biothiols, part a

Equilibrium Formation of Disulfide Bonds Disulfide formation is a formal two-electron oxidation: Biologically, electron donors and acceptors in this process include molecular oxygen, nic

Methods in Enzymology

Volume 2.51

Biothiols Part A Monothiols and Dithiols, Protein Thiols, and

Thiyl Radicals

Lester Packer DEPARTMENT OF MOLECULAR AND CELL BIOLOGY

UNIVERSITY OF CALIFORNIA BERKELEY, BERKELEY CALIFORNIA Editorial Advisory Board Bob B Buchanan Arne Holmgren Enrique Cadenas Alton Meister Carlos Gitler Helmut Sies

ACADEMIC PRESS

San Diego New York Boston London Sydney Tokyo Toronto

P r e f a c e

Biothiols participate in numerous cellular functions, such as biosyn- thetic pathways, detoxification by conjugation, and cell division In re- cent years, studies on oxidative stress have amply documented the key role of thiols more specifically the thiol-disulfide status of the cell in a wide array of biochemical and biological responses Awareness of the great importance of biothiols in cellular oxidative injury has grown along with the recognition of free radicals in biological processes The reactions

of thiols with free radicals are not only of interest in free radical chemis- try: the most abundant nonprotein thiol in the cell, glutathione, is essen- tial for the detoxification of peroxides as cofactors of various selenium- dependent peroxidases The high concentration of glutathione in cells clearly indicates its general importance in metabolic and oxidative detoxi- fication processes In many ways, glutathione may be considered the master antioxidant molecule, a phrase which Alton Meister, one of the pioneers in glutathione research and a contributor to this volume, has used Bolstering of glutathione by other thiols, both natural (such as a-lipoic acid) and synthetic (such as Ebselen and several other drugs), has been investigated as a therapeutic approach to the oxidative component

of various pathologies Moreover, the redox changes of several thiol- containing proteins may be involved in key regulatory steps of the en- zyme as well as in cell proliferation

The contributions to Volumes 251 and 252 of Methods in Enzymology

(Biothiols, Parts A and B) provide a comprehensive and detailed account

of the methodology relating to the molecular mechanisms underlying the multiple functions of biothiols, with emphasis on their interaction at the biochemical and molecular biological levels in cellular reactions, with oxidants and other biological and clinical implications of thiols The con- tributions to this volume (Part A) include methods relating to thiyl radi- cals; chemical basis of thiol/disulfide measurements; monothiols: mea- surement in organs, ceils, organelles, and body fluids; dithiols: a-lipoic acid; and protein thiols and sulfides In Part B (Volume 252) methods are included on glutathione: distribution, biosynthesis, metabolism, and transport; signal transduction and gene expression; thioredoxin and glu- taredoxin; and synthetic mimics of biological thiols and thiols inhibitors Credit must be given to the experts in various specialized areas selected

to provide state-of-the-art methodology The topics and methods included

in these volumes were chosen on the excellent advice of the volume

xiii

xiv PREFACE

advisors, Bob B Buchanan, Enrique Cadenas, Carlos Gitler, Arne Holm- gren, Alton Meister, and Helmut Sies, to whom I extend my thanks and most grateful appreciation

LESTER PACKER

C o n t r i b u t o r s to V o l u m e 251

Article numbers are in parentheses following the names of contributors

Affiliations listed are current

MIGUEL ASENSI (21), Departamento de Fi-

siologla, Facultad de Medicina, Universi-

dad de Valencia, 46010 Valencia, Spain

TAK YEE AW (19), Department o f Physiol-

ogy and Biophysics, Louisiana State Uni-

versity Medical Center, Shreveport, Loui-

siana 71130

AALT BAST (28), Department o f Pharmaco-

chemistry, Division o f Molecular Phar-

macology, Vr(/e University, 1081 HV Am-

sterdam, The Netherlands

INGRID BECK-SPEIER (44), GSF-Forschung-

szentrum fiir Umwelt und Gesundheit,

lnstitut far Inhalations biologie, 85764

Oberschleissheim, Germany

KATJA BECKER (15), Institutfiir Biochemie

II, Universitiit Heidelberg, 69120 Heidel-

berg, Germany

GERREKE P BIEWENGA (28), Leiden~Am-

sterdam Center for Drug Research, De-

partment o f Pharmacochemistry, Divi-

sion o f Molecular Pharmacology, VrUe

Universiteit, 1081 HV Amsterdam, The

Netherlands

WALTER A BL/iTTLER (20), ImmunoGen,

Inc., Cambridge, Massachusetts 02139

MICHAEL BOCKSTETTE, (23), Division oflm-

munochemistry, Deutsches Krebsfors-

chungszentrum, 69120 Heidelberg, Ger-

many

NATHAN BROT (45), Roche Research Insti-

tute, Roche Institute o f Molecular Biol-

ogy, Nuaey, New Jersey 07110

ENRIQUE CADENAS (9), Department o f Mo-

lecular Pharmacology and Toxicology,

School o f Pharmacy, University o f South-

ern California, Los Angeles, California

90033

ALBERT R COLLINSON (20), ImmunoGen,

Inc., Cambridge, Massachusetts 02139

JOHN A C O O K (17), Radiation Biology Branch, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Maryland 20892

ULRICH COSTABEL (44), Ruhrlandklinik, Ab- teilung fiir Pneumologie und Allergologie,

45239 Essen, Germany

CAROLL E CROSS (43), Department oflnter- nal Medicine, U C D Medical Center, Uni- versify of California, Davis, Sacramento, California 95817

HEINI W DIRR (22), Department of Bio- chemistry, University of Witwatersrand, Johannesburg, South Africa

W U L F DROVE (23), Division of Immuno- chemistry, Deutsches Krebsforschungs- zentrum, D-69120 Heidelberg I, Germany

STEVEN A EVERETT (5), Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex

THOMAS FISCHBACH (23), Division o f Im- munochemistry, Deutsches Krebsfors- chungszentrum, 69120 Heidelberg, Ger- many

ROBERT B F R E E D M A N (38), Research School of Biosciences, University of Kent, Canterbury CT2 7 N J, United King- dom

KAZUKO FUJIWARA (32), The Institute for Enzyme Research, University o f To- kushima, Tokushima 770, Japan

ix

X CONTRIBUTORS TO VOLUME 251

DAGMAR GALTER (23), Division oflmmuno-

chemistry, Deutsches Krebsforschungs-

zentrum, 69120 Heidelberg, Germany

HIRAM F GILBERT (2), Department of BiD-

chemistry, Baylor College of Medicine,

Houston, Texas 77030

CARLOS GITLER (25, 35), Department of

Membrane Research and Biophysics,

Weizmann Institute of Science, Rehovot

76100, Israel

HELMUT GMONDER (23), Division of Im-

munochemistry, Deutsches Krebsfors-

chungszentrum, 69120 Heidelberg, Ger-

many

PETER HADDOCK (40), The Rayne Institute,

St Thomas' Hospital, London, United

Kingdom

BARRY HAELIWELL (43), Department ofln-

ternal Medicine, UCD Medical Center,

University of California, Davis, Sacra-

mento, California 95817

DER1CK S nAN (29), Department of Molec-

ular and Cell Biology, University of Cali-

fornia, Berkeley, California 94720

GARRY J HANDELMAN (29), Department of

Molecular and Cell Biology, University of

California, Berkeley, California 94720

HILARY C HAWKINS (38), Research School

of Biosciences, Biological Laboratory,

University of Kent, Canterbury CT2 7N J,

United Kingdom

DANIELA HEINTZ (34), Department of Bio-

physics, Max-Planck Institute for Medical

Resource, D-69120 Heidelberg, Germany

SUZANNE HENDRICH (40), Department of

Food Science and Human Nutrition,

Iowa State University, Ames, Iowa 50011

ROBERT HUBER (22), Abt Strukturfor-

chung, Max-Planck-lnstitut fiir Bioche-

mie, 82152 Martinsried, Germany

CHRISTOPHER HWANG (18), Genzyme Cor-

poration, Framingham, Massachusetts,

01701

E M JACOBY (26), lnstitut fiir Biochemie,

Rheinisch-Westf~ilische Technische Hoch-

schule,AachenKlinikum,D-52057Aachen,

Germany

EDNA KALEF (35), Department of Mem- brane Research and Biophysics, Weiz- mann Institute of Science, Rehovot

76100, Israel

NOBUH[KO KATUNUMA (37), Institute for Health Sciences, Tokushima Bunri Uni- versity, Tokushima 770, Japan

TERUYUKI KAWABATA (30), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

RALF KINSCHERF (23), Division oflmmuno- chemistry, Deutsches Krebsforschungs- zentrum, 69120 Heidelberg, Germany

EIKI KOMINAMI (37), Jutendo University, School of Medicine, Tokyo 113, Japan

EDWARD M KOSOWER (11, 12), Biophysical

Organic Chemistry Unit, TeI-Aviv Univer- sity, Raymond and Beverly Sackler Fac- ulty of Exact Sciences, Ramat-Aviv, Tel- Aviv 69978, Israel

NECHAMA S KOSOWER (11, 12), Depart- ment of Human Genetics, Sackler School

of Medicine, Tel-Aviv University, Ramat- Aviv, TeI-Aviv 69978, Israel

R L KRAUTH-SIEGEL (26), lnstitutfitr Bio- chemie H, Universitiit Heidelberg, 69120 Heidelberg, Germany

SUBHAS C KUNDU (6), Department of Biol- ogy and Biochemistry, Brunel University, Uxbridge, Middlesex UB6 3PH, United Kingdom

SIDNEY R KUSHNER (45), Department of Genetics, University of Georgia, Athens, Georgia 30602

MARTIN KUSSMANN (4 l), Facuhyfor Chem- istry, University of Konstanz, 78434 Kon- stanz, Germany

GuY V LAMOUREUX (14), Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada

WATSON J LEES (14), Department of Bio- logical Chemistry and Molecular Phar- macology, Harvard Medical School, Bos- ton, Massachusetts 02115

CONTRIBUTORS TO VOLUME 251 x i ANr,~-G LENZ (44), GSF-Forschungszen-

trum fiir Umwelt und Gesundheit, lnstitut

far Inhalations Biologie, 85764 Obersch-

leissheim, Germany

HARVEY F LODISH (18), Whitehead Insti-

tute for Biomedical Research, Cam-

bridge, Massachusetts 02142

MAURlClO LONDNER (25), Department o f

Membrane Research and Biophysics,

Weizmann Institute o f Science, Rehovot

76100, Israel

KONRAD L MAIER (44), GSF-Forschungs-

zentrum far Umwelt und Gesundheit, In-

stitut fiir Inhalations Biologic, 85764

Oberschleissheim, Germany

LUISE MAINKA (31), Gustav-Embden-Zen-

trum der Biologischen Chemie, Klinikum

der Johann Wolfgang Goethe Universitiit,

D-60590 Frankfurt am Main, Germany

STEPHEN H McLAUGHLIN (38), Research

School o f Biosciences, Biological Labora-

tory, University o f Kent, Canterbury CT2

7N J, United Kingdom

ALTON MEISTER (1), Department o f Bio-

chemistry, Cornell University Medical

College, New York, New York 10021

DIANA METODIEWA (7), Institute o f Applied

Radiation Chemistry, Technical Univer-

sity, Lodz, Poland

SABINE MIHM (23), Division of Immuno-

chemistry, Deutsches Krebsforschungs-

zentrum, 69120 Heidelberg, Germany

JAMES B MITCHELL (17), Radiation Biology

Branch, National Cancer Institute, Na-

tional Institutes o f Health, Bethesda,

Maryland 20892

JACKOn MOSKOVITZ (45), Roche Research

Center, Roche Institute o f Molecular Bi-

ology, Nutley, New Jersey 07110

YUTARO MOTOKAWA (32), The Institute for

Enzyme Research, University of To-

kushima, Tokushima 770, Japan

REx MONDAY (10), AgResearch, Ruakura

Agricultural Research Centre, Hamilton,

New Zealand

GERALD L NEWTON (13), Department of

Chemistry and Biochemistry, University

o f California, San Diego, La Jolla, Cali- fornia 92093

HANS NOHL (16), Institute o f Pharmacology and Toxicology, Veterinary University o f Vienna, A-I030 Vienna, Austria

KENNETH M NOEL (46), Department of Molecular and Cell Biology, University o f Connecticut, Storrs, Connecticut 06269

CHARLES A O'NEILL (43), Department of Internal Medicine, UCD Medical Center, University o f California, Davis, Sacra- mento, California 95817

KAZUKO OKAMURA-IKEDA (32), The Insti- tute for Enzyme Research, University o f Tokushima, Tokushima 770, Japan

RENI~ Y OLIVIER (24), Unit~ d'Oncologie Viral, D~partment Sida et R~trovirus, In- stitut Pasteur, 75015 Paris, Cedex 15, France

LESTER PACKER (21, 29, 30), Department o f Molecular and Cell Biology, University o f California at Berkeley, Berkeley, Califor- nia 94720

RICHARD N PERHAM (42), Cambridge Cen- tre for Molecular Recognition, Depart- ment o f Biochemistry, University o f Cam- bridge, Cambridge CB2 1QW, United Kingdom

L.L POUESEN (27), Biochemical Institute, Department o f Chemistry and Biochemis- try, The University of Texas at Austin, Austin, Texas 78712

WILLIAM B PRATT (39), Department o f Pharmacology, University o f Michigan Medical School, Ann Arbor, Michigan

48109

MICHAEL PRZYBYLSKI (41), Faculty for Chemistry, University o f Konstanz, 78434 Konstanz, Germany

M ATIQUR RAHMAN (45), Department o f Internal Medicine, Section o f Digestive Diseases, Yale University School of Med- icine, New Haven, Connecticut 06510

PETER REINEMER (22), Bayer AG, Pharma Research, PH-FE/NASP, D-42096 Wup- pertal, Germany

xii CONTRIBUTORS TO VOLUME 2 5 1

FRI~DERIC M RICHARDS (33, 36), Depart-

ment of Molecular Biophysics and Bio-

chemistry, Yale University, New Haven,

Connecticut 06520

STEFFEN ROTH (23), Division of Immuno-

chemistry, Deutsches Krebsforschungs-

zentrum, 69120 Heidelberg, Germany

JUAN SASTRE (21), Departamento de Fi-

siologia, Facultad de Medicina, Universi-

dad de Valencia, 46010 Valencia, Spain

R HEINER SCHIRMER (15, 26), Institut far

Biochemie II, Der Universitiit Heidel-

berg, 69120 Heidelberg, Germany

CHRISTIAN SCHONEICH (4), Department of

Pharmaceutical Chemistry, Malott Hall,

University of Kansas, Lawrence, Kansas

66045

S STONEY SIMONS, JR (39), Steroid Hor-

mones Section, Laboratory of Molecular

and Cellular Biology, National Institute

of Diabetes and Digestive and Kidney

Diseases, National Institutes of Health,

Bethesda, Maryland 20892

RAJEEVA SINGH (14, 20), ImmunoGen, Inc.,

Cambridge, Massachusetts 02139

ANTHONY J SINSKEY (18), Massachusetts

Institute of Technology, Cambridge,

Massachusetts 02139

KLAUS STOLZE (16), Institute of Pharmacol-

ogy and Toxicology, Veterinary Univer-

sity of Vienna, A-1030 Vienna, Austria

JEFFREY STRASSMAN (45), Roche Research

Center, Roche Institute of Molecular Bi-

ology, Nutley, New Jersey 07110

JAMES A THOMAS (40), Department of Bio-

chemistry and Biophysics, Iowa State

University, Ames, Iowa 50011

HANS-JORGEN TRITSCHLER (30), Medical

Research Department, ASTA Medica

AG, Frankfurt-am-Main, D-60314 Ger-

many

HEINZ ULRICH (31), ASTA Medica AG,

Frankfurt-am-Main, D-60314 Germany

ALBERT VAN DER VLIET (43), Department

of Internal Medicine, UCD Medical Cen-

ter, University o f California, Davis, Sac-

ramento, California 95817

Jos~: VIiqA (21), Departamento de Fi- siologia, Facultad de Medicina, Universi- dad de Valencia, 46010 Valencia, Spain

CLEMENS VON SONNTAG (3), Max-Planck- Institut far Strahlenchemie, D-45413 Miilheim an der Ruhr, Germany

PETER WARDMAN (3, 5), Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, England

LEV M WEINER (8, 16), Department of Or-

ganic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

HERBERT WEISSBACH (45), Roche Research Center, Roche Institute of Molecular Bi- ology, Nutley, New Jersey 07110

GEORGE M WHITESIDES (14), Department

of Chemistry, Harvard University, Cam- brige, Massachusetts 02138

ROBIN L WILLSON (6), Department o f Biol-

ogy and Biochemistry, Brunel University, Uxbridge, Middlesex UB6 3PH, United Kingdom

CHRISTINE C WINTERBOURN (7), Depart- ment of Pathology, Christchurch School

of Medicine, Christchurch, New Zealand

RICHARD WYNN (33, 36), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecti- cut 06520

STEPHANIE O YANCEY (45), Department o f

Genetics, University of Georgia, Athens, Georgia 30602

BATIA ZARMI (35), Department of Mem- brane Research and Biophysics, Weiz- mann Institute of Science, Rehovot

76100, Israel

WEI ZHAO (40), Department of Biochemis- try and Biophysics, Iowa State Univer- sity, Ames, Iowa 50011

D M ZIEGLER (27), Biochemical Institute, Department o f Chemistry and Biochemis- try, University of Texas at Austin, Austin, Texas 78712

GUIDO ZIMMER (31), Gustav-Embden-Zen- trum der Biologischen Chemie, Klinikum der Johann Wolfgang Goethe Universit?it, D-60590 Frankfurt am Main, Germany

HOOCCHNH2(CH2)2CONHCHCONHCH2COOH

I

C H z S H Glutathione Glutathione, which is an t~-amino acid as well as a tripeptide, evolved as

a molecule that protects cells against oxidation 2 Glutathione has a number

of important functions in metabolism, catalysis, and transport Its antioxi- dant functions are closely associated with its role in providing the cell with its reducing milieu; this arises from the reducing power of NADPH The enzyme glutathione disulfide reductase (GSSG reductase, EC 1.6.4.2) thus catalyzes an equilibrium that greatly favors formation of GSH It is notable that most of the GSH present in cells is in the thiol form and that most (greater than 90%) of the nonprotein sulfur of the cell is in the form of GSH These points were recognized many years ago by Hopkins 3 Glutathione maintains enzymes and other cellular components in a reduced state Gluta- thione also functions as a storage and transport form of cysteine moieties Glutathione is synthesized within cells and is typically exported from cells The intracellular stability of GSH is promoted by the GSSG reductase system as noted above, and also by the fact that GSH is not a substrate of y-glutamylcyclotransferase (EC 2.3.2.4), nor is it susceptible to the action

of cellular peptidases

1 For reviews, see: D Dolphin, R Poulson, and O Avramovic (eds.), in "Glutathione Chemi-

cal, Biochemical and Medical Aspects, Parts A and B." Wiley, New York, 1989; N Taniguchi,

T Higashi, Y Sakamoto, and A Meister (eds.), in Glutathione Centennial Molecular

Perspectives and Clinical Implications." Academic Press, New York, 1989; A Larsson, S

Orrenius, A Holmgren, and B Mannervik (eds.), in "Functions of Glutathione, Biochemical,

Physiological, Toxicological and Clinical Aspects." Raven, New York, 1983; A Meister

and M E Anderson, Annu Rev Biochem 52, 711 (1983); A Meister, Pharmacol Ther

51, 155 (1991); A Meister, this series, Vol 113, p 571

2 R C Fahey and A R Sundquist, Adv Enzyrnol 64, 1 (1991)

3 F G Hopkins, Biochem J 15, 286 (1921)

Copyright © 1995 by Academic Press, Inc

Metabolism of Glutathione

A summary of the metabolism of GSH is given in Fig 1.4 The reactions

of the T-glutamyl cycle account for the synthesis and breakdown of GSH Glutathione is synthesized by the consecutive action of y-glutamylcysteine synthetase (glutamate-cysteine ligase, EC 6.3.2.2) and GSH synthetase (EC 6.3.2.3) (reactions 1 and 2) y-Glutamylcysteine synthetase is feedback inhibited by GSH 5'6 and therefore does not proceed at its maximal rate

4 A Meister, J Biol Chem 263, 17205 (1988)

5 p Richman and A Meister, J Biol Chem 250~ 1422 (1975)

6 C.-S Huang, L.-S Chang, M E Anderson, and A Meister, J Biol Chem 268, 19675 (1993)

[ 1] GLUTATHIONE METABOLISM 5 under normal physiological conditions The reaction catalyzed by this en- zyme appears to be the rate-limiting step in GSH synthesis; as discussed in Modulation of Glutathione Metabolism (below), this reaction is selectively inhibited by certain agents

The degradation of GSH occurs extracellularly This process involves the activity of y-glutamyl transpeptidase (y-glutamyltransferase, EC 2.3.2.2; reaction 3) and that of dipeptidases (reaction 4), which are bound to the external surfaces of cell membranes Glutathione is exported to the mem- brane-bound enzymes Some GSSG may also be transported normally; the amount exported increases when the intracellular level of GSSG increases S-Conjugates of GSH (see below) are also exported to the membrane- linked enzymes, y-Glutamyl transpeptidase thus acts on GSH, GSSG, and S-conjugates of GSH Transpeptidation, which takes place in the presence

of amino acids, leads to formation of y-glutamyl amino acids 7 Cystine is the most active amino acid acceptor 8 but other neutral amino acids such

as methionine and glutamine are also good acceptors 9 y-Glutamyl amino acids formed in this way are transported into certain cells, y-Glutamyl amino acids, in contrast to GSH, are substrates of the intracellular enzyme y-glutamylcyclotransferase (EC 2.3.2.4), which converts ~/-glutamyl amino acids into 5-oxoproline and the corresponding free amino acids (reaction 5).1° 5-Oxoproline is converted to glutamate in the ATP-dependent reaction catalyzed by 5-oxoprolinase (EC 3.5.2.9; reaction 6) 11

Exported GSH and extracellular cystine interact with y-glutamyl trans- peptidase, leading to the formation of y-glutamylcystine The latter is trans- ported into the cell (reaction 13) and reduced to form cysteine and -y-glutamylcysteine (reaction 10), which are substrates, respectively, of y-glutamylcysteine synthetase and GSH synthetase This constitutes a by- pass of the reaction catalyzed by y-glutamylcysteine synthetase and serves

as a recovery system for cysteine moieties 12 Cysteinylglycine may be split extracellularly or be oxidized and split to form cystine and glycine The dipeptide may also be transported into the cell and hydrolyzed intracellu- larly; this has not yet been studied In some cells transport of 3,-glutamylcys- tine constitutes a major pathway for transport of cysteine moieties Glutathione is used by several GSH transhydrogenases (reaction 10)

7 R D Allison and A Meister, J Biol Chem 256, 2988 (1981)

8 G A T h o m p s o n and A Meister, Proc Natl Acad Sci U.S.A 72, 1985 (1975)

9 S S Tate and A Meister, J Biol Chem 249, 7593 (1974)

10 A Meister, this series, Vol 113, p 438

ii A Meister, O W Griffith, and J M Williamson, this series, Vol 113, p 445; A P Seddon,

L Li, and A Meister, this series, Vol 113, p 451

12 M E Anderson and A Meister, Proc Natl Acad Sci U.S.A 80, 707 (1983)

6 OVERVIEW [11

as well as by GSH peroxidases (reaction 9), and the GSSG formed in these reactions is converted to GSH by GSSG reductase (reaction 12) 13 Conversion of GSH to various S-substituted adducts occurs nonenzymati- cally and may also be catalyzed by various GSH S-transferases (reaction 7) The GSH S-transferases are of increasing interest in relation to the detoxification of certain drugs, m5 There is endogenous formation of GSH S-conjugates as well; for example, such GSH conjugates are formed with leukotriene A 16 and with estrogensY The GSH S-conjugates with drugs as well as those formed with compounds of endogenous origin follow the mercapturic acid pathway, which usually involves conversion to the corre- sponding conjugates of cysteinylglycine The latter are cleaved by dipepti- dase to give the S-conjugates of cysteine (reaction 4) These may be ace- tylated to form mercapturic acids (reaction 8) Other chemical trans- formations of the mercapturic acids and their precursors have also been ob- served TM

Glutathione serves as an antioxidant by reacting directly with free radi- cals (reaction 11) and by providing substrate for the GSH peroxidases and for the GSH transhydrogenases Thus, a variety of reductive reactions that take place within the cell depend on GSH These include reactions that lead to the formation of deoxyribonucleotides and ascorbate (from dehy- droascorbate), and a host of reactions involving conversion of disulfides to the corresponding thiol forms

Modulation of Glutathione Metabolism

Methods for decreasing cellular GSH levels have been reviewed in this series 19'2° In general, the use of buthionine sulfoximine is advantageous because this agent (or similar amino acid analogs) inhibits the first step of GSH synthesis and therefore selectively decreases cellular levels of GSH

as well as the cellular capacity for GSH synthesis 2°"

Methods for increasing cellular GSH levels include administration of compounds that lead to increased cellular levels of cysteine, which is usually

13 See: A Meister (ed.), this series, Vol 113

14 E Boyland and L F Chasseaud, Adv Enzymol 32, 173 (1969)

15 L F Chasseaud, Drug Metab Rev 2, 185 (1973)

16 L Orning, S Hammarstrom, and B Samuelson, Proc Natl Acad Sci U.S.A 77, 2014 (1980)

17 E Kuss, Z Physiol Chem 352, 817 (1971)

18 j L Stevens and D P Jones, in "Glutathione Chemical, Biochemical and Medical Aspects,

Part B," p 45 Wiley, New York, 1988

19 j L Plummer, B R Smith, H Sies, and J R Bend, this series, Vol 77, p 50

20 A Meister, this series, Vol 113, p 571

2oa See: A Meister, this series, Vol 252

[ 1 ] GLUTATHIONE METABOLISM 7 the limiting substrate for G S H synthesis; such c o m p o u n d s include N-acetylcysteine and 2-0xothiazolidine 4-carboxylate 21 G l u t a t h i o n e levels

m a y also b e increased by administration of y-glutamylcysteine or related

c o m p o u n d s , thus providing substrate for G S H synthetase (reaction 2; Fig 1) G l u t a t h i o n e esters, such as G S H mono(glycyl) esters and G S H diethyl ester, p r o v i d e an efficient way of increasing cellular G S H levels in vivo

and in vitro; these esters have b e e n reviewed in this series 22'23

in E s c h e r i c h i a coli h a v e b e e n isolated and used to t r a n s f o r m the wild strain

to one that o v e r p r o d u c e s the synthetases 24 This gene-enriched strain has

a high capacity for G S H synthesis It also exhibits increased radioresistance, which is associated with increased capacity to synthesize G S H C o m p a r a b l e studies with the m a m m a l i a n genes are feasible and in progress (see Refs

6 and 25) O t h e r modulations of G S H m e t a b o l i s m p r o d u c e d by selective inhibition of various enzymes have also b e e n achieved, 13 and m o r e recent

w o r k on the interactions b e t w e e n G S H and ascorbate have b e e n reviewed 26

21 M E Anderson and A Meister, this series, Vol 143, p 313

22 M E Anderson, E J Levy, and A Meister, this series, Vol 234, p 492

23 E J Levy, M E Anderson, and A Meister, this series, Vol 234, p 499

24 W R Moore, M E Anderson, A Meister, K Murata, and A Kimura, Proc Natl Acad Sci U.S.A 86, 1461 (1989)

2s N Yan and A Meister, J Biol Chem 265, 1588 (1990)

26 A Meister, J Biol Chem 269, 9397 (1994)

8 OVERVIEW [2]

[2] T h i o l / D i s u l f i d e E x c h a n g e E q u i l i b r i a a n d D i s u l f i d e

B o n d S t a b i l i t y

B y H I R A M F G I L B E R T

Disulfide bond formation is a versatile oxidation that is used biologically

in such diverse processes as enzyme catalysis, protection against oxidative damage, the stabilization of extracellular proteins, and the regulation of biological activity Because disulfide formation is a reversible process, disul- fide bond stability often plays an important role in the biological utility of disulfide bonds In turn, the ability to form and break a specific disulfide bond under appropriate biological conditions depends on the nature of the oxidant or reductant, the disulfide stability, the kinetics of the forward and reverse reactions, and the nature and redox state of the environment in which the reaction occurs The stability of disulfide bonds in small molecules and proteins spans an enormous range, a factor of approximately 1011, corresponding to a free energy difference of about 15 kcal/mol or a redox potential difference of 0.33 V 1 Several reviews, including many of the chapters in this volume, detail the biology of thiols and disulfides 1-7 The purpose of this chapter is to provide a brief overview of the importance of reversible thiol/disulfide exchange and a discussion of practical considera- tions in measuring disulfide bond stability

Equilibrium Formation of Disulfide Bonds

Disulfide formation is a formal two-electron oxidation:

Biologically, electron donors and acceptors in this process include molecular oxygen, nicotinamide and flavin cofactors, and other thiols and disulfides Reversible thiol/disulfide exchange reactions occur by the nucleophilic attack of a thiol (the thiolate anion is actually the reacting species) on one

of the two sulfurs of a disulfide/

1 H F Gilbert, Adv Enzymol 63, 69 (1990)

2 D M Ziegler, Annu Rev Biochem 54, 305 (1985)

3 B B Buchanan, Annu Rev Plant Physiol 57, 209 (1980)

4 H F Gilbert, this series, Vol 107, p 330

5 N, S Kosower and E M Kosower, Int Rev Cytol 54, 109 (1978)

6 T E Creighton, this series, Vol 131, p 83

7 j M Thornton, J Mol Biol 151, 261 (1981)

8 p C Jocylin, "Biochemistry of the Sulfhydryl Group." Academic Press, New York, 1972

Copyright © 1995 by Academic Press, Inc

To simplify reference to the various thiols and disulfides, the standard thiol/disulfide pair will be termed the "redox buffer." Although any thiol/ disulfide pair could serve as the redox buffer, for illustrative purposes the most abundant biological redox buffer, glutathione 9 (GSH) and its disulfide (GSSG) will be used as the standard redox buffer The other thiol/disulfide pair will be referred to as the "test system." The designations PSH, PSSG, P(SH)2, and P(SS) will be used to represent peptide or protein thiols and disulfide; however, other nonprotein systems will behave similarly

If there is only one sulfhydryl group in the test system and the redox buffer is present in large excess, the only oxidation product of the test system will be the unsymmetrical mixed disulfide:

to GSSG, the higher the value of the equilibrium constant Thus, Kmix represents an oxidation potential for the oxidation of the test thiol by GSSG 1° The equilibrium constant also depends on the relative stabilities

of the test thiol and GSH; however, for test thiols with pKa values near that of GSH, the effects are small 1 The effect of pH on redox equilibria

of thiols and disulfides has been discussed in detail, n Kmi× is unitless, and the extent of mixed disulfide formation, [PSSG]/[PSH], depends only on the equilibrium constant (Kmix) and on the thiol/disulfide ratio of the redox buffer (R = [GSH]/[GSSG])

9 A Meister and M E Anderson, Annu Rev Biochem 52, 711 (1983)

1o D W Walters and H F Gilbert, J Biol Chem 261, 15372 (1986)

11 R P Szajewski and G M Whitesides, J Am Chem Soc 102, 2011 (1980)

10 OVERVIEW [2]

If the test system has two sulfhydryl groups that are in close enough proximity, the initially formed mixed disulfide may be displaced by an intramolecular reaction leading to the formation of an intramolecular disul- fide While the overall reaction is complicated by the potential accumulation

of multiple redox isomers (see Complex Equilibria), in practice, the intra- molecular reaction is often so favorable that negligible mixed disulfide species are present at equilibrium If mixed disulfide intermediates are ignored, the overall equilibrium for formation of the intramolecular disul- fide becomes

of the test system, P ( S S ) / P ( S H ) > will depend on the equilibrium constant, Kox, and the quantity [GSH]2/[GSSG] (which is equivalent to the quan- tity R[GSH])

S t r u c t u r a l S t a b i l i z a t i o n b y D i s u l f i d e B o n d s

T h e cross-link introduced by the formation of a disulfide bond between two cysteine residues provides for increased protein stability T h e stability that a disulfide cross-link contributes to a folded protein depends on the

12 D W Walters and H F Gilbert, J Biol Chem 261, 13135 (1986)

~3 C Abate, L Patel, F J Rausher, and T Curran, Science 249, 1157 (1990)

14 F J Staal, M Roederer, and L A Herzenberg, Proc Natl Acad Sci U.S.A 87, 9943 (1990) ,5 R E Cappel and H F Gilbert, J Biol Chem 263, 12204 (1988)

in disulfide bond stability resulting from the folding of the protein into its tertiary structure,

Kox,fow/Kox,de, Thus, disulfide bonds are stabilized by protein folding to an amount that is equivalent to the contribution of the disulfide bond to stabilizing the folded protein (6)

stability of the disulfide bond itself The intramolecular cross-links intro- duced into an unfolded protein by disulfide bonds organize the unfolded state so that the entropy loss that accompanies folding is significantly smaller than in the protein without cross-links 16 By destabilizing the unfolded protein, this entropic effect stabilizes the folded protein in comparison to the unfolded state An alternative but equivalent view is that folding into the tertiary structure brings distant cysteines into close proximity, making disulfide bonds formed in the folded state more stable (more easily oxidized) than disulfide bonds formed in the unfolded protein 6

Disulfide bond stability and the stability provided to the folded protein are thermodynamically linked and can be described by the closed thermody- namic cycle 6 shown in Fig 1 As the reduced protein folds and brings the two cysteine residues into closer proximity, the oxidation potential increases, that is, Kox,fold >~ Kox,den Because of the thermodynamic linkage, the A G around the closed cycle of Fig 1 must sum to zero, and the increase

in the oxidation potential for disulfide bond formation that results from the protein folding is linked to a corresponding increase in the stability of the folded protein, Kfold,ox/ gfold,red = gox,fold/ gox,den

16 C N Pace, G R Grimsley, J A Thomson, and B J Barnett,J BioL Chem 263,11820 (1988)

12 OVERVIEW [2]

In small peptides and unfolded proteins, the thiol/disulfide oxidation potential depends on the sequence distance between the two cysteine resi- dues Because of geometric constraints, the most stable disulfide bond is formed by residues that have four to five intervening residues and decreases further as the intervening n u m b e r of amino acids increases 17 Kox values for disulfide formation in small peptides and denatured proteins are gener- ally less than 0.1 M In folded proteins, Kox may approach 105 M 1

T h e larger the ratio of Kox for the folded protein to that of the unfolded protein, the greater the stability of the disulfide-cross-linked protein com- pared to that of the reduced protein F o r example, the Kox of the redox active disulfide bond in thioredoxin increases from 26 m M in 8 M urea to

10 M when the protein is stably folded This corresponds to a free energy difference in disulfide stability of 3.5 kcal/mol Experimentally, the disulfide form of thioredoxin is 3.1-3.5 kcal/mol more stable toward urea-induced denaturation than is the dithiol form of the protein, corresponding almost exactly to the free energy change derived from the change in the dithiol oxidation potential that accompanies foldingJ 8 T h e observation that the disulfide bond in the dsbA protein of Escherichia coli is much less stable when the protein is folded lies at the other extreme of the coupling between disulfide bond stability and protein stability; the Kox decreases from 170

m M in the unfolded state to 0.081 m M in the folded stateJ 9 This suggests that the formation of the disulfide bond in dsbA is accompanied by the introduction of strain into the protein, a prediction borne out by experimen- tal measurements showing the disulfide redox state of dsbA is less stable toward denaturation than the dithiol redox state by 3.6 _+ 1.4 kcal/molJ 9

In these cases, the link between disulfide stability is simple: the more stable the disulfide, the m o r e it will contribute to increasing the protein stability

Regulatory Consequences o f Reversible Disulfide Bond Formation

Including Protein S-Thiolation

T h e concentrations of G S H and G S S G in cells and tissues are not constant, and cellular levels of G S H and G S S G change considerably in response to nutritional status, hormones, drugs, and the imposition of oxida- tive stress 2° A change in glutathione redox status (a change in [GSH], [GSSG], or both), if coupled to changes in the redox states of thiols and disulfides in specific proteins, could provide a regulatory signal that affects

17 R M Zhang and G H Snyder, Biochemistry 30, 11343 (1991)

18 T Y Lin and P S Kim, Biochemistry 28, 5282 (1989)

19 A Zapun, J C A Bardwell, and T E Creighton, Biochemistry 32~ 5083 (1993) 2o H Sies, R Brigelius, and P Graf, Adv Enzyme Regul 26, 175 (1987)

[2] THIOL/DISULFIDE EQUILIBRIA 13 the biological activities of enzymes, receptors, transporters, and transcrip- tion factors 1

Disulfide stability and the equilibrium oxidation potential places con- straints on the regulation of biological activity by this mechanism Thiol/ disulfide redox state changes in proteins are usually reversible, so that intracellular disulfide formation is constantly opposed by disulfide reduc- tion If the system is allowed to reach equilibrium, the extent of protein oxidation will be determined by the relationship between the cellular redox buffer and the oxidation potential of the protein At equilibrium, changes

in the glutathione status would be expected to change the oxidation state

of the protein (assuming that the redox state change were fast enough) significantly, only if the oxidation potential of the protein falls within the range of R or R[GSH] maintained by the cellular redox buffer If the oxidation potential of a protein lies significantly outside this range, changes

in the cellular redox buffer will have little effect on the equilibrium redox state of the protein; the protein will be predominantly reduced or oxidized under all conditions, and regulation would be unlikely unless some energy- dependent mechanism maintains the system under nonequilibrium condi- tions

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide (GSSG) The GSH concentration in most eukaryotic cells

is generally in the 2-10 m M range, depending on cell type and metabolic factors 9 GSSG, which is produced from GSH during the destruction of reactive oxygen species including hydroperoxides, is present at much lower concentrations (20-40/zM) owing to the activity of glutathione reductase (GSSG + N A D P H + H ÷ ~ 2GSH + NADP+) 1 Consequently, the ratio

of GSH/GSSG is normally in the range of 100-400 and the quantity R [GSH] varies between 0.2 and 4 M Drugs that are detoxified by the action of glutathione S-transferase, redox-active drugs that increase the production

of reactive oxygen metabolites, or oxidants such as hydrogen peroxide or diamide, may cause GSH levels to fall to less than 20% of normal 2I This may also be accompanied by a significant increase in the levels of GSSG, which may rise to concentrations comparable to GSH Thus, under condi- tions of oxidative stress, [GSH]/[GSSG] ratios may fall to 1-10, and R [GSH] may drop to values below 20 mM

The intrinsically large range of thiol/disulfide oxidation potentials for intramolecular protein disulfides spans the physiological range, and intra- molecular disulfide formation could easily provide a reversible redox-sensi- tive regulatory response to changes in both the ratio of [GSH]/[GSSG] and the concentration of GSH as well The enzyme hydroxymethylglutaryl-

21 D J Reed, Chem Res Toxicol 3, 495 (1990)

14 OVERVIEW [21 CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthe- sis, forms an inactive protein-protein disulfide with a/Cox of 0.6 M, 15 and metabolic changes in glutathione status are correlated with changes in cholesterol levels consistent with regulation of this biological process by reversible thiol/disulfide exchange 22 A number of other proteins including enzymes and transcription factors have been suggested to undergo a similar type of regulation; however, the thiol/disulfide redox potentials of these proteins are not yet known

The formation of mixed disulfides between the intracellular glutathione redox buffer and specific proteins (S-thiolation) has been observed under oxidative stress imposed by the oxidation of the glutatione pool by exoge- nous oxidants such as diamide and hydrogen peroxide 23 Because equilib- rium constants for mixed disulfide formation, Km~x, are usually near one, 1 significant accumulation of mixed disulfides at equilibrium should be low (compared to PSH) under normal physiological conditions where the ratio [GSH]/[GSSG] is greater than 100 However, oxidative stress results in significant oxidation of the glutathione to GSSG such that [GSH]/[GSSG] ratios near one are achieved, and under these conditions the accumulation

of specific mixed disulfides between proteins and glutathione is observed The natural tendency of thiol/disulfide systems to come to redox equilib- rium does not require that the system actually be at equilibrium in vivo

The presence of thiotransferases 24 that catalyze these reactions would make approach to equilibrium faster; however, there are suggestions that protein S-thiolation may occur via alternative reactions that do not involve GSSG and that may maintain a nonequilibrium concentration of S-thiolated pro- teinsY

Dithiols/Disulfides of Catalytic Importance

A number of flavin-dependent reductases, including thioredoxin reduc- tase, glutathione reductase, and lipoyl dehydrogenase (dihydrolipoamide dehydrogenase), 26 have vicinal thiols at the active site that shuttle between dithiol and disulfide redox states to mediate electron transfer between the substrate and flavin cofactor The only enzyme of this group in which the redox potential of the dithiol has been reported is thioredoxin reductase

22 S Kim, P Y Chao, and K G Allen, FASEB Z 6, 2467 (1992)

23 R M° Miller, H Sies, E M Park, and J A Thomas, Arch Biochem Biophys 276, 355 (1990)

24 W W Wells, Y Yang, T L Deits, and Z R Gan, Adv Enzymol 66, 149 (1993)

25 j A Thomas, E M Park, Y C Chai, R Brooks, K Rokutan, and R B Johnston, Adv Exp Med Biol 283, 95 (1991)

z6 C H Williams, in "Chemistry and Biochemistry of Flavoenzymes" (F Muller, ed.), Vol

3, p 121 CRC Press, Boca Raton, Florida, 1992

[2] THIOL/DISULFIDE EQUILIBRIA 15

The Kox for the dithiol depends on the redox state of the flavin cofactor With the flavin reduced the Kox is -270 mV (2.3 M) and it increases to -260 mV (0.63 M) when the flavin is oxidized The redox potential of the dithiol/disulfide is comparable to that of the flavin cofactor (-260 mV for the dithiol form of the enzyme), suggesting that electron transfer between the flavin and dithiol/disulfide center is easily reversible and that the equilib- rium constant for the electron transfer between these two centers is near one (actually 3.1) 27 Efficient catalysis is often associated with internal equilibrium constants that are close to one 28 For lipoyl dehydrogenase, however, the redox potential for transfer of the first two electrons (presum- ably the dithiol/disulfide center) is 66 mV more negative than that for transfer of the next two electrons (presumably the flavin), suggesting a favorable transfer of electrons from the dithiol center to the flavin 29

Magnitude of Thiol/Disulfide Redox Equilibrium C o n s t a n t s

Mixed Disulfides

For alkyl thiols such as the cysteine residues of most proteins, the equilibrium constants for mixed disulfide formation are usually near one (Table I) 3°-36 Electron-withdrawing groups on the test thiol that decrease the pKa will make the equilibrium less favorable and decrease Km~.3° The effects of charge are relatively small For example, the Kmix for forming

a mixed disulfide between cysteamine (NH3+CH2CH2SH) and negatively charged glutathione is 2.4, comparable to that for forming the symmetrical disulfide of negatively charged glutathione (by definition Kmix = 1) 31 In proteins, specific interactions between glutathione and the protein in the mixed disulfide could increase K~n~x; however, the maximum value of Kmix observed to date is 27, for the formation of a mixed disulfide between glutathione and a form of the enzyme hydroxymethylglutaryl-CoA re- ductase? 2

27 M E O'Donnell and C H Williams, J Biol Chem 258, 13795 (1983)

28 j R Knowles and W J Albery, Acc Chem Res 10, 105 (1977)

29 R G Matthews and C H Williams, J BioL Chem 251, 3956 (1976)

3o D A Keire, E Strauss, W Guo, B Noszal, and D L Rabenstein, J Org Chem 57,

123 (1992)

31 R E Cappel and H F Gilbert, J Biol Chem 261, 15378 (1986)

32 R E Cappel and H F Gilbert, J Biol Chem 264, 9180 (1989)

33 p Eyer and D Podhradsky, Anal Biochem 153, 57 (1986)

34 R Zhang and G H Synter, Biochemistry 27, 3785 (1988)

35 S C Tyagi and S R Simon, Biochemistry 31, 10584 (1992)

36 K Konishi and M Fujioka, Arch Biochem Biophys 289, 90 (1992)

16 OVZRVIEW [2]

TABLE I EQUILIBRIUM CONSTANTS (Kmix) FOR FORMATION OF GLUTATHIONE MIXED DISULFIDES

of dithiothreitol have been used

37 R M Zhang and G H Snyder, J Biol Chem 264, 18472 (1989)

38 F Siedler, S Rudolph-Bohner, M Doi, H J Musiol, and L Moroder, Biochemistry 32,

7488 (1993)

39 A Holmgren, this series, Vol 107, p 295

4o H C Hawkins and R B Freedman, Biochem J 275, 335 (1991)

41 M M Lyles and H F Gilbert, Biochemistry 30, 612 (1991)

42 j Lundstrom and A Holmgren, Biochemistry 32, 6649 (1993)

43 D M Rothwarf and H A Scheraga, Proc Natl Acad Sci U.S.A 89, 7944 (1992)

44 M a Chau and J W Nelson, FEBS Lett 291, 296 (1991)

45 F Rebeille and M D Hatch, Arch Biochem Biophys 249, 164 (1986)

46 T E Creighton, in "Functions of Glutathione: Biochemical, Physiological, Toxicological,

and Chemical Aspects" (A Larsson, S Orrenius, A Holmgren, and B Mannervik, eds.),

p 205 Raven, New York, 1983

47 Y C Laurent, E C Moore, and P Reichard, J BioL Chem 239, 3436 (1964)

[2] THIOL/DISULFIDE EQUILIBRIA 17

TABLE II EQUILIBRIUM CONSTANTS (Kox) FOR FORMATION OF INTRAMOLECULAR DISULFIDES

IN GLUTATHIONE REDOX BUFFER

a E o, values are calculated using Eq (12) The E °' values reported here may differ from previously tabulated values 1 because more accurate values for the equilibrium constant

b The equilibrium constant for oxidation of NADPH by GSSG is equivalent to the Kox

5 x 1 0 5 M

The effects of protein structure on thiol/disulfide oxidation potentials are well illustrated by three proteins that have domains with significant sequence homology to thioredoxin Octapeptide analogs of the active sites (CXXC) of thioredoxin, glutaredoxin, thioredoxin reductase, and protein disulfide isomerase exhibit Kox values that vary from 16 to 140 mM at 20 °, depending on the specific sequences, 38 and are comparable to the Kox

18 OVERVIEW [2]

of denatured thioredoxin, 26 mM is Local sequence differences are not

responsible for the large differences in the Kox observed for the folded

proteins In their native states, thioredoxin itself (active site sequence,

W C G P C K ) has a/Cox of 10-16 M, 18,39 while dsbA (WCXXCK) and protein disulfide isomerase (PDI) ( W C G H C K ) , proteins involved in oxidation and rearrangements of disulfide bonds during protein folding, have/Cox values

of 81 /xM 19 and 42-60 /~M, 40'4! respectively However, a higher Kox of

3 m M has been reported for PDI 42 The structural reasons for these large differences in Kox are not yet known; however, the extremely low Kox for dsbA and PDI would be useful in their functions as oxidants in the folding

of disulfide-containing proteins

S t a n d a r d Redox Potentials

Potentiometric methods have not generally been useful for determining redox potentials of thiol-containing systems, principally because of compli- cations resulting from the interaction of the thiol with the electrode system However, one report suggests that electrodes coated with a self-assembled lipid bilayer-modified gold electrode are capable of yielding direct electro- chemical measurements of redox potential that are similar to those mea- sured by thiol/disulfide exchange equilibria 48 However, the standard reduc- tion potential of a disulfde can be calculated from the thiol/disulfide oxidation potential (Kox or Kmix), the equilibrium constant for the glutathi- one reductase reaction [Eq (10)], and the standard reduction potential

of electrons transferred (two), and F is the Faraday constant KCR is the

48 Z Salamon, F K Gleason, and G Totlin, Arch Biochem Biophys 2~), 193 (1992)

49 G Gorin, A Esfandi, and G B Guthrie, Arch Biochem Biophys 168, 327 (1975)

[2] THIOL/DISULFIDE EQUILIBRIA 19 equilibrium constant for the glutathione reductase reaction between GSSG and NADPH The equilibrium constant for this reaction has been critically evaluated in a review by Williams, 26 so that the best estimate for this value

KCR) at 25 ° Put simply, the standard reduction potential, E °', for a given disulfide will be 30 mV more negative than the redox potential of NADPH for each factor of 10 that Kox is greater than the KrR for NADPH oxidation

by GSSG (800 M) A similar conversion can be used to define the reduction potential of glutathione mixed disulfides For the convenience of those who most often deal and think in terms of standard reduction potentials, the values are included in Tables I and II

Practical Considerations in Equilibrium M e a s u r e m e n t s

of Thi'ol/Disulfide Oxidation Potential

Even with the simple formation of a single intramolecular disulfide, the species present at equilibrium may be complex if intermediates accumulate (Fig 2) Fortunately, much simpler models can often be used to describe the change in the redox state of the test system in response to changes in the redox buffer composition As with other equilibrium measurements, there are common problems associated with the measurement of thiol/ disulfide exchange equilibria These include choosing the appropriate redox buffer and the range of redox buffer compositions, verification that equilib-

2 0 OVERVIEW [2]

rium has been reached, m e a s u r e m e n t of the redox state of the test thiol/ disulfide, and the demonstration that any m e t h o d used to assess the redox state of the test thiol/disulfide preserves the concentrations initially present

at equilibrium

Choice of Redox Buffer

While any thiol/disulfide pair can be used as the redox buffer, the redox buffer itself should have a well-defined redox potential The most commonly employed redox buffer is glutathione ( G S H ) and glutathione disulfide (GSSG), the major low molecular weight thiol/disulfide pair in most cellular systems Because of potential ionic strength and solvent effects, the maxi- mum G S H concentration should be limited to 100-200 mM Air oxidation and contamination of G S H with G S S G limits the maximum [GSH]/[GSSG] ratio to approximately 100 Consequently, the maximum R [ G S H ] that can easily be attained for a glutathione r e d o x buffer is approximately 20 M Thus, glutathione redox buffers are useful for test systems with Kox values below 20 M F o r more stable disulfides, dithiothreitol ( D T T ) redox buffers are more useful Dithiothreitol, 5° a dithiol that is easily oxidized to a cyclic intramolecular disulfide, is a much better reducing agent A wide range of values for the Kox of D T T has been reported, but two careful studies have determined a value of 260 M at p H 8, 25o 43'44

Choice of Redox Buffer Composition

With an appropriate choice of redox buffer composition one can deter- mine accurate values for equilibrium constants and, at the same time, often distinguish the type(s) of test disulfide(s) present at equilibrium T h e general strategy is to vary the redox buffer composition, both ratio and concentration, so that the test system undergoes a significant redox state change, from almost fully reduced to fully oxidizedJ The thiol/disulfide species that accumulate at equilibrium can be complex functions of the

so W W Cleland, Biochemistry 3, 480 (1964)

5~ T E Creighton, this series, Vol 107, p 305

r 0.6- 0.4 0.2 0.0 0

If only a mixed disulfide is involved in the equilibrium, a plot of the fraction of the test species present as P S H against the [GSH]/[GSSG] ratio,

R, will be hyperbolic 1° [Eq (14)]

The R value at which the test species is half-reduced will be equal to the Km~ Ideally, the redox buffer composition should be varied so that the test thiol (or dithiol) changes between 20 and 80% of the total test species present For simple mixed disulfides, holding the [GSH]/[GSSG] ratio at 0.25, 0.54, 1, 1.8, and 4 times the Kmix will result in a change in the redox state of the test species from 20 to 80% reduced (PSH) (Fig 4) Note that the values of R are chosen to give an equal spacing of the fraction reduced rather than the [GSH]/[GSSG] axis If only a mixed disulfide is involved, changing the concentration of G S H while holding the ratio constant will not affect the extent of PSSG formation

W h e n dealing with intramolecular disulfides that do not accumulate significant mixed disulfides at equilibrium (a simple two-state system), plots

of the fraction of the test system that is present in the reduced, dithiol form, against the quantity R [ G S H ] are hyperbolic,

of 0.4, 1, 2, 4, and 12 to give five equally spaced points along the vertical axis between 0.2 and 0.8 B Redox equilibrium involving the formation of an intramolecular disulfide The R values is varied at several fixed GSH concentrations as indicated The curves are calculated for a Kox of 0.2 M C Redox equilibrium involving the formation of both a single mixed disulfide intermediate and an intramolecular disulfide The curves were calculated from Eq

16 using a Kox of 0.2 M and a Kmix of 2 The lower R values and higher GSH concentrations favor the accumulation of mixed disulfides

P ( S H ) 2 _ R [ G S H ]

a n d t h e v a l u e o f R [ G S H ] a t w h i c h t h e t e s t s y s t e m is h a l f r e d u c e d is e q u a l

t o Kox 15 P l o t s o f P(SH)2/etot a g a i n s t R a t a c o n s t a n t [ G S H ] s h o u l d a l s o

[9.] THIOL/DISULFIDE EQUILIBRIA 23

be hyperbolic and the R value at which the test system is half reduced is equal to Kox/[GSH] (Fig 4) The [GSH]/[GSSG] ratio should be varied at several different [GSH] concentrations so that R [GSH] varies around Kox Using high [GSH]/[GSSG] ratios (10-50) and lower concentrations of GSH may minimize the formation of mixed disulfide intermediates (see below)

An appropriate change in the redox state of the test system can be obtained

by using equally spaced R values, varied over a fivefold range, and choosing several constant GSH concentrations so that the test system will vary from

15 to 85% reduced The highest GSH concentration should be chosen to bring the system to 85% reduced at the highest value of R, and the lowest GSH concentration should be chosen to reach 15% reduction at the lowest value of R When an intramolecular disulfide is formed, holding the [GSH]/ [GSSG] ratio constant while changing the [GSH] concentration will result

in a change in redox state of the test system Thus, performing equilibrium experiments at several different fixed [GSH] concentrations is useful for distinguishing the behavior of an intramolecular disulfide from that of a simple mixed disulfide

Depending on the test system under investigation, mixed disulfides may accumulate at equilibrium When monomixed disulfides accumulate, the fraction of the test system present in the dithiol redox state is 52

Ptot R[GSH] + Kmix[GSH] + Ko×

Kox/[GSH] A plot of R0.5 against 1/[GSH] will have slope of Kox and

an intercept of Kmix When mixed disulfides are present, changing the concentration of [GSH] has a smaller effect on P(SH)2 than when mixed disulfides do not accumulate (Fig 4) To maximize the detection of mixed disulfides by their effect on the concentration of P(SH)2, the [GSH] concen- tration should be held n e a r Kox/Kmix and R values should be near Kmix

24 OvERvlzw [21 when equilibrium is approached from both directions Initiating the equili- bration with the test thiol and with the test disulfide should yield the same equilibrium constant In addition, the value of the derived equilibrium constant should be independent of the incubation time For an accuracy

of 5%, this requires that the m e a s u r e m e n t must be made after at least five half-lives based on the rate constant for approach to equilibrium Bimolecular thiol/disulfide exchange reactions between unhindered thiols and disulfides with pKa values of 8.6 occur with rate constants near 20 M-1 min -1 at p H 7.0 H F o r an equilibrium constant of one and with a redox buffer consisting of 10 m M R S H and 10 m M RSSR, equilibrium would be expected afte} 8.6 min at p H 7 and 1.0 min at p H 8 Because the rate constants for approaching equilibrium depend on the redox potential of the test disulfide, the pKa of both attacking and leaving thiolates, the pH, and steric factors, a the verification that equilibrium has been attained should

be made experimentally Particularly for proteins, steric, electronic, and entropic factors make it virtually impossible to predict the rate constants for thiol/disulfide exchange, and verification of equilibrium must be made experimentally

Quenching the Reaction

B e f o r e using chromatographic or electrophoretic methods to separate the various redox states of the test species it is necessary to quench the equilibrium mixture to preserve the distribution of species that was present initially T h e most c o m m o n quenching m e t h o d involves alkylation of all free thiols (including the redox buffer) or quenching the reaction mixture with acid to prevent subsequent thiol/disulfide exchange from altering the equilibrium distribution T h e rate of the quenching reaction must be faster than all thiol/disulfide interconversions, including intramolecular reactions; otherwise, the distribution of redox species will be shifted toward the species that reacts fastest with the particular quenching agent, s3

Quenching agents differ widely with respect to the rate at which they react with thiols A t p H 8, the second o r d e r rate constant for reaction of

a typical thiol (pKa of 8.6) with iodoacetamide is 4 6 M -1 sec 1.6 N-Ethylmaleimide ( N E M ) reacts much faster (k = 1 x 104 M -1 s e c - l ) , 54

and quenching with acid occurs at near the diffusion controlled limit (109

M -~ min-1) 53 Because intramolecular thiol/disulfide rearrangements may

be fast (on the millisecond time scale), using a high concentration of alkylat- ing agent does not necessarily ensure adequate quenching

53 j s Weissman and P S Kim, Science 253, 1386 (1991)

54 y M Torchinski, in "Thiol and Disulfide Groups of Proteins" (H B F Dixon, ed.),

p 24 Consultants Bureau, New York, 1974

[2] THIOL/DISULFIDE EQUILIBRIA 25 Acid quenching is fast and effective; however, raising the pH may result

in rearrangement so that analytical techniques must be performed at low

pH As an alkylating agent, the greater reactivity of NEM makes it superior

to iodoacetamide or iodoacetate At a concentration of 0.1 M NEM the half-time for the alkylation of a typical thiol at pH 8 would be about 0.7 msec, while 0.1 M iodoacetate would react with a half-life of 1.5 sec Iodoacetamide alkylation is generally irrreversible, but NEM alkylation products are less stable To verify the effectiveness of the trapping reagent,

it should be demonstrated that doubling (or halving) the concentration

of the trapping agent does not affect the measured equilibrium con- stants

Monitoring Redox State Changes

Any analytical method that results in quantitative observations of the distribution of redox species without significant alterations of the equilib- rium mixture can be used to follow thiol/disulfide redox equilibria For small molecules including peptides and small proteins, high-performance liquid chromatography (HPLC) separation and quantitation of quenched redox species 37'~3 or nuclear magnetic resonance (NMR) u,3° is often the method of choice, and the ability to quantitate all of the redox isomers present may simplify determination of the equilibrium constants

Gel electrophoresis has been a useful technique for measuring redox potentials of proteins Intramolecular disulfide cross-linking will increase the mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gels because

of the relatively smaller volume of the cross-linked protein; however, such effects are variable from protein to protein Creighton has described several useful electrophoretic methods to detect and quantitate disulfide formation

to detect the effects of oxidation on label incorporation by measuring the incorporation of radioactivity A more useful approach in this situation is

to alkylate the protein with a high concentration of unlabeled alkylating

Analyzing Results

If the concentrations of all redox species can be measured indepen- dently, equilibrium constants can be determined by constructing plots of the appropriate ratios of species against the appropriate redox buffer com-

of the intramolecular disulfide in the dsbA protein by equilibrating dsbA with a series of glutathione redox buffers and measuring the concentration

of dithiol, disulfide, and mixed disulfide intermediates A plot of the ratio

of the concentration of fully reduced dsbA to that of the disulfide redox

used to determine Kmi×

It may be possible to directly detect only one of the redox species in equilibrium, for example, an enzyme that is active only in the dithiol redox state or in the detection of thiols by alkylation with a radiolabel In such cases, the presence of intermediates can often be inferred by the effect of intermediate accumulation on the concentration of the species that can

be detected directly Simple graphical methods have been described to analyze the data; however, fitting the data to an appropriate model by nonlinear least squares 55 is preferable It is relatively easy to distinguish

55 p R Bevington, "Data Reduction and Error Analysis for the Physical Sciences." McGraw- Hill, New York, 1969

[2] THIOL/DISULFIDE EQUILIBRIA 27 mixed disulfide formation from intramolecular disulfide formation by the dependence on the concentration of GSH at a constant [GSH]/[GSSG] ratio; however, if a GSH dependence is observed, it may be more difficult

to decide whether a two-state model is appropriate or if a more complex model including mixed disulfide species should be used 56 More complex models should be used only if they give a statistically better description of the dataY

In some cases, for example the detection of redox state changes by the incorporation of radiolabeled sulfhydryl reagents into species with free sulfhydryl groups, the experimentally measurable quantity will involve the sum of more than one species 56 Similar problems may be encountered in analyzing data from the dependence of enzyme activity measurements

on redox buffer composition when one or more species in the equilib- rium mixture are active In such cases, the experimentally observable quantity is given by the sum of equations describing the fraction of each individual species at equilibrium For example, the incorporation of a radiolabeled alkylating agent into an equilibrium mixture of a test dithiol will depend on the quantitites of each species at equilibrium and the number of free sulfhydryl groups (assuming that all sulfhydryl groups are sterically accessible, which may not be a valid assumption in many cases) For a dithiol system in which the dithiol incorporates two labels per mole

of protein and the mixed disulfide incorporates one label per mole of protein, the radiolabel incorporated per mole of proteins will then be given by

where the terms in the numerator represent the incorporation of label into dithiol and the mixed disulfide intermediate

Complex Equilibria

The description of the equilibrium behavior of test thiols and dithiols provided above is based on simplifications and assumptions regarding the types of disulfides formed at equilibrium These simpler models are usually sufficient to describe the experimental behavior; however, the equilibrium distribution of all possible species that can be formed in a dithiol system (Fig 2) is given by Eqs (19)-(22)

56 R E Capperl and H F Gilbert, J BioL Chem 268~ 342 (1993)

These distribution equations appear relatively complex; however, they do simplify to the expressions that describe the simpler two- and three-state models For example, Eq (19) simplifies to Eq (15) describing the forma- tion of a simple intramolecular disulfide when the values of Kmix and Kmix2 are set to zero Note that there are two different mixed disulfide intermedi- ates that could form, and the value of Kmix actually is equal to the sum of the individual equilibrium constants for mixed disulfide formation Using the more complex model it is also possible to show that the maximum level of monomixed disulfide that can be observed at equilib- rium is

Kma

2 N/Kmix2 + Kmix + Kox/[GSH]

and that this maximum will be independent of GSH and occur at an R value of vl/2 Ix mix2

Acknowledgments

Work in the author's laboratory was supported by NIH Grants GM-40379 and HL-28521

"repaired" by thiols by hydrogen (or electron/proton) donation:

It is only recently that reactions of the form of Eq (1) were recognized to

be equilibria because the reverse reaction occurred, that is, thiyl radicals could abstract hydrogen atoms from suitably activated C - H groups, for example, from alcohols and ethers, 1-3 or from polyunsaturated fatty acids 3-5 This has renewed interest in the fate of thiyl radicals in cells, because previously the "repair" or "protective" properties of thiols had received far greater attention than the chemistry of the thiyl radicals inevita- bly formed in the radical-repair reactions of thiols

Thiols can also act as cellular antioxidants by electron transfer to oxidiz- ing species, producing thiyl radicals Two general types of thiol-reactive oxidants are illustrative Radicals derived from D N A bases, such as those produced on reaction of guanine moieties with • OH, are "repaired" (but not restituted) by thiols6:

1 M S Akhlaq, H.-P Schuchmann, and C von Sonntag, Int J Rad&t Biol 51, 91 (1987)

2 C Sch6neich, M Bonifacic, and K.-D Asmus, Free Radical Res Commun 6, 393 (1989)

3 C SchOneich, M Bonifacic, U Dillinger, and K.-D Asmus, in "Sulfur-Centered Reactive

Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 367 Plenum, New York, 1990

4 C Sch6neich, K.-D Asmus, U Dillinger, and F von Bruchhausen, Biochern Biophys Res Commun 161, 113 (1989)

5 C SchOneich, U Dillinger, F von Bruchhausen, and K.-D Asmus, Arch Biochem Biophys

292, 456 (1992)

6 p O'Neill, Radiar Res 96, 198 (1983)

Copyright © 1995 by Academic Press, Inc

The fate of thiyl radicals in cells will reflect the kinetics of reactions that produce and remove them Overviews of the chemistry of thiyl radicals and their detection in biological systems have been published 13-23 The main experimental technique for measuring the kinetics of thiyl radical

7 M Bando, H Obazawa, and T Tanikawa, J Free Radicals Biol Med 2, 261 (1986)

8 D Ross, E Albano, U Nilsson, and P Mold6us, Biochem Biophys Res Commun 125,

109 (1984)

9 D Ross, R Larsson, K Norbeck, and P Mold6us, Life Chem Rep 3, 112 (1985)

10 D Ross, K Norbeck, and P Mold6us, J Biol Chem 260, 15028 (1985)

11 p j O'Brien, Free Radical Biol Med 4, 169 (1988)

12 L G Forni, J M6nig, V O Mora-Arellano, and R L Willson, J Chem Soc., Perkin Trans

2, 961 (1983)

13 O Ito and M Matsuda, in "Chemical Kinetics of Small Organic Radicals Volume III Reactions of Special Radicals" (Z B Alfasi, ed.), p 133 CRC Press, Boca Raton, Flor- ida, 1988

14 p Wardman, in "Glutathione Conjugation Mechanisms and Biological Significance" (H Sies and B Kenerer, eds.), p 43 Academic Press, London, 1988

15 K.-D Asmus, this series, Vol 186, p 168

16 B C Gilbert, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 135 Plenum, New York, 1990

17 D A Armstrong, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 121 Plenum, New York, 1990

is D A Armstrong, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 341 Plenum, New York, 1990

19 C von Sonntag, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 359 Plenum, New York, 1990

20 C Dunster and R L Willson, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 377 Plenum, New York, 1990

21 p Wardman, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C Chatgilialoglu and K.-D Asmus, eds.), p 415 Plenum, New York, 1990

22 C Chatgilialoglu and K.-D Asmus (eds.) "Sulfur-Centered Reactive Intermediates in Chem- istry and Biology." Plenum, New York, 1990

23 R P Mason and D N R Rao, this series, Vol 186, p 318

[3] KINETICS OF THIYL RADICALS IN CELLS 33

reactions is pulse radiolysis, also reviewed extensively 17,24-2s This chapter therefore concentrates on a discussion of the kinetic factors controlling the reaction pathways of thiyl radicals in cells, the experimental problems in quantitation, and the outstanding questions

Thiol Ionization

Equation (2) is written to reflect the higher reactivity of the thiolate anion (RS-) compared to the undissociated thiol 6 Other examples involving higher thiolate reactivity, such as a-oxoalkyl radicals, are known 29 This is

a general feature of many reactions of thiols as nucleophiles, a differential reactivity that may also be a feature of reactions such as those represented

by Eq (3), in which the reaction is written in the form shown without implication as to the most reactive form of the thiol In contrast, Eq (1) requires the thiol to be undissociated for hydrogen transfer

Thus many reactions with thiols may vary with pH because of the ion- ization:

and any other prototropic equilibria occurring over the pH range of interest

In addition, the rates of reaction involving charged species may vary with ionic strength, and rate constants quoted here, unless otherwise indicated, refer to measurements at ambient temperature

Thiyl Radicals from Different Thiols

The main cellular thiol is glutathione [GSH, see Fig 1, (1)] Other thiols commonly studied in free-radical research include cysteine (2) and its N-acetyl derivative (3), penicillamine (4), cysteamine (5), 2-mercaptoethanol (6), and 2-mercaptopropionylglycine (7) WR 1065 (8)

is the dephosphorylated and active form of the radioprotector ethiofos (WR 2721 or Amifostine, the phosphate ester of WR 1065); WR 2721,

24 K.-D Asmus, this series, Vol 105, p 167

25 M G Simic, this series, Vol 186, p 89

26 y Tabata (ed.) "Pulse Radiolysis." C R C Press, Boca Raton, Florida, 1991

27 R V Bensasson, E J Land, and T G Truscott, "Excited States and Free Radicals in Biology and Medicine Contributions from Flash Photolysis and Pulse Radiolysis." Oxford Univ Press, Oxford, 1993

28 C von Sonntag and H.-P Schuchmann, this series, Vol 223, p 3

29 M S Akhlaq, S A1-Baghdadi, and C von Sonntag, Carbohydr Res 164, 71 (1987)

FIG 1 Structures of some thiols important in biology (see text)

while disappointing as a clinical radioprotector, 3° is now attracting clinical interest as a chemoprotector in cancer chemotherapy 31 Other medical uses

of antioxidant thiols, particularly (3) and (7) and the antihypertensive agent, captopril (9), have been reviewed 32

Although most properties and reactions of cysteine [CySH, (2)] involv- ing free radicals are similar to those of glutathione, there is at least one important exception (involving an intramolecular reaction), which is dis- cussed below Rate constants involving thiyl radicals from other thiols will frequently differ quantitatively from those involving GSH or CySH, particularly in those thiols in which the carbon a or/3 to the thiol function carries electron-donating or -withdrawing functions Such substituents will also influence the pKa for the ionization of the S - H function in the ground state [Eq (4)], influencing the proportion of thiol in the ionized (thiolate) form at physiological pH This has important consequences because the thiolate anion is an important reactant for thiyl radicals, as described below

30 T Liu, Y Liu, Z Zhang, and M M Kligerman, Cancer 69, 2820 (i992)

3] S Walder, J J Beitler, J S Rubin, H Haynes, F McGill, A Rozenblit, G Goldberg, C

Cohen, J Speyer, and C Runowicz, J Clin Oncol 11, 1511 (1993)

32 C A Rice-Evans and A T Diplock, Free Radical Biol Med 15, 77 (1993)

[3] KINETICS OF THIYL RADICALS IN CELLS 35

In dithiothreitol [Fig 1, (10)], t h e r e are two sulfhydryl centers O n e -

e l e c t r o n o x i d a t i o n results in the f o r m a t i o n of an i n t r a m o l e c u l a r cyclic radi- cal, with an S - S t h r e e - e l e c t r o n b o n d 17,33 A similar radical is p r o d u c e d o n

o n e - e l e c t r o n r e d u c t i o n o f the disulfide, lipoic acid (11) T h e p r o p e r t i e s o f

t h e thiyl radicals f o r m e d f r o m such dithiols or disulfides will also differ significantly f r o m that f r o m G S H or C y S H M e t h i m a z o l e (12), a l t h o u g h also a " p r o t e c t i v e " thiol, 34 is best v i e w e d as an a r o m a t i c thiol O x i d a t i o n yields a radical a n a l o g o u s to an arylthiyl radical, with p r o p e r t i e s different

f r o m those o f thiyl radicals f r o m aliphatic - S H functions 35'36 S o m e p r o p e r - ties o f arylthiyl radicals h a v e b e e n reviewed 13,37

S t u d y i n g R e a c t i o n K i n e t i c s o f T h i y l R a d i c a l s

Pulse radiolysis is the m o s t c o n v e n i e n t m e t h o d to g e n e r a t e a d e t e c t a b l e

" i n s t a n t a n e o u s " c o n c e n t r a t i o n o f thiyl radicals, a n d m o n i t o r their reactions

f o r kinetic analysis A s d e s c r i b e d previously, 15,22 thiyl radicals are c o n v e - niently o b t a i n e d o n r e a c t i o n of thiols ( R S H ) with h y d r o x y l radicals [Eq (5)] or o n e - e l e c t r o n oxidants such as Br2 ~ [Eq (6)]:

R S H + • O H -> R S + H 2 0 (5)

R S H + Br2- ~ R S + H + + 2 B r - (6)

R a t e c o n s t a n t s f o r these ( a n d t h o u s a n d s o f o t h e r reactions involving free radicals) h a v e b e e n e v a l u a t e d a n d collated 38,39 F o r R S H = G S H , k5 = 1.3 × 101° M -1 sec -1 a n d k6 = 2.5 × 108 M -1 sec -1 at a r o u n d p H 7

E x p e r i m e n t a l c o n d i t i o n s vary, b u t with a typical pulse radiolysis installa- tion, a b s o r b a n c e signals o f <0.005 at w a v e l e n g t h s in t h e range 2 2 0 - 8 0 0 n m

c a n be easily analyzed, particularly after a v e r a g i n g signals f r o m 5 - 1 0 pulses

T h u s the p r o d u c t o f radical c o n c e n t r a t i o n , [RS ], a n d a b s o r p t i o n coefficient,

e, m u s t be > 0 0 0 2 cm -1 f o r facile o b s e r v a t i o n (if the a b s o r p t i o n coefficient

is e x p r e s s e d in units o f M -1 c m 1 a n d the p a t h length is 2 cm) W h i l e the thiyl radical f r o m penicillamine has ~ = 1.2 × 10 3 M -1 c m -1 at 330 nm, 15

o t h e r thiyl radicals have significantly l o w e r values T h o s e d e r i v e d f r o m

33 M S Akhlaq and C von Sonntag, Z Naturforsch C: Biosci 42, 134 (1987)

34 j j Taylor, R L Willson, and P Kendall-Taylor, F E B S Letr 176, 337 (1984)

35 M G Simic and E P L Hunter, J Free Radicals Biol Med 2~ 227 (1986)

36 L G McGirr, S D Jatoe, and P J O'Brien, Chem.-Biol Interact 73, 279 (1990)

37 D A Armstrong, Q Sun, G N R Tripathi, R H Schuler, and D McKinnon, J Phys

38 p Wardman and A B Ross, Free Radical Biol Med 10, 243 (1991)

39 A B Ross, W G Mallard, W P Helman, G V Buxton, R E Huie, and P Neta,

"NDRL-NIST Solution Kinetics Database: Version 2." National Institute of Standards and Technology, Gaithersburg, Maryland, 1994

3 6 THIYL RADICALS [3]

GSH, cysteine, and cysteamine have extinction coefficients in the range 300-600 M -1 cm -I at - 3 3 0 nm 4° If e = 500 M 1 cm 1, then [RS.] must

be > 4 / x M (or thereabouts) for accurate kinetic analysis of its reactions

by direct observation Accelerators or radiation generators used in pulse radiolysis can easily generate radical concentrations 10 times this value in much less than 1/~sec However, there are kinetic constraints that in practice limit pulse radiolysis experiments to lower radiation doses (i.e., lower radical concentrations)

Many radicals are unstable with respect to combination or dispropor- tionation In the case of thiyl radicals, reactions to form disulfides are rapid:

Values of 2k7 of 1-3 x 109 M 1 sec 1 for c o m m o n thiols have been re- ported 41 F o r such a second order reaction, - d [ R S .]/dt = 2k7[RS.]2, and the first half-life for loss of thiyl radicals via Eq (7) is 1/(2kv[RS "]0), where the subscript zero denotes the initial concentration Thus with [RS "]0 =

4 / z M (for easy direct detection) and 2k7 = 2 x 109 M -1 sec -l, the first half-life is - 1 3 0 / z s e c ; - 1 0 % of RS will be lost in - 1 4 / z s e c T h e study of

a reaction of thiyl radicals with a substance must therefore be designed such that reaction is essentially complete in a few microseconds if the competing reaction, Eq (7), is not to occur to a significant extent This will not always be possible The concentration of the second solute (i.e., other than the thiol used to generate thiyl radicals) is constrained by the need

to avoid • O H or Br2: radicals reacting directly with the solute rather than

by Eqs (5) and (6)

These kinetic constraints, and the weak optical absorption of most thiyl radicals, together result in much kinetic information concerning thiyl radical reactions being obtained not by directly monitoring the thiyl radical, but

by observing some secondary product This must be produced in a reaction that is not itself rate-limiting

Two approaches are common The first has been n o t e d above T h e equilibrium shown in Eq (3) is well over to the left, with k-3 = 6 x 108

M -1 sec -1 for P Z = chlorpromazine and R S H = G S H (see below) [We denote the rate constant or reaction for the " b a c k " or " r e v e r s e " reaction

in an equilibrium by preceding it with the negative sign, e.g., Eq ( - 3 ) ]

T h e formation of thiyl radicals can be monitored by observing the intense

c h r o m o p h o r e of the phenothiazine radical cation With chlorpromazine, e

is - 1 0 x 104 M -1 cm -1 at 525 nm O t h e r oxidizable solutes yielding conve- nient c h r o m o p h o r e s in reactions analogous to Eq ( - 3 ) include A B T S [2,2'-

40 M Z H o f f m a n and E Hayon, J Am Chem Soc 94, 7950 (1972)

4i M Z Hoffman and E Hayon, J Phys Chem 77, 990 (1973)

[3] KINETICS OF THIYL RADICALS IN CELLS 37 azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] 42 and aminopyrine 43 The high extinction coefficients result in observations being possible with submi- cromolar radical concentrations, so that the potential contribution of Eq (7) is much reduced compared to experiments involving direct observation

Eq (8b), neglecting Eqs (8a) and (9), although in the case of the "cyclic" thiyl radicals from dithiols, e.g., Fig 1, (10, 11), the protonated disulfide radicals are stabilized 17,19 The importance of Eqs (8a) and (9) is that thiyl radicals can decay through Eq ( - 8 a ) followed by Eq (7) Hence (RSSR): from simple monothiols is normally stable only at high pH values, at which

a negligible fraction of the thiol is in the undissociated form.]

In addition to observing the absorption of (RSSR)- at ~420 nm directly, this powerful reductant can be reacted with an oxidant to yield a chromo- phore detectable in a different spectral region (which is sometimes desir- able) or with a higher extinction coefficient Examples that have been used include 4-nitroacetophenone, which forms a nitroarene radical anion on reduction, 2 or viologens (1,1'-dialkyl-4,4'-bypyridinium compounds), 21 which form the highly characteristic, intense absorption of the stable radical cation on reduction

Because most common thiols ionize with pK4 values of - 8 - 1 0 , stabiliz- ing thiyl radicals through Eq (8b) is only partially effective at pH values

42 B S Wolfenden and R L Willson, J Chem Soc., Perkin Trans 2, 805 (1982)

43 I Wilson, P Wardman, G M Cohen, and M d'Arcy Doherty, Biochem Pharmacol 35,