Bioluminescence and Chemiluminescence: Light Emission: Biology and
Scientific Applications, Proceedings of the 15th International
SymposiumBIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission:
Biology and Scientific Applications
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NEW JERSEY * ~ O ~ O O N * SINGAPORE BElJlNG * SHANGHAI * HONG KONG
* TAIPEI * CHENNAI
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
Published by
USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ
07601
UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
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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
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ISBN-13 978-981-283-957-2 ISBN-I0 981-283-957-7
PREFACE
These are the Proceedings of the 15th 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
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), Jin Ling 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. 13th
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 981- 02-4679-X.
viii Preface
10th 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. 9th 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. 8th 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. 3rd
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. 2nd 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
real time 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,
CONTENTS
PART 1. BASIC BIOLUMINESCENCE
Plenary lecture - Progress, perspectives and problems in basic
aspects of bioluminescence 3
HastingsJW
Bioluminescence of sharks, a case study: Etmopterus spinax 15 Claes
JM and Mallefet J
Chemiexcitation mechanism for Cypridina (Vargula) and Aequorea
bioluminescence 19
Hirano T, Ohba H, Takahashi Y, Maki S, Kojima S, Ikeda H
andNiwaH
Site-directed mutagenesis of Lampyris turkestanicus luciferase: The
effect of conserved residue(s) in bioluminescence emission spectra
among firefly luciferases 23
Hosseinkhani S, Tafreshi N Kh, Sadeghizadeh M, Emamzadeh R, Ranjbar
Band Naderi-Manesh H
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 27
Kagenishi T, Yokawa K, Lin C, Tanaka K, Tanaka R and KawanoT
pH-tolerant mutants of Luciola mingrelica luciferase created by
random mutagenesis
Koksharov MI and Ugarova NN
xi
31
Krasnova 01, Tyulkova NA and Doroshenko 10
New method of measuring bacterial bioluminescence 39 Krasnova 01,
Tyulkova NA and Doroshenko 10
Enhancement of thermostability of Luciola mingrelica firefly
luciferase by mutagenesis of non-conservative residues CYS62 and
CYS146 43
Lomakina GY, Modestova YA and Ugarova NN
Web-resource: "Bioluminescence and luminous organisms" of the IBSO
culture collection 47
Medvedeva SE, Kotov DA and Rodicheva EK
Chemistry of symplectin bioluminescence with
fluorodehydrocoelenterazine 51 Nakashima Y, Kongjinda V, Tani N,
Kuse M and Isobe M
Mechanisms of heavy atom effect in bioluminescent reactions 55
Nemtseva EV, Kirillova TN, Brukhovskih TV and Kudryasheva NS
Theoretical analysis on the absorption spectra of intermediates of
firefly luciferin in deoxygenated dimethyl sulfoxide 59
Sakai Hand Wada N
Biophoton emission of biological systems in terms of odd and even
coherent states 63
Kun SI, Liu C and Jia H- Y
Study on ATP-dependent luminescence reaction of the arm light
organs of the luminous squid Watasenia scintillans 67
Teranishi K and Shimomura 0
Mechanism of bacterialluciferase: Energetic and quantum yield
Considerations 71
TuS-C
Mechanism responsible for the spectral differences in firefly
bioluminescence 75
UgarovaNN
Luminous mushrooms 79 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 83
Yokawa K, Kagenishi T and Kawano T
PART 2. APPLIED BIOLUMINESCENCE
Bioluminescent assay of antibiotic susceptibility of clinical
samples 89 Frundzhyan VG and Ugarova NN
BART: Smart biochemistry, bright bioluminescence, low-cost hardware
93 Gandelman GA, KiddIe G, McElgunn CJ, Rizzoli M, Murray JAH and
Tisi LC
BART applications in medical and food diagnostics 97 Gandelman GA,
KiddIe G, Rizzoli M, Murray JAH and Tisi LC
Change of expression efficiency of natural and cloned lux-operon in
conditions of famine . 101
GusevAA
Construction of recombinant luminescence bacteria vector to
evaluate genetoxic environmental pollutants 105
Huang X-X; He M, Shi H-C and Cai Q
Development ofa novel bioluminescent assay for nitric oxide by
using soluble guanylate cyclase 109
Sano Y, Seki M, Suzuki S, Abe S, Ito K and Arakawa H
xiv Contents
Mass spectrometric approach to elucidation of chemiexcitation of
dioxetanes 115
Ijuin HK, Ohashi M, Tanimura M, Watanabe Nand MatsumotoM
Theoretical considerations on the roles of hydrogen bonding in
thermal decomposition of peroxides 119
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 123
Kazakov DV, Safarov FE, Schmidt Rand Kazakov VP
Study of novel aryloxalate chemiluminescence reaction without
addition of hydrogen peroxide 127
Kishikawa N, Ohyama K, Nakashima K and Kuroda N
Nucleophilic acylation catalysts effect on luminol
chemiluminescence 131 Marzocchi E, Grilli S, Della Ciana L,
Mirasoli M, Simoni P, Prodi Land Roda A
Effect of surfactants on peroxyoxalate chemiluminescence reaction
135 Nakashima K, Abe K, Nakamura S, Wada M, Harada S
andKurodaN
Solvent-promoted chemiluminescent decomposition of bicyclic
dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl
139
Tanimura M, Watanabe N, ljuin HK and Matsumoto M
Synthesis and characterization of near-infrared chemiluminescent
probes 143 Teranishi K
Contents xv
Generation of high-energy chemiluminophores in ambient light 147
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 151
Watanabe N, Kakuno F, Hoshiya N, Ijuin HK and Matsumoto M
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
Plenary lecture - Molecular imprinted polymer-based
chemiluminescence sensors
Zhang Z
Baezzat MR and Izadpanah M
Study on gold-sensitised chemiluminescence for the determination of
norfloxacin
Bao J-F, Jiang Z-H and Yu X-J
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
Chemiluminescence determination of rutin based on a
micelle-sensitizing N-bromosuccinimide-H20 2 reaction
Du JX, Hao Land Lu JR
Luminol-dependent chemiluminescence increases with formation of
phenothiazine cation radicals by horseradish peroxidase
Hadjimitova VA, Traykov T and Bakalova R
157
161
173
177
181
185
189
Hadjimitova VA, Traykov Tand Bakalova R
Simultaneous mUltiplex bio- and chemiluminescent enzyme immunoassay
for PCR products derived from genetically modified Papaya 197
Ito K, Tanaka Y, Maeda M, Gomi K, Inouye S, Akiyama H and Arakawa
H
Effect of sugars on aluminum-induced oxidative burst and cell death
in suspensions of tomato cells 201
Kadono T, Kawano T, Yuasa T and Iwaya-Inoue M
Chemiluminescence determination of sparfloxacin using
Ru(bipY)32+-Ce(IV) system 205
Karim MM, Choi JH, Alam SM and Lee SH
Flow injection analysis with chemiluminescence detection:
Determination of gatifloxacin using the KMn04-formaldehyde system
209
Khan MA, Alam SM and Lee SH
Determination of ciprofloxacin in pharmaceutical formulation by
chemiluminescence method 213
Khan MA, Lee SH, Alam SM, Wabaidur SM and Chung HY
Chemiluminescence flow-through biosensor for hydrogen peroxide
based on enhanced HRP activity by gold nanoparticles 217
Lan D and Li B
Flow injection chemiluminescence determination of thiamine by the
enhancement of luminol- K3Fe(CN)6 system 221
Li YH, Yang Y and Lu JR
Chemiluminescent and electron spin resonance spectroscopic
measurements of reactive oxygen species generated in water treated
with Titania-coated photocatalytic fibers 225
Lin C, Tanaka K, Tanaka L and Kawano T
Contents xvii
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
237
Motoyoshiya J, Tanaka T, Kuroe M and Nishii Y
Effects of l,4-butanediol dimethacrylate on HL-60 cells metabolism
241 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 245
Nozaki 0, Munesue M, Momoi H, Shizuma M, Kawamoto H and Ikeda
T
Chemiluminescence study on the regulation of NADPH oxidase activity
by thioredoxin reductase in vascular endothelial cells 249
Shen X and Liu Z-B
Quantitative detection of singlet oxygen with a chemiluminescence
probe during photodynamic reactions 253
Wei Y, Xing D, Luo S, Xu Wand Chen Q
Flow-injection chemiluminescence determination of human serum
albumin based on fluoresceinyl Cypridina luciferin analog-'02
reaction 257
Xu W, Wei Y, Xing DA, Luo S and Chen Q
Charge-transfer-induced luminescence (CTIL) mechanisms of chemi-
and bioluminescence reactions 261
Yamaguchi K, Isobe H, Yamanaka S and Okumura M
xviii Contents
A novel synergistic enhancer for HRP-Luminol-H20 2 based
chemiluminescence and its application in immunoassay 265
Yang X and Sun X
Separation and detection of amino acids with a novel capillary
electrophoresis chemiluminescence system 269
Yin DG, Xie CJ, Liu BH and Wu MH
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
273
Yin DG, He YF, Liu YB, Shen DC, Han SQ, Luo ZF, Xie CJ, Zhang L,
Liu BH and Wu MH
Determination of ascorbic acid by a flow injection
chemiluminescence method with a novel rhodanine 277
Yu J, Zhang C, Tan Y, Ge S, Dai P and Zhu Y
Study of superweak luminescence in plants and application to salt
tolerance in alfalfa 281
Zhou H, Yang Q and Liu Y
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 287
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 291
Zomer G and Hamzink M
PART 5. APPLIED ELECTROLUMINESCENCE
Wei J and Zhang L 297
Contents xix
Capillary electrophoresis - electrochemiluminescence detection of
ciprofloxacin in biological fluids
Zhou X and Jia L
301
305
A novel multicolor fluorescent protein from the soft coral
Scleronephthya gracillima Kuekenthal 311
Kato Y, Jimbo M, Sato C, Takahashi T, lmahara Yand Kamiya H
Fluorescence from STlevel of complexes of tryptophan with europium
(III) in water-ethanol solution 315
Osina 10, Ostahov Sand Kazakov V
Identification of developmental enhancers using targeted regional
electroporation (TREP) of evolutionarily conserved regions
319
Pira CU, Caltharp SA, Kanaya K, Manu SK, Greer LF and Oberg
KC
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 325
Hu S, Yang H, Cai R, Zhang Q and Yang X
Synthesis and photoluminescence of green-emitting
X2-(Y,GdhSiOs:Tb3+ phosphor under VUV excitation 329
Zhang ZH, Wang YH and Li XX
Luminescent properties of Na2CaMg2Si401s:Tb3+ nano-sized phosphor
333 Zhou L-Y, Yi L-H, Huang J-L, Wei J-S and Gong F-Z
xx Contents
The measurement of cytosolic ATP during apoptosis: Bioluminescence
imaging at the single cell level 339
Akiyoshi R and Suzuki H
Bioluminescence imaging of bacteria-host interplay: Interaction of
E. coli with epithelial cells 343
Brovko LY, Wang H, Elliot J, Dadarwal R, Minikh 0 and Griffiths
MW
Ultrasensitive chemiluminescent immunochemicallocalisation of
protein components in painting cross-sections 347
Dolci LS, Sciutto G, Rizzoli M, Guardigli M, Mazzeo R, Prati S and
RodaA
Development of a new device for ultrasensitive
electrochemiluminescence microscope imaging 351
Dolci LS, Rizzoli M, Marzocchi E, Zanarini S, Della Ciana L and
RodaA
Visualization of sequential response in intra cellular signal
transduction cascade by fluorescence and luminescence imaging in
the same living cell 355
Hatta-Ohashi Y, Takahashi T and Suzuki H
Bioluminescence imaging of intracellular calcium dynamics by the
photoprotein obelin 359
The! MM, Sugiyama T and Suzuki H
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
367
Zhang Land Wen F
PART 9. ASPECTS OF FLUORESCENCE AND PHOSPHORESCENCE
The interaction of Tb3+-protocatechuic acid complex with nucleic
acids and its application in determination of nucleic acids based
on fluorescence quenching 373
Chen Y, Yang Yand Yang J
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
381
385
pharmaceutical formulations 389 Lee SH, Kim WH, Meea K and Khan
MA
Improvement in carbaryl assay by fluorescence in a micellar medium
393 Lee SH, Jean CW, Kim WH, Chung HY, Wabaidur SM, Park HW, Suh YS
and Khan MA
Study of the interaction between human serum albumin and
7-ethyl-1O- hydroxycamptothecin 397
Li G and Liu Y
Resonance Rayleigh scattering method for determination of alginic
sodium diester with methylene blue 401
Liu Yand Li G
Effects of metal ions on peroxynitrite nitrifying protein 405 Luo
Y, Cui S, Zhang L and Zhong R
xxii Contents
Mechanism and properties of bio-photon emission and absorption of
protein molecules in living systems 409
Pang X-F
The mechanism of photon emission of bio-tissues and its properties
415 Pang X-F and Cao X-Y
Synthesis of a novel fluorescence probe of P-CD and cuprous iodide
pyridine and its application 421
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 425
Shen QJ, Zou WS, Jin WJ and Wang Y
Study on the interaction between methyl blue and HSA in the
presence of P-CDIHP-P-CD by molecular spectroscopy 429
Song S, Hou X, Shuang S and Dong C
Study on the interaction of kaempferol with human serum albumin by
spectroscopy and molecular modeling 433
Tian J, Liu J, Hu Z and Chen X
Selection of salt-tolerant rice variety using light-induced delayed
fluorescence 437
Wang J, Xu W, Xing D and Zhang L
Effects of LMWOA on biodegradation of phenanthrene studied by
fluorimetry 441
Wei XY, Sang LZ, Zhu YX and Zhang Y
Alleviation effects of salicylic acid and lanthanum on ultra weak
bioluminescence in maize leaves under cadmium stress 445
Wei ZL, Jiao CZ, Su YN and Tian ZH
Rhodamine B-quinoline-8-amide as a fluorescent "ON" probe for
Fe3
+ in acetonitrile 449 Xiang Y, Li ZF and Tong AJ
Contents xxiii
Studies on determination of deoxyribonucleic acid by second order
scattering with a novel rhodanine 453
Yu J, Li B, Zhu Y, Cheng X and Zhang L
Fluorescence characteristics of novel
chlorophenyl-arsenoxylphenylazo rhodanines and application in the
determination of thallium (I) 457
Yu J, Cheng X, Ge S, Tan Y and Li B
Molecular recognition of amino acids by hematoporphyrin and
metallohematoporphyrin receptors 461
Zhang Y, Lei Y-C and Liu D-S
Determination of BSA by its enhancement effect on second order
scattering of 3-( 4'-methyl phenyl)-5-(
4'-methyl-2'-sulfophenylazo) rhodanine 465
Zhu Y, Yu J, Dai P, Zhang C and Li B
Index 469
PARTl
Cambridge, MA 02138, USA
INTRODUCTION It is a great pleasure to participate in this 15th
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 EDT A
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 subsequent
reactant has been withheld, as confirmed by its crystal
structure.6
• 7 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.
1\ ,~ \~ "., a 4
\
~\ . \
o 1 2 3 4 5 6 7 8 9 10 msec (x 10 Z)
Fig. 1. Kinetics of the reaction of aequorin with calcium mixed in
a stopped-flow apparatus at 230 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 discovered8
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.
cold water
f 5 10
Firefly: both 19.(:;i19[91H1 & IYGiJ~ril) remain; A TP
exhausted
combiY Time (min.)
5 10 lime (min.)
Firefly: luciferir & ATP remain
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 A TP 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.
_Flashes c
TlME- MINUTES
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, Strehler16 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 system20 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
AMP+PP NAOP'
FMNHz
IOH very fast t 0
IGHT + H 0 + FMN h!d(er~~~:-flaYi!l hYclron L 2 the emItter
Fig. 5. Pathways and intermediates in the bacterialluciferase
reaction.
8 Hastings JW
10
1.0
0.1
.01
t:..O.D.- 660 NM o IN VIVO LUM. o IN VITRO LUM . • CRM
234 5 Time - Hours
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
km2
, 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 be concentrated on a
substrate, perhaps a filamentous of some autoindueer could
accumulate. Future studies should give the answer.
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
by which have been shown to be involved in the
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 responsible for regulation by pH.33 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. Schram E, Stanley P. eds. International Symposium on
Analytical Applications
of Bioluminescence and Chemiluminescence. Westlake, CA: State
Printing & Publishing, Inc. 1979: 696 pp.
2. Hastings JW, Johnson C. Bioluminescence and chemiluminescence.
Meth Enz. 2003;360:75-104.
3. Wilson T, Hastings JW. Bioluminescence. Annu Rev Cell Devel BioI
1998;14:197-230.
4. 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.
5. Shimomura 0, Johnson F. Regeneration of the photoprotein
aequorin. Nature 1975;256:236-8.
6. 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.
7. 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.
8. McElroy WD. The energy source for bioluminescence in an isolated
system. Proc Natl Acad Sci 1947;342-5.
9. 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.
10. Case J, Strause L. Neurally controlled luminescent systems. In:
Herring P. Ed Bioluminescence in Action. London: Academic Press,
1978:331-45.
II. 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.
12. Ghiradella H, Schmidt J. Fireflies at 100: A new look at flash
control. Integrat Comp BioI 2004;44:202-12.
13. 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.
14. 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.
15. McElroy WD, Hastings JW. Initiation and control of firefly
luminescence. In: Prosser C. Ed. Physiological Triggers. New York,
NY:Ronald Press, 1956:80-4.
16. Strehler B. Luminescence in cell-free extracts of luminous
bacteria and its activation by DPN. J Am Chern Soc
1953;75:1264.
17. Haas E. Bioluminescence from single bacterial cells exhibits no
oscillation. Biophys J 1980; 31: 301-12.
18. Hastings JW, Gibson Q. Intermediates in the bioluminescent
oxidation of reduced flavin mononucleotide. J BioI Chern
1963;238:2537-54.
Progress, Perspectives and Problems in Basic Aspects of
Bioluminescence 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 pH regulated 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; 1 04:696-70 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 Hastings JW
36. 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.
37. 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 MALLEFET1,2 1 Laboratory 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.
10000
1000
100
10
Shark length (em)
l. Maximum light emission of the ventral zone by hydrogen peroxide
in relation to the size
of the sharks. Dashed line separates embryos from free-swimming
fish. Values are expressed as mean ±SEM.
s:>.. 120
1 20 ~
Fig. 4. Drugs triggering light in E. spinax. SNP = Sodium
nitroprusside (NO-donor). Mt Melatonin. Pt = prolactin. N 7 (except
for 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;14:197-
230. 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 NIWA1
J Dept 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 (<PBL =:: 0.3). This indicates that
the chemiexcitation process in the bioluminescence reaction
produces an excited molecule with a high efficiency. To learn how
to design efficient chemiluminescent molecules from nature, it is
important to clarify the chemiexcitation mechanism in
bioluminescence. For this purpose, we investigated the
chemiluminescence of a series of 6-arylimidazopyrazinones 1 as a
bioluminescence model. From the results, we explained the mechanism
for the highly efficient chemiexcitation in Cypridina
bioluminescence: thermal decomposition of the N-H form of
dioxetanone intermediate gives the singlet-excited state (Sl) of
oxyluciferin with intramolecular charge transfer (lCT) character
via an ICT transition state (TS). The similarity of the strong ICT
character of Sl and TS leads the chemiexcitation process to be
efficient with the charge transfer-induced luminescence (CTIL)
mechanism. 1
• 2 In this paper, we apply the chemiexcitation mechanism to
the
Aequorea bioluminescence system.
coelenteramide (phenolate anion)
Light emission from chemiluminescence reactions was monitored with
a luminometer (PMT: Hamamatsu R5929) at 25 ± 1°C. Chemiluminescence
quantum yields (<Pcd were determined as values relative to
<PCL (0.0l3) of luminol in DMSO containing t-BuOK under air.
Product-analyses with HPLC were carried out with a Merck
19
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 chemiluminesces with a high <Del.3 Thus, it has been
predicted that the mechanism of the chemiluminescence reaction of
Cypridina luciferin in diglyme/acetate buffer is similar to that of
the bioluminescence reaction. Under this condition, 1 having an
electron-donating aryl group at C6 chemiluminesces with light
emission arising from the N-H form of 12*. Further, it was
clarified that the reaction mechanism includes the chemiexcitation
process from the N-H form of dioxetanone 3 to 12* (Figure 1).1 We
reported the chemiluminescent property of la,b in diglyme/acetate
buffer, which have electron-donating 4-(dimethylamino)phenyl and
3-indolyl groups, respectively. 1 In addition, we investigated
chemiluminescence of lc, which is a prototype for coelenterazine.
Chemiluminescence of lc in diglyme/acetate buffer showed light
emission arising from 12c* (Am ax 403 nm), but not from phenolate
anion of 12c*. The <Del for Ic was 0.0041. A <Del consists of
the product of three efficiencies: <Del == <DR X <Ds X
<DF, where <DR is the efficiency of producing 2; <Ds is
the chemiexcitation quantum yield; and <DF is the fluorescence
quantum yield of2. To estimate the <Ds for Ic, the <DR (0.70)
was determined by HPLC analyses of the product 2c and the <Dr
(0.27) of 2c was measured in diglyme/acetate buffer. Then, the
<Ds for lc was calculated as 0.021. The result that the <Ds
for Ic was smaller than that for la (0.07) corresponds to the
difference of the electron donating ability of the substituents (R)
on the phenyl groups (OH < NMe2).1
0t{ ~J(
~"9 . chemiexcitation
+ + 0""'r 0Yl * ~~~ N NH -co2 • N NH __ _ jV jV a:Ar=~M" Ar ~ ~ Ar
~ b: Ar = 3-indolyl 3 1
2 "
2 + hv
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--C6H4) 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 <I>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
<I>s. To reproduce the high efficiency of Aequorea biolumi
nescence, 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 D.E J.1ID qDo qN~)t qAr state I kcal mol-! (D.J.1t
(D.qt (M: a (D.qt 3c(OH) 0.00 2.35 0.049 -0.181 0.074
3c(OH)-TS 29.32 5.85 -0.128 -0.062 0.152 (+3.50) (-0.177) (+0.119)
(+0.078)
3c(O Lij 0.00 9.21 0.037 -0.219 0.130
3c(O-Lt)-TS 25.07 18.79 -0.244 -0.058 0.296
(+9.58) (-0.281) (+0.161) (+0.165) 3c(O Na+) 0.00 12.68 0.031
-0.238 0.158
3c(O-Na+)-TS 22.42 23.90 -0.285 -0.066 0.358
(+ 11.22) (-0.316) (+0.172) (+0.200) a Differences between the
values for 3-TS and the corresponding 3. The results of the
previous fluorescence study on coelenteramide phenolate
anion4
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-- 81 route in the CTIL
mechanism.
$ B·H
[ ~':'O]\ O?'f-R
Nx.NH
1.'1 E9 11'''(- CH,C,H, O-,R B-H 6 0..Q,.!T , N NH transition state
(T5)
d ;r" (strong ICT) ~, N~H,C.H5 ~
9 0
_ coelenteramide 51 + CO
2 phenolate anion
d o' ,Nx.NH
SO + cO2 E9 "" I N' CH,C,H, (weak ICT) B·H eo ,..
Fig. 2. The ICT TS---+S 1 route in the CTIL mechanism for Aequorea
bioluminescence [R = 4-hydroxyphenyl]
REFERENCES 1. 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.
2. 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.
3. Goto T. Chemistry of bioluminescence. Pure Appl Chern 1968; 17:
421-41. 4. 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, CO2 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 firefly Photinus pyralis. 6
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 PhRE 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 A TP 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. 1 A). Moreover H245, S284 and
H433 are in the conserved regions (data not shown). Bioluminescence
spectra. As is depicted in Fig 1 B 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 1 B,
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.
Mutants
Asterisks identity minor peaks. Error associated with Km ±
10%
L 'f,' ATP Quantum Specific . Yield *1014 activity*IOI3 RelatIve
UCI enn Km
Km(IlM) (IlM)
ITPEG.,GWLHS
(RLU/s/mg) ~tlVlty
1.5±0.IS 100 0.36 ± 0.04 24 1.15±0.14 76.6 0.25 ± 0,07 16.6 0.73 ±
0.14 81
Amax(nm) Optimum
pH = 7.8 pH = 5.5 temperature ("C)
555 560 24 618 619 30
572*,617 617 24 564*,612 619,564* 24 558*,616 618,560· 34
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
Ti02-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 Ti02. The exPCA
Wl.2 is also equipped with two ultrasonic (USW) generating devices
for mixing. When required, O2 alone or O2 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). Cell death was assessed
by staining cells with Evans Blue. Quantitative was
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 29
~L 10min
• ,I 0 _j'~USW J' / • I NO Control ~
• •
Q) (.)
~ 8000 u 7000 ~ 6000 c: 5000 .- 4000 § S' 3000 - i: 2000 'E - 1000
Q) 0 J: u « ..J U
o without fiber ~ with fiber
Control UV +
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, O2 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.
~ ~ •
1~ .. uv+ usw + 02
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.
Control uv + usw uv + USW + 02
B 45
~ 20 'j§ 15
Control 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 wa
