are increasingly being recognized as possessing a broad spectrum of biological activities and important therapeutic applications.' The microenvironments of the binding sites of such flavonoid molecules with the living cell and proteins are expected to be complex and are an unexplored area until now. Previously, we studied the interaction of flavonoids 6 ,7 with serum albumin, and the results indicated that these natural drugs can interact with protein (main binding site is the site II of the serum albumin). In this paper, the interaction of kaempferol (4H-l-benzopyran-4-one), which is the active component of the Chinese traditional herbal medicine Kaempferia galanga L. and Diphylleia sinensis Ii, with HSA was studied at pH 7.40 by spectroscopic methods including circular dichroism (CD), fluorescence spectroscopy, and Fourier transform infrared spectroscopy (FT-IR) spectra. MATERIALS AND METHODS Human serum albumin (HSA, essentially fatty acid-free) was obtained from Sino-American Biotechnology Company and used without further purification. Kaempferol (analytical grade) was obtained from the National Institute for Control of Pharmaceutical and Products, China. O.S moIlL NaCI solution was used to keep the ion strength at 0.1. Tris-HCl buffer was selected to keep the pH 5 of the solution at 7.40. HSA solution of l.Sxl0· moIlL was prepared in pH 7.40 3 Tris-HCl buffer solution. Kaempferol (1.0xl0· moIlL) solution was obtained by dissolving it in SO mL ethanol. All other chemicals were of analytical reagent Fluorescence spectra were recorded using a RF-S301 PC grade. spectrofluorophotometer (Shimadzu) with a ISO W Xenon lamp and a 1 cm 433

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quartz cell. The excitation and emission bandwidths were both 5 nm. Circular diachronic (CD) measurements were made on a Jasco-20c automatic recording spectropolarimeter (Japan), using a 2 mm cell at 296K. FI-IR measurements were carried out at room temperature on a Nicolet Nexus 670 FT-IR spectrometer (America) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter. 8 Binding parameters and thermodynamic parameters. 3 mL solution containing 1.5 x 10-6 mollL HSA was titrated by successive additions of kaempferol solution and the fluorescence intensity was measured (excitation at 280 nm). The binding parameters have been calculated using the Scatchard's procedure. '0. Quenching data were also analyzed according to the Stern-Volmer equation. " The thermodynamic parameters were calculated from the Van't Hoff equation. Molecular modeling study. The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes Ih9z). The potential of the 3D structures of HSA was assigned according to the Amber 4.0 force field with Kollman-all-atom charges. The initial structures of all the molecules were generated by molecular modelling software Sybyl 6.9. AutoDock3.05 program was used to calculate the interaction modes between the drug and HSA. All calculations were performed on Silicon Graphics Ocatane2 workstation. RESULTS AND DISCUSSION Spectral studies. The conformational changes of HSA were evaluated by the measurement of intrinsic fluorescence intensity of protein before and after addition of drug. Fluorescence measurements give information about the molecular environment in a vicinity of the chromophore molecules. When a molar excess of kaempferol was titrated into a fixed concentration of HSA, a remarkable fluorescence decreasing of HSA was observed. To confirm that kaempferol-HSA interactions lead to unfolding of the protein, CD and FT-IR method was used to analyze the interactions. The CD spectra of HSA were characterized by the presence of two minima at 208 and 217 nm, characteristic of a+p structure of proteins 12 The intensity of these bands was slightly decreased by adding kaempferol to the protein solution, indicating that the binding of kaempferol to HSA perturbed the secondary structure and induces a slight decrease in the helix structure content of the protein. The FT-IR spectrum of free HSA in Tris-HCl buffer and the difference spectra after binding with kaempferol were also studied. The peak position of amide I moved from 1645 to 1652.7 cm-Iand amide II moved from 1550.5 to 1542.8 cm- I in HSA infrared spectrum after interaction with kaempferol, furthermore, a new peak appears I at 1743.4 cm- , which indicate that the secondary structure of HSA has been changed because of the interaction ofkaempferol with HSA. Therefore, the change of amide I and amide II indicated that kaempferol can interact with the group of C=O and C-N-H, that is, polypeptide chain of HSA and the hydrogen bond may be formed between the kaempferol and HSA. Binding parameters. Using the fluorescence decrease the association constants K

Interaction of Kaempferol with Human Serum Albumin by Spectroscopy

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for the of kaempferol with HSA at different temperatures were calculated in Table 1. The binding constants decreased with the increasing rthpTn"m·p. the binding parameter is consistent with the molecular modelling study of the interaction between HSA and kaempferol.

°

Table 1 show the values of liHo and liS obtained for the binding site from the slopes and ordinates at the origin of the fitted lines and lists the corresponding value of for 296K. From Table I, the negative sign for !lOo means that the binding process is spontaneous. For drug-protein interaction, positive entropy is frequently taken as evidence for hydrophobic interaction, but it has been pointed out that may also be a manifestation of electrostatic interaction. 13 the main source of !lOo value is derived from a large contribution of term with little contribution from the liHo factor, so the main interaction is hydrophobic contact, but the electrostatic interaction can not be excluded.

parameters for

296

1.79

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Interaction mode between kaempferol and HAS.

As shown in I, kaempferol is situated within subdomain IIA in Sudlow's site I formed six helices. The benzopyrone moiety is located within the binding

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and the A- and C- rings are practically coplanar. The interaction between kaempferol and HSA is not exclusively hydrophobic in nature since there are several ionic and polar residues in the proximity of the bound ligand playing important role in stabilizing the negatively charged kaempferol molecule via H-bonds and electrostatic interactions.There are hydrogen interactions between 4'-OH, 7-0H, 3-0H, 5-0H of the kaempferol and the residues Arg-222, Leu-260, His-242 and Arg-257 of HSA, respectively. The hydrogen bonds were formed by four hydroxyl groups of kaempferol donates hydrogen to the amino acids residues of HSA. Site I is large enough to accommodate the kaempferol molecule. The calculated binding free energy is -33.3 kJ/mol and the corresponding binding constant is 6.944 x l0 5 , which are approximately consistent with our experimental data.

REFERENCES 1. 2 3

4

5 6

7

8

9 10 11 12

13

Kragh-hansen U. Molecular aspect of ligand binding to serum albumin. Pharmacol Rev 1981;33:17-53. Peter TJ. All about albumin. Biochemistry, genetics, and medical applications. San Diego:Academic Press, 1996. Urien S, Nguyen P, Berlioz S, Vacherot FB, Tillement IP. Characterization of discrete classes of binding sites of human serum albumin by application of thermodynamic principles. Biochem I 1994;302:69-72. Tian IN, Liu IQ, Zhang IY, Hu ZD, Chen XG. Fluorescence studies on the interactions of Barbaloin with bovine serum albumin. Chern Pharm Bull 2003;51 :579-82. Rusznyak S, Gyorgyi AS. Vitamin: flavonols as vitamins. Nature 1936;138,:27. Tian IN, Liu JQ, He WH, Hu ZD, Yao Xl, Chen XG. Probing the binding of Scutellarin to human serum albumin by circular dichroism, fluorescence spectroscopy, FTIR, and molecular modeling method. Biomacromolecules 2004;5: 1956-61. Tian IN, Liu IQ, Xie IP, Yao Xl, Hu ZD, Chen XG. Binding of wogonin to human serum albumin: a common binding site of wogonin in subdomain IIA,I Photochem. Photobiol B 2004;74:39-45. Lu ZX, Cui T, Shi QL. Applications of circular dichroism (CD) and optical rotatory dispersion (ORO) in molecular biology, 151 edn. Science Press, Beijing, 1987. Dong AC, Huang P, Caughey WS. Protein secondary structure in water from second-derivative amide infrared spectra. Biochem 1990;29:3303-6. Scatchard G. The attractions of protein for small molecules and ions. Ann NY Acad Sci 1949;51:660-73. Eftink M. R., Ghiron C. A., Fluorescence quenching studies of proteins, Anal. Biochem.,1981;114,199-227 Dockal M, Carter DC, Ruker F. Conformational transitions of the three recombinant domains of human serum albumin depending on pH. I Bioi Chern 2000,275:3042-50. He XM, Carter DC. Atomic structure and chemistry of human serum albumin. Nature 1992;358:209-15.

SELECTION OF SALT-TOLERANT RICE VARIETY USING LIGHT-INDUCED DELAYED FLUORESCENCE JUNSHENG WANG,I WENHAI XU,I DA XING,2 LINGRUI ZHANG 2 of Information Technology, Dalian Maritime University, Dalian 116026, China; 21nstitute of Laser Life Science, South China Normal University, Guangzhou 510631, China, [email protected]

JCollege

INTRODUCTION Salt (NaCI) stress is a major factor limiting crop production. Real-time detection of the physiological status of plants under salt stress is important for identifYing salt-tolerant species and providing specific guidance for agricultural production. I.2 Delayed fluorescence (DF) is photon emission by a living system after stimulation 3 by visible radiation. DF has many practical applications and it can be used as a sensitive indicator of many stress factors. 4,5 In this paper, a new method and biosensor for selection of salt-tolerant rice varieties based on quantitative measurement of delayed fluorescence (DF) is presented. A set of Oryza sativa L. indica, a variety with known ability for salt-tolerance was used as experimental materials. Changes of DF with different NaCI concentration stress were studied using a custom-built DF detection system and comparison with Pn measured using LI-6400 was completed. Compared with common methods for evaluating rice salt-tolerance, DF technique can quantifY the change of plant photosynthetic physiological status more accurately and faster. MATERIALS AND METHODS Preparation of sample. For every plant (Oryza sativa L.) (Pokkali, Peta) seedlings were divided into three groups (2 seedlings per plant). NaCI concentration of the nutrition solution was 0 (control), 50, 100, 150, 200, 250, 300 and 350 mM in the first group and 0 (control) and 250mM in the second and third group. DF intensity and Pn were measured once every 6 h in 108 h. Each experiment was repeated at least 5 times. Measurement of Pn. Pn was measured directly under CO 2 concentration of 400 ppm and a saturating irradiance of 1000 f.!mol photons m,2 S,I in the leaf chamber using a commercially available system (LI-6400; LI-COR, Inc., USA). DF biosensor system. DF emission (0.26 to 5.26 s after being irradiated) was recorded with a custom-built DF biosensor system described previously. 6 RESULTS AND DISCUSSION Effects of different NaCI concentrations stress on the DF intensity and Pn of rice leaves. Effects of different NaCI concentration stress on the DF intensity and Pn of rice was investigated and the results are shown in Fig.I. 437

438

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Effects ofNaCI stress for 24 h on the DF intensity and Pn leaves of rice and between them. (a) Pokkali; (b) Peta; (c) correlation between DF intensity and Pn.

The results show that lower NaCI concentrations (50 150mM) the DF and Pn of Pokkali and Peta leaves, and at 100mM reached amaximum. At the 150 mM NaCI, the improvement effect began to DF and Pn of Pokkali leaves are higher than the control and those of Peta leaves are lower than the control. Further increase in the NaCI concentration above 150 mM had a negative effect on the DF intensity and Pn of Pokkali and Peta leaves. But it should be noted that the dependence of DF intensity on NaCI is identical to that of the Pn. Further analysis of the results show that there is correlation between DF intensity and Pn of rice leaves at different NaCI concentration and DF intensity can reflect the change of plant photosynthetic status more accurately under different NaCI concentration conditions.

Selection of Salt-Tolerant Rice Variety Using Light-Induced Delayed Fluorescence

439

Effects of NaCI treatment on DF intensity and Pn in rice as a function of time. DF intensity and Pn of Pokkali and Peta leaves measured as a function of hours at 250 mM Nacl concentration treatment for 108 h were further studied and the results are shown in Fig. 2. DF intensity and Pn of two rice plant leaves both declined as treatment time was extended at 250 mM NaCI. The rate of decline of DF intensity and Pn of Peta leaves is faster than those of Pokkali leaves. DF intensity and Pn are 49.9% and 47.9% of contrast of Peta leaves under NaCI stress for 24 h, respectively, however those of Pokkali leaves are 75.7% and 76.2%. The results show that change of plant photosynthetic physiological status under stress due to different NaCI concentrations can be reflected by DF intensity. 120 0-

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Fig. 2. Effect of200 mM NaCI treatment on DF intensity (a) and Pn (b) in rice. CONCLUSIONS Effects of stress due to different NaCI concentrations on the DF intensity and Pn of rice were studied. The results show that there is good correlation between the DF intensity and Pn, and that DF intensity can quantify the change of plant photosynthetic physiological status more accurately and faster under different NaCI concentration conditions. Therefore, it is likely that the DF technique will provide a potentially useful approach for non-invasive and real-time monitoring of the effects of NaCI stress on plant growth and photosynthetic metabolism and a powerful means in the selection of salt-tolerance of plants. ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (Grant No.: 60774056). REFERENCES 1. Lu Z, Liu D, Liu S. Two rice cytosolic ascorbate peroxidases differentially

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improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep 2007;26: 1909-17. 2. Van XL, Tan KZ. Screening rice varieties for salt tolerance in greenhouse. IRRN, 1991 ;16:16-7. 3. Guo ZY, Zhu VB, Ma JF, et al. The spectra distribution properties of ultraweak photon emission from biological system and biophoton coherence. Acta Photon Sin 2000;29:890-3. 4. Wang J, Xing D, Zhang L, Jia L. A new principle photosynthesis capacity biosensor based on quantitative measurement of delayed fluorescence in vivo. Biosens Bioelectron 2007;22:2861-8. 5. Zhang LR, Xing D,. Wang JS. A non-invasive and real-time monitoring of the regulation of photosynthetic metabolism biosensor based on measurement of delayed fluorescence in vivo. Sensors 2007;7: 52-66. 6. Wang JS, Xing D, Xu WHo Rapid and compact optical measurement system for photosynthesis ability using LED excitation. J Optoelectron Laser 2006;17:377-80.

EFFECTS OF LMWOA ON BIODEGRADA nON OF PHENANTHRENE STUDIED BY FLUORIMETRY XY WEI,' LZ SANG,2 YX ZHU/ Y ZHANG l 1State Key Lab 0/ Marine Environmental Science, Environmental Science Research Center; 2Department a/Chemistry, Xiamen University 361005, PR China Email: [email protected]

INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the environment and are potential health hazards because of their carcinogenic, mutagenic, toxic, and genotoxic properties. l Microbial degradation is believed to be one of the principal means of successfully removing PAHs from natural environments? PAHs are refractory to biodegradation and persist in the natural environment because of their hydrophobic nature, resulting in low water solubility and a tendency to be adsorbed to soil and sediment. There are two forms of PAHs, 3 dissolved PAHs and solid PAHs, in aquatic environment. It is accepted that PAHs dissolved in aqueous solution is available to microorganisms. 4 A better understanding of how microbial biodegradation proceeds for low concentrations of PAHs in aqueous solution is of great importance. However, studies on the biodegradation rate and capability of PAHs-degrading bacterial strains have commonly used higher PAH concentrations, due to the lack of a method for determining low concentrations of PAH. This can lead to misunderstanding of the process of PAH decomposition in natural environments. Fluorescence assays can be used for PAHs at low concentration. 3,5 Low molecular weight organic acids (LMWOA) are produced in the environment by various biological and chemical processes and they are very reactive. 6-8 Thus, in the environment, LMWOA may effect biodegradation of PAHs dissolved in aqueous solution. We studied butylic acid, malic acid and citric acid as models of LMWOA with one, two and three carboxyl groups, and their effects on the biodegradation of phenanthrene dissolved in aqueous solution using fluorimetry. MATERIALS AND METHODS Chemicals. Phenanthrene stock solution (Aldrich, purity>99%)(O.lOS g in SO mL dichloromethane) was diluted to prepare working solutions just before use. Mineral salts medium (MSM) comprised - (NH4)2S04, 1000mg; Na2HP04, 800mg; K2HP0 4, 200mg; MgS0 4 '7H 20, 200mg; CaCh'2H 20, 100mg; FeCI 3 'H 20, Smg; CNH4)6M07024'H20,1 mg; 1000ml ofMilli-Q water; pH=7]. 2.682g DL-malic acid CAR, Shantou Xilong Chemical, China), 4.203g citric acid monohydrate CAR, Sinopharm Chemical Reagent, China) and 1.94 mL butylic acid CAR, Shantou Xilong chemical, China) were dissolved in 100 mL MSM solution respectively to 441

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prepare as stock solutions, of which the concentration were all 200 mmol/L. Phenanthrene-degrading microorganisms were from School of Life Science, Xiamen University. Fluorescence was measured using a Cary Ellipse spectrofluorimeter (Varian, USA)( ex and em slits 5.0 nm). UV and visible absorption spectra obtained on an Ultrospec 2100 pro UV-Visible spectrophotometer (Amersham Bioscience) (xenon lamps as light source). 600 249.06,495.33(1

164.00,484.813

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150

300 350 Wavelength (urn)

400

Fig. 1. Excitation (a) and emission (b) spectra of phenanthrene in Milli-Q water

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Fig. 2. Excitation (a) and emission (b) spectra of phenanthrene in MSM solution

Fluorescence method for phenanthrene. Fig. I and 2 show that the excitation and emission fluorescence spectra of phenanthrene in Milli-Q water and MSM solution are similar, maximal excitation and emission peaks at 249.06 and 364.00 nm respectively, and signal of phenanthrene was determined at this wavelength. Serial concentrations of phenanthrene prepared in MSM were used to determine the fluorescence signa!.3 The experimental results shown that in the range of 60 Ilg/L to 1000 IlglL, a good relationship between the concentration (x) and fluorescence signal (y) value of phenanthrene in MSM was obtained (y=1351.6x (r2 =0.9978). The detection limit was 44.9 Ilg phenanthrene/L (B + 3SD), with a relative standard deviation of less than 1.42% Cn = 11). The fluorescence signal blank was measured throughout the experiment; the average value was 1.23 (n = II), which was much lower than the fluorescence signal of phenanthrene. Fluorescence spectra of phenanthrene in LMWOA solution Control experiments showed that malic acid, citric acid and butylic acid had little effect on the spectral characteristics of phenanthrene or its fluorimetric determination. Measurement of phenanthrene biodegradation by fluorescence method. 50.0 mL MSM were added to each incubation flask; the final concentration of phenanthrene was 1000 j.l giL. After sterilization at 121"C for 15 min, the bacterial strain was inoculated into individual incubation flasks and incubated on a rotary shaking incubator at 25 'C and 150 rpm in the dark. The biodegradation rate of phenanthrene was monitored directly by the fluorescence method without solvent extraction. Quantification of LMWOA by UV-VIS method. A series concentration of malic acid, citric acid and butylic acid were prepared in MSM respectively and their absorption signals were determined all at 205 nm 9 to obtained absorption calibration

Effects of LMWOA on Biodegradation of Phenanthrene Studied by Fluorimetry

443

standard curves. All of the standard curves for these three LMWOA have good linear relationship in experiment demanded concentration range. RESULTS AND DISCUSSION Effects of different LMWOA on the degradation of phenanthrene. Biodegradation of phenanthrene in MSM solution with or without malic acid, citric acid and butylic acid (LMWOA 1600 ilmol/L) are shown in Fig. 3. 800

>-

700

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0 It -100~~~~~~~~ 6 8 10 12

time (h)

Fig. 3. Effects of malic acid, citric acid and butylic acid on degradation of phenanthrene (LMWOA concentration 1600 ilmoIlL). From Fig. 3, it can be seen that biodegradation rate of phenanthrene were increased within the presence of both malic acid and citric acid, but decreased within the presence ofbutylic acid. The biodegradation rate of phenanthrene under the effect of citric acid was a little bit higher than that under the effect of malic acid. Effects of different concentrations of malic acid and citric acid on the degradation of phenanthrene. Since malic acid and citric acid can improve the biodegradation rate of phenanthrene, variety of the biodegradation rate with different concentration of LMWOA were investigated to understand how the degradation processes were affected. Relationship between concentration of phenanthrene and biodegradation time were obtained and shown in Fig. 4 and Fig. 5 respectively. It can be found that the biodegradation rate can be expedited with increase of concentration of these two LMWOA. The measurement of LMWOA during the degradation process showed that there is no obvious change in concentration of LMWOA. So, LMWOA had been co-metabolism with phenanthrene cannot be the reason for increased biodegradation rate. The reason for positive effect of malic acid on bio-degradation of phenanthrene and mechanism of biodegradation process is still require further research. CONCLUSIONS Effects of LMWOA on phenanthrene biodegradation using a fluorescence method are reported in this work. It can be concluded that both malic acid and citric acid have positive effect on biodegradation of pheneanthrene. However, butylic acid restrained the biodegradation of phenanthrene. Further research is needed to explain the results in these experiments and discover the mechanism in it.

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Fig. 5. Effects of CItrIC acid on phenanthrene biodegradation (malic acid a, 400 b, 800 c, 1600 f1mollL)

ACKNOWLEDGEMENTS The authors acknowledge financial support by Natural Science Foundation of China (20577037,20777062 and 405210003), and thank Professor Zheng Tian-ling and Dr Tian Yun for providing bacteria strains and suggestions on our experiments. REFERENCES 1. Mastral AM, Callen MS. A review on polycyclic aromatic hydrocarbon (P AH) emissions from energy generation. Environ Sci TechnoI2000;34:3051-7. 2. Yuan SY, Wei SH, Chang BV. Biodegradation of polycyclic aromatic hydrocarbons by a mixed culture. Chemosphere 2000;41 :1463-8. 3. Yong Z. Novel method for determining pyrene biodegradation using synchronous fluorimetry. Chemosphere 2004;55:389-94. 4. Sikkema J, de Bont JAM, Poolman B. Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 1995;9:201-22. 5. Zhang Y. Detection of biodegradation of pyrene by synchronous fluorometry. China Environmental Science 2002; 22:289-92. 6. Huang PM, Schnitzer M. Eds. Interactions of soil minerals with natural organics and microbes. Soil Sci Soc Am Spec Publ 1986;17: 160-21. 7. Stevenson FJ. Humus chemistry - Genesis, composition, reactions. New York: Wiley, 1994. 8. Blank RR, Allen F, Young JA. Extractable anions in soils following wildfire in a sagebrush-grass community. Soil Sci Soc Am J 1994;58:564-70. 9. Lu HL. Low molecular weight organic acids in mangrove wetlands and their impact on heavy metals bioavailability. Xiamen University: Doctoral thesis, 2006.

ALLEVIATION EFFECTS OF SALICYLIC ACID AND LANTHANUM ON ULTRAWEAK BIOLUMINESCENCE IN MAIZE LEAVES UNDER CADMIUM STRESS ZL WEll CZ HAO 1 YN SU 1,2 ZH TIAN 1 I Biology Departmen't, DeZhou Universit;, DeZhou, China, 253023 2Life Science School, HeBei University, HeBei, China, 071002 [email protected]

INTRODUCTION Cadmium (Cd) is a highly toxic heavy metal element, that in plants may cause oxidation damage or even death,' Research indicated salicylic acid (SA) and lanthanum, may adjust plant physiology pathways or change ion channels to reduce Cd absorption and repair damage caused by Cd stress. SA has been reported to induce a number of defense responses to abiotic stress,' and La 3+ can decrease the accumulation of Cd in crops/however, whether the mixture of SA and La3+ could alleviate Cd stress is still unclear, and this is the focus of these studies. In plants, reactive oxygen species (ROS) generation and plant metabolism was considered as two primary factors on ultraweak bioluminescence (UBL)."s At present, UBL is applied in plant anti-stress research, but the ultraweak bioluminescence (UBL) under Cd stress has not been studied. In this report, the alleviating effects of SA and La 3+ on Cd-stress was analyzed by detecting UBL. METHODS Growth conditions and treatment. Seeds of maize (Feng-Liao 008) were surface sterilized, then incubated at 25°C in the dark to germination, with a day/night regime of 16 h / 8 h. At the stage of completely developed first trifoliolate, they were transferred to fresh nutrient solution containing SA (10,20, 40, 80 and 120 mg/L) or LaCI3 (20,40, 60, 80 and 100 mg/L) and CdCIz (0 and 50 mg/L). The treatment period was 48 h. Determination of SOD activity, MDA, protein and ROS. SOD activity was assayed as described by Zhang et al: ROS, MDA and total protein content was analyzed as described by Li et at. 7 Determination of UBL intensity. UBL was determined at 18°C in a darkroom. The BPCL produced by the Institute of Biophysics Chinese Academy of Sciences was used to detect UBL. Delayed luminescence detection was perfomed as discribed previously."· RESULTS Assessment of SOD and MDA. As shown in Fig.l, Cd stress raised the SOD activity and MDA content. SA (20 mg/L) significantly reduced this effect. The

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SOD activity was increased 10.1 %, and MDA increased 24.6%. After 60 mg/L LaCh treatment, SOD activity increased l3.2%, MDA content increased 27.4%. When 20 mg/L SA and 60 mg/L LaCh were used together, SOD activity increased 5.8%, MDA content increased 9%, compared with the individual treatments, hence, the mixture was more effectively under the experimental conditions. 10

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Determination of delayed luminescence. As shown in Fig. 2, when 20 mg/L SA and 60 mg/L LaCl 3 were used alone, the delayed luminescence curves were similar to that of control; when 120 mg/L SA or 100 mg/L LaCh were used alone, the curves were similar to that of Cd treatment, indicating no alleviation of the effect of Cd. When the mixture of 20 mg/L SA and 60 mg/L LaCh was used, the delayed luminescence curves were similar to that of control. The curves also indicated Cd stress can gradually decrease UBL intensity of maize leaves and accelerate the decay rate of the curve. On the other hand, delayed luminescence curve showed there were more notable differences among curves in the first 20 seconds in this experiment. Assessment of ROS and protein compared with UBL. ROS content was significantly higher than control, but protein content did not change significantly, while the photon number was significantly lower than control. As shown in Table 1, the photon number and delayed luminescence were decreased, but ROS content was increased in maize leaves. The determination of protein showed that the different concentrations of SA alleviated Cd stress (48 h), all of protein content were similar to the control, but there was no correlation with the photon number and delayed luminescence values. The results showed that the photon number and delayed luminescence parameter were decreased in maize leaves. To alleviate Cd stress, SA and LaCl 3 were used separately. The results showed that 20 mg/L SA or 60 mg/L LaCh could raise UBL, and the mixture of 20 mg/L SA or 60 mg/L LaCl 3 (SLl) gave the best result. It also indicated the photon number and delayed luminescence parameter had no obvious link with ROS and protein content in maize leaves. It implied that the main source ofUBL under these conditions was metabolism.

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Fig. 2. Delayed luminescence curve. Table 1. Control Cd SI S2 Ll L2 SLl SL2 LSI LS2

Assessment ofROS and Protein Compared with UBL

Photo Counts 6574 5574 6071 4960 6359 5233 6497 5421 5152 5325

ROS (mg/mUm in) 3.56±0.OSa 5.16±0.0Ic 4.62±0.OSb 6.26±0.lld 4.57±0.15 b 5.77±0.07 d 4.43±0. 12b 5.73±0.13 d 5.16±0.32c 6.78±0.05 d

Protein(llg/gFW) 2.66±0.05 a 2.55±0.04a 3.2S±0.OSb 3.32±0.OSb 2.71±0.04 a 3.24±0.05 b 3.1l±0.04 b 2.92±0.04a 3.l2±0.02 b 3.29±0.02 b

Data are the mean ± S.E.M. of two independent experiments, with three replicates for each treatment. treatment. Different letters within columns indicate significant differences (P < 0.05), according to T-test

DISCUSSION The results of this study showed that short term Cd treatment can stimulate both SOD activity and MDA content, and the ROS increased at the same time, indicating that excessive ROS content could aggravate oxidative damage to the cell membrane. Low concentrations of SA and LaCI 3 could alleviate Cd stress, and the combination of 20 mg/L SA and 60 mg/L LaCI 3 gave the best results (better that either

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individually). SA and La have different mechanisms of alleviating Cd stress. Our study did not give the details of mechanisms for SA and La to resistance to Cd stress. This will be the topic of further research. This study also showed that UBL is a sensitive and convenient method to estimate the status of a plant under Cd stress. After 48 h of Cd stress, UBL intensity decreased and ROS content increased, indicating that ROS was not the key factor to affect UBL, while plant metabolism was the key source for UBL. This maybe largely due to: 1. ROS induced cells apoptosis, thus reducing plant metabolism; and 2. ROS interfered with plant metabolic pathways (including DNA and RNA synthesis), thus affecting normal redox reaction, leading to weakening of metabolism.

REFERENCES 1.

2.

3.

4. 5.

6. 7. 8. 9.

Siedlecka A, Krupa Z, Samuelsson G, 6quist G, Gardestrom P. Primary carbon metabolism in Phaseolus vulgaris plants under Cd/Fe interaction. Plant Physiol Biochem 1997;35:951-7. Kang HM, Saltviet M. Chilling tolerance of maize, cucumber and rice seedling leaves and roots are differentially affected by salicylic acid, Physiol Plant 2002;115:571-6. He ZY, Li JC, Zhang HY, Ma M. Different effects of calcium and lanthanum on the expression of phytochelatin synthase gene and cadmium absorption in Lactuca sativa. Plant Sci 2005;168:309-18. Cheng HP, Xue JH, Wang JH. Ultraweak luminescence in organisms. Bulletin of Biology 2002;34:15-7. Bolwell G.P., Blee KA, Butt VS, et al. Recent advances in understanding the origin of the apoplastic oxidative burst in plant cells. Free Radic Res 1999;31: 137-45. Zhang ZL, Qu WQ. Experiments in plant physiology. Beijing: Higher Education Press, 2003. Li JW, Xiao NG, Yu RY. Principles and methods of biochemical experiments. Beijing: Peking University Press,1994. Wei ZL, Su YN, Jiao CZ, Yao CL. Effects of La on Superweak Luminescence of Rose Leaves under Cd Stress. Anhui Agric Sci 2007;32:10213-4. Popp FA, Li KH, Gu Q. Recent advances in biophoton research and its applications. Singapore: World Scientific, 1992: 1-46.

RHODAMINE B-QUINOLINE-8-AMIDE AS A FLUORESCENT "ON" PROBE FOR Fe3+ IN ACETONITRILE Y XIANG, ZF LI, AJ TONG Department o/Chemistry, Tsinghua University, Beijing 100084, PR China Email: tongaj@mail. ts inghua. edu. en

INTRODUCTION The development of fluorescent probes which can selectively sense metal ions has received increasing interests in recent years.! The significant attention attracted in this research field is not only because of the important roles of metal ions in biological, clinical and environmental chemistry, but also due to the advantages of fluorescence technology: simple instrumentation suitable for field analysis, and optical signals observable by the naked eye. Many examples of fluorescent probes exhibiting an excellent response to various metal ions have been reported during the recent decades. Most probes underwent a photo-induced electron transfer (PET),2 intramolecular charge transfer (lCT),3 or fluorescence resonance energy transfer (FRET)4 mechanism upon binding with metal ions. Alternatively, spirolactam amide derivatives of rhodamine dyes are utilized as fluorophores for design of fluorescent probes for metal ions recently.5,6 These probes showed fluorescence enhancement response to metal ions by a very interesting chelating-induced "ring closed" to "ring opened" transformation. Owning to their advantages in high sensitivity of fluorescence enhancement and long wavelength emission (> 500 nm), these "metal ion responsible" fluorescent probes are more and more frequently studied, In this paper, we report a fluorescent "ON" probe (1, rhodamine B-quinoline-8amide) for Fe3+, which was a metal ion involved in many biochemical processes at the cellular level. EXPERIMENTAL Apparatus. Fluorescence spectra were obtained on a Jasco FP-6500 spectrofluorimeter. NMR spectra were recorded using a Joel JNM-ECA300 spectrometer (300MHz), ESI MS spectra were measured on a HP 1100 LC-MS spectrometer, Synthesis of compound 1. Rhodamine B acid chloride was synthesized according to a reported procedure,5 and then reacted with 8-aminoquinoline to yield 1 (Scheme 1): To a 100 mL flask was added 0.288 g (2 mmol) 8-aminoquinoline, 2,5 mL triethylamine and 30 mL acetonitrile. Then, under vigorous stirring, 1.2 g Rhodamine B acid chloride (2.4 mmol) in 30 mL acetonitrile was added dropwise to the flask under an ice bath. The reaction mixture was left to stand at room temperature for another 3 h after the addition. The resulting solid was filtered out 449

450

Xiang Yet ai.

from the mixture and then washed by saturated NaHC0 3 (15 mL x 3) and acetone (15 mL x 5). After drying in air, 1 was obtained as purple solid (0.S31 g, yield 73 %). ESI-MS: m/z = 569.4 ([M+Hf), calculated mlz for M+ 568.3. IH-NMR (CDCI 3), b (ppm): 1.08 (t, 12H), 3.25 (q, SH), 6.09 (s, 2H), 6.23 (d, 2H), 6.74 (d,IH), 6.94 (d, 2H), 7.21 (m, 3H), 7.53 (m, 2H), 7.59 (d, IH), 7.92 (d,lH), 8.01 (m, lH), 8.69 (m, IH). 13 C_NMR (CDCI 3 ) b (ppm): 12.6,44.4,68.5,97.4,106.8, 107.7, 120.8, 123.8, 124.2, 125.7, 128.2, 128.8, 128.9, 130.6, 130.7, 132.2, 132.6, 134.7, 135.5,144.8,148.7,149.6,153.3,153.4,167.8.

Et,N

Et,N

NEt,

Scheme 1. Synthesis of compound 1

RESULTS AND DISCUSSION 3 Spectral characteristics of compound 1 in the absence and presence of Fe +. In acetonitrile, a solution of 1 (10 flM) free of metal ions was completely colourless and non-fluorescent, and nearly no absorbance or fluorescence could be observed in the visible range, suggesting the predomination of its closed ring state. It was also supported by the characteristic peak in the I3C-NMR spectrum of 1 at 68.5 ppm, which indicated the existence of quaternary carbon at the 9-position. 7 However, upon the addition of Fe 3+ (up to 2 equivalents), a dramatic enhancement of both absorbance and fluorescence at 5611594 nm occurred as a result of chelationinduced spirolactam ring opening of 1 by Fe3+.

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Rhodamine B-Quinoline-8-Amide as a Fluorescent "ON" Probefor

in Acetonitrile 451

As illustrated in l(a), the absorbance of 10 /lM 1 in acetonitrile at 561 nm increased gradually with the increasing of Fe3+ concentration, and reached its 3 when Fe + added was more than 2 equivalents. It was very astonishing to find that the first portion (about 0.5 equivalents) of caused only very mild enhancement of absorbance at 561 nm of 1. This phenomenon was probably the cheiating by N atom of quinoline moiety without ring opening of the result of rhodamine spiroiactam amide. Nevertheless, in the presence of additional amounts of , the closed form of 1 transformed to its ring opened state by further , and the absorbance at 561 nm was greatly enhanced. Job's plot interaction with 8 analysis showed that the binding mode of I-Fe 3+ complex in acetonitrile most chelated by probably exhibited a metal-to-Iigand ratio of 2:1, with each rhodamine amide and the quinoline moiety respectively. 1(b) exhibits the fluorescence spectra changes of 10 /lM 1 in acetonitrile upon the addition of various amounts of Fe3+. In accordance with absorption the fluorescence intensity at peak wavelength of 594 nm increased with addition gradually and reached its maximum when Fe3+ added was more than equivalents of 1, with also very little fluorescence change for the first 0.5 of . A fluorescence enhancement of more than 100-fold was observed for when fully chelated by Fe3+. This significant enhancement factor of fluorescence enabled the sensing of Fe3+ in acetonitrile by 1 (10 /lM) with a high 3 Da.'D'.""'.... , in range of 5~20 /lM Fe +. (a)

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2. (a) Fluorescence spectra of 1 (10 /lM) in the presence of different metal ions (20 /lM) in acetonitrile. (b) Fluorescence intensity at 594 nm of 1 (10 /lM) in the presence of different metal ions (20 /lM) without (black bar) or with (gray bar) (20 /lM) in acetonitrile. ""611~~,...,a,, of 1 toward over other metal ions. Fluorescence spectra changes of 1 upon the addition of other metal ions in the absence and presence of were studied to evaluate the specific response of 1 to Fe3+. As depicted in Fig. 2(a), only induced significant enhancement of fluorescence of 1 at 594 11m, and Cu 2+ and caused much milder effect, while other metal ions investigated showed no

452

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response. In addition, when these foreign metal ions were added to a solution of I in the presence of Fe3+ in acetonitrile, the fluorescence of I-Fe 3+ was hardly changed (Fig. 2(b This outstanding selectivity of I to Fe 3+ was probably due to the high charge to radius ratio ofFe 3 + (highest among the metal ions investigated), which was crucial for the affinity of metal ions and I. In conclusion, we describe the fluorescent "ON" behaviour of a new fluorescent probe (I) toward Fe3+ in acetonitrile with high selectivity. The fluorescence of I at 594 nm was enhanced by more than 100-fold when 2 equivalents of Fe3+ was added to a solution of I (10 J.lM) in acetonitrile, and 5.0-20 J.lM Fe 3+ could be sensed by monitoring this fluorescence change with high sensitivity.

».

ACKNOWLEDGEMENT We are very grateful for the financial support from the National Natural Science Foundation of China (NSFC, No. 20675043) and the National High Technology Research and Development Program of China (No. 2006AA09Z171). REFERENCES l. de Silva AP, Gunaratne HQN, Gunnlaugsson T, Huxley A1M, McCoy CP, Rademacher lT, Rice TE. Signaling recognition events with fluorescent sensors and switches. Chern Rev 1997;97:1515-66. 2. Zeng L, Miller EW, Pralle A, Isacoff EY, Chang Cl. A selective turn-on fluorescent sensor for imaging copper in living cells. 1 Am Chern Soc 2006; 128: 10-1. 3. Peng Xl, Du 11, Fan lL, Wang lY, Wu YK, Zhao lZ, Sun SG, Xu T. A selective fluorescent sensor for imaging Cd 2+ in living cells. 1 Am Chern Soc 2007; 129: 1500-l. 4. Coskun A, Akkaya ED. Signal ratio amplification via modulation of resonance energy transfer: proof of principle in an emission ratiometric Hg(II) sensor. 1 Am Chern Soc 2006;128:14474-5. 5. Dujols V, Ford F, Czarnik A W. A long-wavelength fluorescent chemodosimeter selective for CU(Il) ion in water. J Am Chern Soc 1997; 19;7386-7. 6. Yang Y-K, Yook K-J, Tae 1. A rhodamine-based fluorescent and colorimetric chemodosimeter for the rapid detection of Hg2+ ions in aqueous media. 1 Am Chern Soc 2005;127:16760-l. 7. Anthoni U, Christophersen C, Nielsen P, Puschl A, Schaumburg K. Structure of red and orange fluorescein. Structural Chern 1995;3: 161-5. 8. Vosburgh WC, Cooper GR. Complex ions. I. The identification of complex ions in solution by spectrophotometric measurements. 1 Am Chern Soc 1941 ;63:43742.

STUDIES ON DETERMINATION OF DEOXYRIBONUCLEIC ACID BY SECOND ORDER SCATTERING WITH A NOVEL RHODANINE YU JINGHUA, LI 80, ZHU YUANNA, CHENG XIAOLIANG, ZHANG LINA School of Chemistry and Chemical Engineering, University ofJinan, Jinan 250022, Email: [email protected]

INTRODUCTION Quantitative determination of nucleic acids is important in the diagnosis and prevention of disease. Generally, methods for nucleic acids are based on spectrophotometry' and spectrofluorimetry,' but these methods are relatively insensitive and subject to interference. Second order scattering (SOS) is a type of non-linear scattering produced by resonance Rayleigh scattering. In fluorimetry, second order scattering is a common phenomenon and this peak at double the excitation wavelength is regarded as an interference peak. Liu first reported SOS and applied it to the determination of trace selenium (III)' with a high sensitivity. Furthermore, Liu found that SOS can provide useful information on the structure and reactivity of a substance and predicted it could become a new analysis method. However, at present, SOS is seldom applied in the determination of nucleic acids. Rhodanine is an important organic reagent which is commonly used in the determination of noble metals with spectrophotometry.' Recently, many rhodanine derivatives with multifarious active groups have been synthesized and applied in the determination of many metal particles with fluorescence spectrometry.' We synthesized 3-( 4'-methylphenyl)-5-(2'-sulfophenylazo) rhodanine (4MRASP), and used it as a probe for determination of DNA and report the second-order scattering spectrums of interaction between deoxyribonucleic acid (DNA) and 4MRASP in the presence of surface active substance, sodium dodecyl sulfate. A new method for DNA determination with high sensitivity and good selectivity was established. The possible mechanism and nature of the interactions in the presence of sodium dodecyl sulfate was studied using ultraviolet spectroscopy and thermodynamics.

MATERIALS AND METHODS Perkin-Elmer LS55 (Perkin-Elmer, USA) fluorescence spectrophotometer; The Electronic Balance (Mettler Toledo, Shanghai, China); PHS-3C digital pH-meter (Shang Hai Lei Ci Device Works, Shanghai, China). 4 Stock 3-(4'-methylphenyl)-5-(2'-sulfophenylazo) rhodanine (4MRASP, 2.0 x 105 moIlL). The working concentration was 2.0 x 10- mollL by diluting the stock concentration with water. DNA standard solution (100 Jlg/mL), stored at 0-4 DC, the working concentration was 10 Jlg/mL. 2.0 mollL HCI solution; 5 % (m/m) sodium dodecyl sulfate micromulsion (SDS). All chemicals were of analytical reagent grade 453

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or above. Doubly distilled demineralized water is used throughout Procedure. Solutions were added in a calibrated flask in order of 0.025 mL of SDS, 1.5 mL of 2.0 xlO,5mollL 4MRASP, 2.0 mL of 0.5 mol/L HCI solution and appropriate amounts of 10 I1g/mL DNA solution, then diluted to 10 mL with water and mixed well. The SOS spectra of the systems were recorded with scanning at Aex=1I2A.em and the SOS intensity of the reaction product (l) and that of the reagent blank (10) were measured at the maximum SOS wavelength AexlAem: 310 nm/620 nm, M=I-Io.

RESULTS AND DISCUSSION SOS spectrum of different systems. Second order scattering spectrums of different systems were recorded at Aex=31 0 nm in the wavelength range of 600 nm-640 nm according to the experimental method (Fig. 1) .

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620 )Aun

Fig. 1. SOS spectrum of 4MRASP-DNA system 1.4MRASP+SDS;2.4MRASP;3.4MRASP+DNA;4.4MRASP+SDS+DNA(5.0I1g); 5.4MRASP+SDS+DNA(10.0 I1g);6. 4MRASP+SDS+DNA(l5 I1g). It can be seen from Fig. 1 that SOS intensities of 4MRASP+HCI, 4MRASP-SDS+HCI and 4MRASP-DNA+HCI were very weak; SOS intensity of 4MRASP-DNA+HCI was enhanced obviously by the presence of SDS which had a

Studies on Determination of DNA by Second Order Scattering with a Novel Rhodanine

455

positive effect in the determination of DNA. The probable reason was that under the experimental conditions, 4MRASP and DNA existed as cations because of the protonation of amines and carbonyl groups and can not combine easily with each other. 4MRASP, DNA and SDS could form an ion-association complex by electrostatic force, the volume of which enlarged and resulted in the enhancement of SOS intensity. As it can also be seen from Fig. 1 that SOS intensity gradually increased with the addition of DNA, based on the phenomena, a new determination method of trace DNA was established. Optimum conditions. We studied probable conditions of the determination and selected respectively 2.0 mL, 0.025 mL, 1.5 mL as the optimum amounts of HCI, SDS and 4 MRAS P. Relation between SOS intensity and concentration of DNA. When concentration of DNA was in the range of 0.2-1.4 f,lg/mL, SOS intensity was directly proportional to the concentration of DNA. The linear regression equation of the method wasM = 8 -16.87+385.02 P (f,lg/mL), the detection limit was 3.07 x 10- g/mL (3slslope). Selectivity of the method. It is known that the intensity of SOS is affected by the coexistence of other chemicals. The tolerable concentration ratios of foreign species on the determination of 10.0 f,lg of BSA in 10 mL of BSA-4MRASP system are as follow (the relative error is under ±5 %; fold): Cd 2+ (20), Ni2+ (10), Mg2+ (100), C0 2+ (2), Ca 2+ (100), cr (numerous), N0 3- (numerous), SO/- (numerous), D-phenylalanine (50), DL-phenylalanine (l00), L-cysteine hydrochloride (50), DL-a-amino acid (l0), L-leucine (50), L-lysine (60), L- tyrosine (50), L-histidine (50), L-glutamic acid (30), L-methionine (60), DL-lactamine (100), Lphenylalanine (numerous), DL- methionine (70). It can be seen that most of amino acids, common metal ions and acid radical anions do not interfere the determination except Ni 2+, C0 2+, and DL-a-amino acid. It is concluded that the method had good selectivity. Applications. To test the proposed method, we synthesized two samples (f,lg/mL): 1. Ca2+ (20), A1 3 + (10), DL-ethionine (20), L-Iysine (20) and DNA (10); 2. K+ (40), Mg 2+ (20), L-phenylalanine (30); DL-phenylalanine (30) and DNA (10); from which we took 1.0 mL DNA and detected it according to the procedure. The recovery rates of the method is 97.5 % and 109.8 %; RSD was 1.26 and 1.28. The assay results indicated the results were satisfactory, and there were no obvious differences between the results ofUV- Vis and those of this method. CONCLUSION A new method for the determination of DNA with high sensitivity and selectivity was established based on the significant enhancement of SOS intensity of 4MRASP-SDS-DNA ion-associatIOn complexes. These investigations have expanded the applications of rhodanine spectroscopy.

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ACKNOWLEDGMENTS This work is financially supported by Science Research Foundation of Shandong Province, China (Y2007B07), and National eleventh- five year plan, China. REFERENCES 1. Huang CZ, Li KA, Tong S Y. Spectrophotometry of nucleic acids by their of cobalt (II) with effect on the complex 4-[(5-chloropyridyl)azo ]-13-diamino-benzene. Anal Chim Acta 1997;345:235-42. 2. Xucong L, Zenghong X, Liangqia G, et al. Studies on a new fluorescence-enhanced system of nucleic acids-Morin-AI. Spectr Sp A 2004; 10: 1230-4. 3. Shaopu L, Zhongfang L. Spectra of resonant luminescence and double scattering of selenium(IV)-iodide-crystal violet system. Chern J Chin Univ 1996;8:1213-5. 4. Jinghua Y, Qingyu 0, Shiye P, et al. Study on photometric determination of nikel with a new rhodanine reagent. PTCA Part B Chern Anal 2004;2:75-6. 5. Jinghua Y, Qingyu 0, Van L, et al. Study on the synthesis of a new reagent 3-(4' -fluorophenyl)-5-(2' -arsenoxylphenylazo)-rhodanine and the fluoresence determination of bismuth. Spectr Sp A 2004;9:1093-5.

FLUORESCENCE CHARACTERISTICS OF NOVEL CHLOROPHENYL-ARSENOXYLPHENYLAZO RHODANINES AND APPLICATION IN THE DETERMINATION OF THALLIUM (I)

YU JINGHUA, CHENG XIAOLIANG, GE SHENGUANG, TAN YUN, LI BO School of Chemistry and Chemical Engineering, University ofJinan, Jinan 250022, P R China, Email: [email protected] INTRODUCTION Thallium is a highly toxic element.' Methods for its determination include differential pulse anodic stripping voltammetry/ field desorption mass spectrometry, inductively coupled plasma mass spectrometry (ICP-MS)3 and furnace atomic absorption spectrophotometry.4 These methods have drawbacks: spectrophotometry uses basic dyes (e.g., brightgreen,5 methylviolet) as color reagents and has low sensitivity, and aqueous spectrophotometry requires micelles. Flame and flameless atomic absorption spectra measurements need chelating agents to improve detection limits, avoid interference or enrich the concentration of the analyte. Direct analysis by graphite furnace atomic absorption spectrophotometry and ICP-MS can have a high sensitivity. However, the volatility of the metallic elements restricts the use of high charring temperature for thermal treatment in the graphite furnace and as a consequence, matrix interference may appear. Recently, the catalytic kinetic fluorescence analysis (CKFA) method has shown high sensitivity and good selectivity in the determination of trace elements, but has not been used for the determination of Tl. To investigate the possibility of applying the CKFA method in analyzing Tl, a suitable fluorescence reagent has to be found. The well known rhodanine dye is a photometric reagent and an arsenoxyl group has good complexing performance. We first successfully introduced arsenoxyl and active -Cl group into rhodanine. It was found that this new rhodanine reagent has good fluorescence characteristics. MATERIALS AND METHODS All fluorescence measurements were performed with a Perkin-Elmer (USA) Model LS-55 spectrofluorimeter with 1.0 cm quartz cell. The excitation and emission bandwidths were set at 10 nm throughout the experiment. Stock thallium solution (Img/mL) was prepared by dissolving purified metal thallium. Working standard solutions were freshly prepared by appropriate dilution with doubly distilled demineralized water. 3-(4'-chlorophenyl)-5(2'-arsenoxyl phenylazo) rhodanine(4ClRAAP) (2 x lO- 4 mollL) was prepared by dissolving 0.0944 g of the reagent in 1000 mL absolute ethanol. Working solution was freshly prepared by appropriate dilution with doubly distilled demineralized water. Determination of TI by the CKFA method 1.0 mL standard thallium solution

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(0.1 mg/mL) or the sample solution is put into a 10 mL color comparison tube, followed by a sequential addition of 1.0 mL of 4ClRAAP solution (2 x 10-5 moIlL), 1.5 mL of potassium hydrogen phthalate-sodium hydroxide buffer solution (pH=5.2) and 1.0 mL of 0.03% hydrogen peroxide. The solution was then placed in boiling water bath for 20 min. After being quickly cooled down to room temperature by flowing water, it was diluted to the mark with doubly distilled demineralized water. The fluorescent intensity (F t ) of the solution was measured at excitation and emission wavelengths at 309 and 406 nm, respectively. The fluorescent intensity (Fo) of correspondent non-catalyzed solution was also measured. Fluorescence quenching value, M F, the difference between Fo and Fb was then correlated with the TI concentration.

RESULTS AND DISCUSSION Fluorescence spectra. Fluorescence spectra of the 4ClRAAP show that maximal fluorescence quenching occurs at excitation and emission wavelengths of 309 nm and 406 nm, respectively (Fig. 1). Hence, AeJAem=309 nm/406 nm is used in the following tests. In addition, it can also be seen that the addition of H2 0 2 into the 4ClRAAP solution results in a significant increase in the fluorescence intensity, while a further addition ofTlleads to a decrease in the fluorescence intensity.

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Optimization. The optimal pH range was between 5.0 and 5.4. Among the buffer solutions studied (potassium hydrogen phthalate-sodium hydroxide, acetic acid-sodium acetate, disodium hydrogen phosphate- citric acid and tartaric acid-sodium tartrate), potassium hydrogen phthalate-sodium hydroxide was found to be best. The fluorescence intensity increased with increasing the amount of the buffer fluorescence intensity (MF) and the heating time had a linear relationship MF=1.85t+49.33 (regression coefficient r is 0.9866), reaction rate constant k

Fluorescence Characteristics of Novel Chlorophenyl-Arsenoxylphenylazo Rhodanines

459

=0.031 S-I. After 21 min, the fluorescent intensity decreased. 20 min was then set as the optimal heating time. A linear relationship between the fluorescence intensity and thallium(l) concentration was found in the range 0-10 ng/mL (regression equation ~h=-15.74+16.03p (ng/mL) - correlation coefficient was 0.9930). The detection limit, as defined by IUPAC, was 2.64 x 10-9 g/mL. Interference. The criterion for judging interference was fixed at ±5 %. Maximal allowed folds of interfering ions at 10 [lg/L thallium were: N?+(60), Zn2+(200), Cu 2+(80), Zr4 +(200), V5 +(l00), Bi3 \20), Ca2+(l000), Cr 3+(500), Ba2+(l00), Sb3+(40), Au3+(300), Mn2+(lOO), Fe3\80), Cd 2+(l00), Ag \20), Pd 2+(50), La3+(lOO), Sn 2+(50), Pb 2 +(300), C0 2 \50), Ni2+(60); K+, Na+, F, B{, cr, sot, N0 3-, NH/ in a large excess did not interfere. Therefore, this method has good selectivity. Different wines and waters, in which known amounts of Tl were added, were analyzed and good recovery was obtained. solution up to 1.3 mL. Further increase the amount 1.3 mL to 1.8 mL, the fluorescence intensity remained constant. Thus, 1.5 mL was selected to ensure a sufficient excess of the reagent throughout the tests. Varying the volume of 4ClRAAP (2 x 10-5 mollL) from 0.8 mL to l.2 mL, no significant difference in the fluorescent intensity was observed. 1.0 mL was then used in subsequent tests. Various oxidized reagents (hydrogen peroxide, potassium periodate and potassium bromate), were tested and maximal and constant net fluorescence values were found using hydrogen peroxide in the range of 0.8 mL to l.2 mL - the optimal volume of hydrogen peroxide (0.03 %) was then set at l.0 mL. 100°C was adopted in the tests, due to its easy accessibility. In addition, using the ~h measured at different temperatures, In(~h) versus liT curve can be plotted. It gives a linear regression equation -lnL~h = 25.78+ 7.82 X 10 3 IT (regression coefficient 0.9801). According to the Arrhenius equation, the apparent activation energy of the used reaction system is: E c• t=7.82 x 10 3 x 8.314=65.00 kJ/mo!. ACKNOWLEDGMENTS Supported by Science Research Foundation of Shandong Province, China (Y2007B07), and National eleventh- 5 year plan, China. REFERENCES 1. Sanchez-Chardi A. Tissue, age, and sex distribution of thallium in shrews from Dofiana, a protected area in SW Spain. Sci Total Envirn 2007;383:237-40. 2. Spano N, Panzanelli A, Piu PC, et a!. Anodic stripping voltammetric determination of traces and ultratraces of thallium at a graphite microelectrode Method development and application to environmental waters Analtica Chimica Acta, 2005; 553: 201-207. 3. Maia SM, Pozebon D, Curtius AJ. Determination of Cd, Hg, Pb and Tl in coal and coal fly ash slurries using electrothermal vaporization inductively coupled plasma mass spectrometry and isotopic dilution. J Anal At Spectrom

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2003;18:330-7. Welz B, Becker-Ross H, Florek S, Heitmann U, Vale MGR. Braz J. High-resolution continuum-source atomic absorption spectrometry - what can we expect? Chern Soc 2003;14:220-9. Ariel M, Bach D. The determination of thallium in urine. Analyst 1963;88:30.

MOLECULAR RECOGNITION OF AMINO ACIDS BY HEMATOPORPHYRIN AND METALLOHEMATOPORPHYRIN RECEPTORS YONG ZHANG, YA-CHUN LEI, DIAN-SHENG LIU Dept of chemistry, Shanxi University, Taiyuan, 030006, P.R. of China Email: [email protected]

INTRODUCTION Molecular recognition plays important roles in natural processes such as conversion of energy, reaction in cells. Porphyrins are believed to have a considerable potential as receptors in molecular and chiral recognition research. Molecular recognition of amino acids and their derivatives is essential in the mechanism of aminoacyl-transfer RNA synthase reactions. Various studies on the interactions between amino acid esters and porphyrin or metallohematoporphyrin (MHP) have been reported. Mizutani,I,2 and his co-workers have studied the zinc (II) porphyrin complex as a receptor for amino acid esters and other molecules in chloroform, and described that there are three- recognition sites: a Lewis acidic site (Zn), a salt bridge site (COO' group), and a hydrophobic binding pocket. Konishi 3 designed and synthesized a novel anion-binding chiral receptor based on metalloporphyrin, which was used to recognize amino acids derivatives, Hematoporphyrin (HP) is widely used as photosensitizer in photodynamic therapy because of its photosensitivity, It has been successfully used in the therapy for different forms of leukemia,4 In this paper we describe the interactions between amino acids and Zn(II)-HP, Cu(II)-HP, Co(II)-HP, and Ni(II)-HP by fluorescence and absorption spectra in aqueous solution. The binding constants were calculated and the mechanism of recognition discussed, MATERIALS AND METHODS Fluorescence spectra were obtained on an LS 50B fluorometry (Perkin-Elmer), The excitation wavelength of L- (or D-) tyrosine (Tyr), and D-, L- (or L-) tryptophan (Trp) is 275 and 278 nm, respectively, Absorption spectra were recorded on a UV-265 spectrometer (Shimadzu), Hematoporphyrin (HP) was purchased from Fluka Inc, L(or D-) tyrosine, L-tryptophan were from the Shanghai Institute of Biochemistry CAS. All amino acids were dissolved in distilled water and a few drops of 0.5mo1iL HCI were added to accelerate dissolution, All other reagents were AR, Doubly distilled water was used throughout. An appropriate amount of amino acid of the stock solution was transferred into a 5 mL volumetric flask, and the various concentrations of MHP or HP added. Then the pH was adjusted by addition of phosphate buffer solution. The mixture was diluted to the final volume with the distilled water and shaken thoroughly, following equilibration for 20 min at room temperature. Preparation of metallohematoporphyrin. According to the published procedure,5 461

462

Zhang Yet al.

in a 100 mL round-bottomed flask, fitted with a mechanical stirrer and a reflux condenser, was placed 30 mL of dimethylformamide. To the solution was added 0.0015 g HP and 0.0033 g zinc acetate (or CuCIz, NiCIz, CoCIz,), and the mixture was stirred and heated. The reaction was continued until the red fluorescence characteristic of free base porphyrin was no longer detectable. Removal the solvent yielded a red crystalline product. The excess metallic salt was removed by water washing and the product was purified using dilute ammonia. RESUL TS AND DISCUSSIONS The effect of pH. The effect of pH on fluorescence intensity of amino acids in the absence and presence of MHP was studied. It revealed the changes in fluorescence intensity of amino acids in the pH 3.5, 7.4, and 10.0 buffer solutions. The fluorescence intensity of tyrosine is stronger in acidic and neutral media. Hence, phosphate buffer solution (pH=7.4) was selected for controlling pH in the following studies. Effect of concentration of amino acids. The concentration of MHP was fixed at 10-6 mollL, the concentration of amino acids was varied from 0 to 4.0 x 10-5 mol/L. As the concentration of amino acids increased, the excitation and emission intensity of fluorescence gradually increased. Under the optimum conditions described above, the calibration graphs were linear in the range of 0 - 2.8 x 10-5 mollL L-tyrosine in the absence of MHP. In the presence of MHP, the linear range was 0 - 4.6 x 10-5 mollL. This two-fold increase may be due to the porphyrin preventing the formation of the polymer. The effect of HP and MHP concentration on fluorescence intensity. The amino acid concentration was fixed, and the MHP concentration varied. This revealed that the fluorescence intensity of the amino acids was remarkably quenched, and the emission wavelength shifted to a shorter wavelength. It was especially remarkable to D, L- (or L-) tryptophan, but the excitation wavelength was unchanged (Fig. 1). 800

600

400 ll-

200

0 245

270

295

320

345

370

395

Wavelength(nm)

Fig. 1. The fluorescence spectra of D-tyrosine in the presence of different concentration ofZn(II)-HP. D-tyrosine: 5.0xlO-6 mollL. The volume ofZn(II)-HP (3.2 x lO-5 mollL) (mL): 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8,2.0

Molecular Recognition of Amino Acids

463

The effect of the concentration of MHP on absorption spectra. With increasing concentration of MHP, the absorbance of amino acids increased and the maximum peak was blue shifted slightly. We concluded that interactions between amino acids and MHP had occurred,6 and the absorbance enhancement was due to strengthened conjugative effect. Molecular recognitions of amino acids. The binding constant is a measure of recognition interactions of different amino acids with metallohematoporphyrin. It can be estimated by Benesi-Hildebrand equation (the double-reciprocal method):

1

F'o -F

1

1

KkQ[por] [AA]

kQ[AA]

------+---

ilF (Fo-F) is the change of fluorescence intensity in the presence of MHP, k is an instrumental constant, [por] is the concentration of MHP, Q is the quantum yield for complex, [AA] is the concentration of amino acid, K is the bonding constant. K can be calculated from a plot of lIilF vs. l/[por]. The bonding constants of amino acids with different HP are shown in Table 1. Since the line of lIilF vs. 1 / [por] has a good liner correlation coefficient; the ratio of binding is 1: 1.7 Table 1. The bonding constants of amino acid to different hematoporphyrin and metallohematoporphyrins

D- tyrosine L- tyrosine D,L-tryptophan L-tryptophan

Zn(II)-HP

Cu(II)-HP

Co(II)-HP

Ni(II)-HP

HP

3.33xI04 6.15 x lO4 5.83xI04 2.I4 x lO5

I.7IxI04 4.7IxlO4 4.80x103 4.85xlO4

9.19xI03 3.60xlO4 4.I3x104 6.56xlO4

6.17xI03 4.62xlO4 4.46x 10.3 2.52xlO4

2.0Ix103 3.20xI03 7.I9xlO4 7.78xlO4

Mechanism of recognition. The values of binding constants reflect the extent of binding between molecules. It shows that the binding constants of the L-isomer are larger than that of D-isomer or the racemic mixture. The possible reason is that the residual group of the L-isomer is directed away the host C0 2 H group in the complexes, i.e., the aromatic group of purine interacts with the core of the porphyrin. This is also the reason why the binding constants ofTrp with MHP are larger than that ofTyr with porphyrin. The order of the binding constants between amino acids and metallohematoporphyrin were found to be: KZn(II)-HP> KCu(II)-HP~KNi(II)-HP> KCo(II)-HP. The mechanism of recognition in MHP involves three recognition groups: metal ion (a metal coordination site), the hydroxy groups (a hydrogen bond donor site), and carboxyl groups (steric repulsion site). We have found that the aromatic group is one of the most important recognition groups, and that recognition is achieved by cooperative functions of three recognition groups, the hydroxy groups, 89 carboxyl groups and aromatic group, and this is in agreement with other studies. •

464

Zhang Yet al.

CONCLUSION In the present paper, MHP was prepared according to published procedures. Studies of recognition between amino acids and HP showed that metallohematoporphyrin exhibits significant recognition toward amino acids in phosphate buffer solution (pH=7.4). The binding constants were in the rank order: KZn(II)-HP> KCu(II)-HP;:: KNi(II)-HP> KCo(II)-HP. Binding constants of the L-isomer are larger than that of D-isomer or no racemic mixtures. The binding constants of Trp with porphyrin are larger than that of Tyr with porphyrin. Recognition is achieved by cooperative functions of three recognition groups: carboxyl groups, hydroxyl groups, and aromatic groups.

ACKNOWLEDGEMENTS The work was supported by Grants for Shanxi Youth Technical Foundation of China (No.20021008), and Institute of Modern Chemistry, China.

REFERENCES 1. 2.

3.

4. 5. 6.

7. 8. 9.

Mizutani T, Wada K. Porphyrin receptors for amines, amino acids, and oligopeptides in water. J Am Chern Soc 1999;121:11425-31. Mizutani T, Ema T, Yoshida T, Kuroda Y, Ogoshi H. Recognition of a.-amino acid esters by zinc porphyrin derivatives via coordination and hydrogen bonding interactions. Evidence for two-point fixation from thermodynamic and induced circular dichroism spectroscopic studies. Inorg Chern 1993;32:2072-7. Konishi K., Yahara K. A novel anion-binding chiral receptor based on a metalloporphyrin with molecular asymmetry. Highly enantioselective recognition of amino acid derivatives. J Am Chern Soc 1994;116:1337-44. Stredansky M, Pizzariello A. Selective and sensitive biosensor for theophylline based on xanthine oxidase electrode. Anal Biochem 2000;285:225-9. Liu J, Xu D.H. Anticancer activity and mechanism of water-soluble porphyrin and metalloporphyins. Chern J Chin Univ 2001;22:6-9. Mizutani T, Wada K. Molecular recognition of amines and amino esters by zinc porphyrin receptors: binding mechanisms and solvent effects. J Org Chern 2002;65 :6097-1 06 Catena GC, Bright FY. Thermodynamic study on the effects ofp-cyclodextrin inclusion with anilinonaphthalenesulfonates. Anal Chern 1989;61:905-9. Imai H, Misawa K. Water-soluble zinc porphyrins as receptors for amino carboxylates. Chern Lett 2001 :688-9. Kuroda Y, Kato Y. Chiral amino acid recognition by a porphyrin-based artificial receptor. J Am Chern Soc 1995;117:10950-8.

DETERMINATION OF BSA BY ITS ENHANCEMENT EFFECT ON SECOND-ORDER SCATTERING OF 3-(4'-METHYL PHENYL)-5-(4'-METHYL-2'-SULFOPHENYLAZO) RHODANINE ZHU YUANNA,I YU JINGHUA,2 DAI PING,2 ZHANG CONGCONG,2 LI B02 i School of Material Science and Engineering, University of Jinan, 2School of Chemistry and Chemical Engineering, University ofJinan, Jinan 250022, P R China, i Email: [email protected] [email protected]

INTRODUCTION Proteins are important in many biological processes and deficiency can cause growth failure, loss of muscle mass, decreased immunity, weakening of the heart and respiratory system and even death. Quantitative determination of protein has become the most familiar analytical measure in food and medicine quality inspection. There are many methods for the determination of protein including nephelometry, Kjeldahl determination,! spectrophotometry,' spectrofluorimetry3 and resonance Rayleigh light scattering.' Resonance Rayleigh light scattering has developed rapidly in recent years because of its good sensitivity and selectivity in determination of protein. In spectrofluorimetry, the second order scattering (SOS) presenting at double the excitation wavelength is a common phenomenon, and it has been regarded as a detrimental interference. The characteristics and cause of this scattering has not been extensively researched until Liu s investigated the determination of trace Se using SOS, which had excellent sensitivity and provided clues for the further study of substance structure and reactions. Subsequently, SOS has become an emerging and powerful analytical tool for determination of trace substances. Rhodanine and its derivatives are an important type of reagent mainly applied for determination of metal ions by spectrophotometry. To improve the analytical performance of rhodanine reagent, a sulfonic group with good complexing ability was introduced to the reagent to increase its solubility. We prepared 3-(4'-methylphenyl)-5-(4'-methyl-2'-sulfophenylazo) rhodanine (M4MRASP) and used it as probe in the investigation of interaction between BSA and M4MRASP. The SOS spectrum of BSA-M4MRASP system was studied, and it was found that in acidic medium, the SOS intensity of M4MRASP itself was quite weak, but it was sharply enhanced when the dye molecule associated with the protein. Furthermore, the SOS intensity was found to have linear relationship with concentration of protein under specified conditions. Based on this, a new method for determination of protein was established and applied to determine protein in milk samples with satisfactory results. Compared with resonance Rayleigh scattering, second order scattering has better sensitivity. 465

466 Zhu Yet al.

MATERIALS AND METHODS The following instrumentation was employed in our studies: Perkin-Elmer LS55 (America) fluorescence spectrophotometer (Perkin-Elmer, America); Electronic balance (Mettler Toledo, Shanghai, China); PHS-3C digital pH-meter (Shang Hai Lei Ci Device Works, Shanghai, China); SYZ-550 quartz sub-boil high-purified water distiller (Jiang Su Jin Tan, Jiang Su, China). A standard solution of BSA (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) (50 I1g/mL); a standard solution of 3-(4'-methylphenyl)-5-(4'-methyl-2'- sulfophenylazo) rhodanine (M4MRASP) (2.0 x 10-4 mollL); Britton-Robinson buffer solution: pH 1.62; A sodium dodecylsulphonate (SDS) micromulsion. All chemicals were of analytical reagent grade or better. Procedure. 2.0 x 10-5 mollLof M4MRASP (2.5 mL) was measured into a 10 mL of color comparison tube with a cover, 50 I1g/mLof BSA standard solution (1.0 mL), 2.0 mL of B-R buffer solution (pH=I.62), and 0.03 mL of SDS micromulsion were added in turn, and the mixture then diluted to the standard volume. The scattering intensity of blank solution (10) and sample or BSA standard solution (11) were measured at Aex/Aem=260 nm/520 nm (slit=15/8). The concentration of BSA was quantified via the peak height (relative scattering intensity), which was obtained by subtracting the blank solution scattering intensity from that of the sample or BSA standard solution. RESULTS AND DISCUSSION SOS spectrum. SOS spectra of different components are shown in Fig. I. This shows that the scattering intensity of BSA or M4MRASP itself was quite weak, either in the acidic medium or with SDS. However, when BSA was injected into M4MRASP in the presence of SDS, the scattering intensity was enhanced sharply (curve 5). The scattering intensity became stronger with an increase in the BSA concentration, and there was a functional relationship between the concentration of BSA and scattering intensity. Optimum conditions. Initially it was found that the BSA solution scattering intensity was enhanced the most in the B-R buffer solution. So we choose pH 1.62 to control solution pH. It was found that the highest sensitivity was obtained in the presence of the SDS microemulsion. The optimum reagent amounts of buffer solution, SDS microemulsion and M4MRASP were 2.0 mL, 0.03 mL and 2.5 mL. Finally, 0.02 mollLofNaCI was chosen to control the ionic strength of the solution.

Determination of BSA by Its Enhancement Effect on Second Order Scattering

467

t.~4

5(!() .

.til(l .

.......

3'1J{I

~

:~oo

.

, 'j

,, ,I ,T

]{'t
0·

)Jum Fig.1. SOS spectrum ofM4MRASP-BSA system. B-R+M4MRASP; 2. B-R+M4MRASP+SDS; 3. B-R+M4MRASP +BSA; 4. B-R+SDS+BSA; 5. B-R+M4MRASP+SDS+BSA (0.2 mL) 6. B-R+M4MRASP+SDS+BSA (0.25 mL). Effect of coexisting species. The tolerable concentration ratios of foreign species on the determination of 10.0 J.lg of BSA in 10 mL of BSA-M4MRASP system are as follows (the relative error is under ±5 %; fold): L-cysteine, DL-u-aminoisovaleric acid, D-phenylalanine (60); L-leucine, DL-alanine (40); L-tryptophan, L-cysteine, L-tyrosine, DL-methionine (50); glycine (60); L-arginine (45); L-methionine, L-lysine, DL-malic acid (70); DL-phenylalanine (100); ascorbic acid, L-arginine (200) Zn 2+, Ae+ (40); K+, Fe 2+(30); Fe3+ (5); Mg2+, Ca2+ (l00). It can be seen from the data that the common ions and amino acid did not interfere with the determination of BSA except for Fe3+. To elimiante the Fe 3+ interference, ascorbic acid was added to reduce Fe3+ to Fe2+. It is conclusioned that the new type rhodanine is good spectrum probe with good analytical performance, and the BSA-M4MRASP system has good selectivity. Analytical characteristics. The proposed SOS method was studied for linearity, precision, and sensitivity. Under the optimum conditions, a linear relationship between BSA concentration and enhanced scattering intensity was obtained over the range of 0-2.5 J.lg/mL, with a regression equation of Ai= -166.39 + 429.34 P (J.lg/mL) and correlation coefficient (r) 0.9972. The low detection limit of BSA was found to be 2.55 x 10-8 g/mL based on 11 replicates analyses of the blank solution. A linear relationship between BSA concentration and enhanced scattering intensity was obtained over the range of 0-2.5 J.lg/mL, with a regression equation of Ai= 1.57 + 139.65 P (J.lg/mL) and correlation coefficient (r) of 0.9972. The low detection limit ofBSA was found to be 1.78 x 10-7 g/mL

468

Zhu Yet al.

Applications. A milk sample was diluted to 100 mL with water, and then determined by the proposed method. The recovery rates of the method were 98.6% and 97.2 %; RSD was 3.58 and l.61 which indicated the results were satisfactory. As we can conclude that the recoveries of added BSA can be quantitative and I-tests indicated that there was no significant difference between recovery efficiency and 100 % at a confidence level of95 %. ACKNOWLEDGMENTS This work is financially supported by Key Subject (Laboratory) Research Foundation of Shandong Province, China (XTD0705) and Science Research Foundation of Shandong Province, China (Y2007B07). REFERENCES 1. Wei Q, Wu D. The trend of development of analytical techniques for protein. J Jinnan Univ 2003;17:312-20. 2. Ma W, Li Y. Photometric features of protein-dibromocarboxyarsenazo supermolecular complexes and its analytical applications. Chin J Anal Lab 2007;6: 48-5l. 3. Gao F, Zhu Y. Synthesis of a novel fluorescence reagent of bis(l-H-benzotriazole)-ethyldione-thiosemicarbazone and its application in the determination of proteins. Chern Reag 2004;26:168-70. 4. Zhang Y, Zhexuan L. Study of protein conformation in solution by tyrosine residues RRS spectrum. Spectro Spectral Anal 2006; 11 :2089-92. 5. Shaopu L, Zhongfang L, Ming L. Analytical application of double scattering spectra of ion-association complex. Acta Chim Sin 1995;12: 1178-84.

INDEX

1,4-Butanediol dimethacrylate 241 10 2 reaction 257 2-Bromoquinoline-3-boronic acid 425 3-(4'-Methylpheny 1)-5-(2'sulfophenylazo) rhodanine 381 7-Ethyl-l O-hydroxycamptothecin 397 9-Benzy lid ene-l O-methylacridans 237 ABE K 135 ABE S 109 Absorption spectra 59 Acetone 123 Acetonitrile 449 Acridinium ester 273 Acridiniumx-BSA-anti-HCV core 181 Aequorea 3, 19 Aequorin 3 AKIY AMA H 197 AKIYOSHI R 339 ALAM SM 205,209,213 Alfalfa 281 Alginic sodium diester, assay 401 Aluminum-induced oxidative burst 201 Amine hydrochloride 173 Amino acids 269, 461 Conserved 23, 319 ANTENUCCI M 241 Antibiotic susceptibility 89 Antibody-antigen 325 Antioxidant activity 193 Apoptosis 339 ARAKAWA H 109,197 Arm light organs 67

Artificial acid rain stress 363 Aryloxalate 127 Ascorbic acid 277 ATP-dependent luminescence 67 Au nanoparticles 217 Azacrown 237 Bacteria-host interplay 343 Bacterial bioluminescence 35 luciferase, mechanism 7, 71 BAEZZA T MR 173 BAKALOVA R 189,193 BAO J-F 177 BART 93, 97 BELOVANV79 Benserazide assay 389 Beta-CD 421, 429 Bicyclic dioxetanes 139, 151 Biodegradation 441 Biological systems 63 Bioluminescence 3,19,51,93,197, 339 analysis 27,89,109 emission spectra 23 imaging 343, 359 organisms 43 reactions 55,261 Biomedical diagnostics 157 Biophoton emission 635, 409 Bio-tissues 415 Bovine neonates 233 BROVKO LY 343 BRUKHOVSKIH TV 55 BSA 381 BSA assay 465 BURVENICH C 233 Cadmium stress 445 469

470

Index

CAl Q 105 CAl R325 CALTHARP SA 319 CAO X-V 415 Capillary electrophoresis 269, 305 Carbaryl assay 393 Carbohydrates, recognition 425 Cascade 355 Cell death 201 CHANG CD 181 Charge-transfer-induced luminescence 261 Chemiexcitation 19 Chemiluminescence 127,185,205, 209,213,217,225,237,245, 249,265 assay 27,193,197 immunoassay 273 probe 143,253 reaction 123,261 sensors 161 Chemiluminophores, high-energy 147 CHEN G 301 CHEN Q 253, 257 CHEN X433 CHEN Y 373 CHENG KY 181 CHENG X 453,457 Chitosan 301 Chloropheny I-arsenoxy Ipheny lazorhodanine 457 CHOIJH 205 CHUNG HY 213, 393 Ciprofloxacin 213,305 CLAES JM 15 Coelenterazine 51 Conjugate 181 Conserved residue(s) 23

Coral311 CORSALE P 241 Crataegus oxicantha 193 CTIL 261 CUI S 405 Cuprous iodide 421 Cypridina 19 Cypridina luciferin analog 83 CYS14643 CYS6243 Cytosolic ATP, assay 339 DADARW AL R 343 DAI H 301 DAI P 277, 381, 465 DEHAAN A287 DE PALMA F 241 DE SOLE P 241 Delayed fluorescence 363, 367 DELLA CIANA L 131 Developmental enhancers 319 Devices 157 Diagnostics, food 97 Dimethyl sulfoxide 59 DING H378 Dinoflagellates 10 Dioxetanes, chemiexcitation 115 DNA 453 DOLCI LS 157,347,351 DONG C 421, 429 DONG R421 Doroshenko 10 35 Dot blot 287 DU JX 185 E. coli 343 Electrochemiluminescence 297,305 microscope imaging 351 sensor 301 Electron spin resonance spectroscopy 225

Index

Electron-donating groups 237 Electroporation 319 ELISA 287 ELLIOT J 343 EMAMZADEH R 23 Endothelial cells 249 Enhanced chemiluminescence 151 Enhanced fluorimetric determination 389 Enhancement 43, 221, 381 Enhancer 265 Enzyme immunoassay 197 Epithelial cells 343 Etmopterus spinax 15 Europium (III) 315 nitrate 123 Evolutionarily conserved regions 319 Expression efficiency 101 Extracellular chemiluminescence 233 Famine 101 Firefly bioluminescence 75 luciferase 23,43 luciferin intermediates 59 Flavin mononucleotide 35 Flow injection analysis 173,209,229 chemiluminescence 221,257, 277 Flow-through biosensor 217 Fluoresceinyl Cypridina lucifer in analog 257 Fluorescence 315, 381, 393, 441, 457 assay 385 imaging 355 "ON" probe 449

471

probe 421 quenching 373 resonance energy transfer donors 325 Fluorodehydrocoelenterazine 51 Food microbiology 97FRUNDZHY AN VG 89 Gandelman OA 93, 97 Gatifloxacin 209 GE S 277, 381,457 Genetoxic environmental pollutants 105 GIARDINA B 241 GITELSON JI 79 Gold-sensitized chemiluminescence 177 GOMI K, 197 GONG F-Z 333 Green fluorescent protein 3 GREERLF 319 GRIFFITHS MW 343 GRILLI S 131 Guanylate cyclase 109 GUARDIGLI M 157, 347 GUSEV AA 79,101 GZII 287,291 HADJIMITOVA VA 189, 193 HAMZINK M 287, 291 HAN SQ273 HAO L 185 HARADA S 135 Hardware 93 HASTINGS JW 3 HATTA-OHASHI Y 355 HeV core antigen 181 HE YF 273 Heavy atom effect 55 Helical copper-binding motif 83 Hematoporphyrin receptors 461

472

Index

High temperature 367 HIRANO T 19 HL-60 cells 241 Horseradish peroxidase 189 HOSHIYA N 151 HOSSEINKHANI S 23 HOUX429 HRP 217 HRP-H 20 2 265 HSA 429 HU S 325 HU Z433 HUANG l-L 333 HUANG X-X 105 Human prion protein 83 Human serum albumin, assay 257, 397,433 Hydrogen bonding 119 Hydrogen peroxide 127,245 hydrogen peroxide assay 217, 301 IBSO culture collection 47 IJUIN HK 115, 139, 151 IKEDA H 19 IKEDA T245 Imaging 339 IMAHARA Y 311 Imidazole 245 Immunoassay 265 Immunochemicallocalisation 347 Inclusion complex 421 INOUYE S 197 Intracellular calcium, dynamics 359 Iron 449 ISOBE H 119,261 ISOBE M 51 ITO K 109, 197 IWA Y A-INOUE M 201 IZADP ANAH M 173 JEON CW 385, 393

JI Q 281 JIA H-H 63 JIA L 305 JIANG L 181 JIANG Z-H 177 JIAO CZ445 JIMBO M 311 lIN WI 425 KADONOT201 Kaempferol433 KAGENISHI T 27,83 KAKUNO F 151 KAMIYAH 311 KANAYAK319 KARIM MM 205,385 KATO Y 311 KA WAMOTO H 245 KAWANOT27, 83,201,225 KAZAKOV DV 123 KAZAKOV V 315 KAZAKOV VP 123 KERSTEN G 287 KHAN MA 209, 213, 389, 393 KIDDLE G 93, 97 KIM WH 389, 393 KIRILLOV A TN 55 KISHIKAWA N 127 KMn04-formaldehyde system 209 KOJIMA S 19 KOKSHAROV MI 31 KONGJINDA V 51 KOTOVDA47 KRASNOV A OJ 35, 39 KUDRY ASHEV A NS 55 Kuekenthal 311 KUN S 63 KURODA N 127, 135 KUROE M237 KUSE M 51

Index

Label 273 Lampyris turkestanicus luciferase 23 LAND 217 Lanthanum 445 Laser confocal scanning 363 LEE SH 205,209,385,389,393 LEI Y-C 461 LI B 217, 381, 457,453,465 LI D 421 LI G 397, 401 LI XX 329 LI YH 221 LI ZF 449 Light-induced delayed fluorescence 437 LIN C 27,225 LIU BH 269, 273 LIU C 63 LIU D-S 461 LID J 433 LIU Y 397, 401 LIU YB 273 LIU Z 281 LIU Z-B 249 Living cell 355 systems 409 LMWOA441 LOMAKINA GY 43 LU JR 185, 221 Luciola mingrelica 31, 43 Luminescence imaging 355 Luminol chemiluminescence 131 Luminol- K3Fe(CN)6 system 221 Luminol-dependent chemiluminescence 189 Luminous mushrooms 79 organisms 47

squid 67 LUO S 253, 257 LUOY 405 LUO ZF 273 LUPI A 241 Lux-operon 101 MA H229 MAEDA M 197 Maize leaves 445 MAKI S 19 MALLEFET J 15 MANU SK319 MARTORANA GE 241 MARZOCCHI E 131,351 Mass spectrometry 115 MATSUMOTO M 115,139,151 MAZZEO R347 MCELGUNN CJ 93 Medical diagnostics 97 MEDVEDEVA SE 47, 79 MEEAK389 MEHRZADJ 233 Metal ion 151, 405 sensing 237 Metallohematoporphyrin receptors 461 Methacrylate 241 Methyl blue 429 Methylene blue 401 MIAO L229 Micellar medium 393 Micellar-enhanced 229 Micelle 389 sensitized 185 MICHELINI E 157 Microemulsion 381 Microscope 363 Milky Sea 6 MINIKH 0131,343

473

474

Index

MIRA SOLI M 157 MODESTOV A YA 43 MOHRIM233 Molecular imprinted polymer 161 Molecular modeling 433 recognition 461 spectroscopy 429 MOMOI H245 Monoamine oxidase-like superoxide-generating activities 83 MORIN-fsDNA 381 MOTOYOSHIY A J 237 Multicolor fluorescent protein 311 Multiplex assay 197 MUNESUE M 245 MURRAY JAH 93,97 MUSIANI M 157 Mutagenesis 43 Na2C<4Mg2Si4015:Tb3+ 333 NADERI-MANESH H 23 NADPH oxidase 249 NAKAMURA S 135 NAKASHIMA K 127, 135 NAKASHIMA Y 51 Nano-sized phosphor 333 N-bromosuccinimide-H 20 2 185 Near-infrared 143 Necrosis 233 NEMTSEV A EV 55 Neutrophils 233 NISHI! Y 237 Nitric oxide 109 NIWA H 19 N-methylimidazole 35 NOCCA G 241 Noctiluca 10 Non-conservative residues 43 Norfloxacin 177

NOZAKI 0 245 Nucleic acids 373 Nucleophilic acylation catalyst 131 Obelin 359 OBERG KC 319 Odd and even coherent states 63 Ofloxacin assay 229 OHASHI M 115 OHBA H 19 OHYAMAK 127 OKUMURA M 119 Optical molecule imaging 367 OSINA 10 315 OSTAKHOV S 315 Oxygen 237 Painting cross-sections 347 PANG X-F 409, 415 Papaya 197 PARK HW 393 PASHENOV A NV 79 PCR 197 Peroxynitrite nitrifying protein 405 Peroxyoxalate chemiluminescence 135 Pharmaceutical formulation 213, 389 Phenanthrene, biodegradation 441 Phenothiazine cation radicals 189 Phenyl-l0-methyl-acridinium-9carboxylate methosulfate 273 Phosphor 333 Phosphorescence 425 photocatalytic fibers 27, 225 Photodynamic reactions 253 Photoluminescence 329 Photon emission, mechanism 415 Photoprotein 359 pH-tolerant mutants 31 PIRA CU 319

Index

Plant cell responses 27 Plants 281, 363, 367 Point-of-care testing 157 Polio D-antigen 291 Polio vaccine, inactivated 287 Polymer 301 Polymerase chain reaction 297 Potassium monoperoxysulfate 123 PRATI S 347 Probe 253 PROm L 131 Protein 347, 409 PSURTSEVA NV 79 Pyrogallol assay 245 QIAO J 421 Quantitative detection of singlet oxygen 253 Quantum dots 325 Quantum yield 71 Quenching 37 Quorum sensing 6 Rain 363 Random mutagenesis 31 RANJBARB23 Rayleigh scattering 381 Reactive oxygen species 27, 225 Recombinant luminescence bacteria 105 Residual host cell proteins 287 Resonance rayleigh scattering 401 REUBSAET K 287 Rhodamine B-quinoline-8-amide 449 Rhodanine 277, 453, 465 Rice, salt-tolerant 437 RIZZOLI M 93, 97,347,351 RODAA 131,157,347,351 ROmCHEVA EK47, 79 ROSSI C 241

Ru(bipY)3 2+-Ce(IV) system 205 Rutin 385 Rutin assay 185 Rutin-Fe(III) 385 Sz-level315 SADEGHIZADEH M 23 SAFAROV FE 123 SAKAI H59 SALBILLA VA 181 Salicylic acid 445 Salt tolerance 281, 437 SANG LZ441 SANOY 109 SATO C 311 SCHMIDT R 123 SCIUTTO G 347 Scleronephthya gracillima 311 Second order scattering 453, 465 SEKI M 109 SHAH DO 181 Shark 15 SHEN DC273 SHEN QJ 425 SHEN X 249 SHI H-C 105 SHIMOMURA 0 67 SHIZUMA M 245 SHUANG S 421 SHUANG S 429 SIK63 Signal reagent 287, 291 SIMONI P 131 Single cell 339 Singlet oxygen 237, 253 Site-directed mutagenesis 23 Sodium deoxycholate 425 Soft coral 311 Solvent-promoted chemiluminescence 139

475

476

Index

SONG S 429 Spartloxacin assay 205 Spectra 23, 59, 75 Spectral differences 75 Spectroscopy 433 Stress response 367 SU YN 445 Sugars 201 SUGIYAMA T 359 SUHYS 393 SUN X 229, 265 Superoxide 83 Superweak luminescence 281 Surfactants 135 SUZUKI H 109,339,355,359 Symplectin 51 Synergistic agent 265 TAFRESHI NKH 23 TAKAHASHI T 311,355 TAKAHASHI Y 19 TAN Y 277, 381, 457 TANAKA K 27, 225 TANAKA T237 TANAKA Y 197 TANI N 51 TANIMURA M 115,139 Tb 3+-protocatechuic acid complex 373 TERANISHI K 67,143 Thallium(I) assay 457 Theoretical analysis 59 Thermal decomposition, peroxides 119 Thermostability 43 THETMM359 Thiamine assay 221 Thioredoxin reductase 249 TIAN J 433 TIAN ZH 445

TISI LC 93, 97 Titania 27,225 Tomato cells 201 TONG AJ 449 Total thyroxine 273 TRA YKOV T 189, 193 TREP 319 TROFIMOV AV 147 Tryptophan complexes 315 TSAPLEV YB 147 TU S-C 71 TYULKOV ANA 35 UGAROVA NN 31, 43, 89 Ultrasensitive chemiluminescence 347 Ultraweak bioluminescence 445 Vargula see Cypridina Vascular endothelial cells 249 VASIL'EV RF 147 Visualization 355 Vitamin C 233 VUV excitation 329 VYDRYAKOV A GA 79 WABAIDUR SM 213,385,393 WADA M 135 WADAN 59 WANG F 381 WANG H343 WANG J 437 WANG Y 301, 425 WANG YH 329 WATANABE N 115, 139, 151 Watasenia scintillans 67 Water 225 conditioning 27 Web-resource 47 WEI J 297 WEI J-S 333 WEI XY 441

Index 477 WEI Y 253, 257 WEI ZL445 WEN F 363, 367 Western blot 287 WU MH 269, 273 WU X 301,381 X2-(Y,Gd)2Si05:Tb3+ phosphor 329 Xanthomonas oryzae pv. Oryzicola 297 XIANGY 449 XIE CJ 269 XING D 253, 257 XING DA437 XU W 253, 257, 437 YAMAGUCHI K 119,261 YAMANAKA S 119,261 YANGH325 YANGJ 373,381 YANG Q281 YANG X 265,325 YANG Y 221, 373 YI L-H 333 YIN DG 273 YIN DG 269 YOKAWA K 27,83 YU J 277,381,453,457,465 YU X-J 177 YUASA T201 ZANARINI S 351 ZHANG C 277,465 ZHANGH 363 ZHANG L 273, 297,367,405,437, 453 ZHANG Q 325 ZHANG Y 229, 441, 461 ZHANG Z 161 ZHANG ZH329 ZHONG R405

ZHOU H 281 ZHOU L-Y 333 ZHOU X 305, 363 ZHU Y 277, 453, 465 ZHU YX 441 ZOMER G 287, 291 ZOU WS425

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