biothiols, part a

Methods in Enzymology
Volume 2.51
Biothiols
Part A
Monothiols and Dithiols, Protein Thiols, and
Thiyl Radicals
EDITED BY
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
0
m
ACADEMIC PRESS
San Diego New York Boston London Sydney Tokyo Toronto
Preface
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
Contributors to Volume
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 of Physiol-
ogy and Biophysics, Louisiana State Uni-
versity Medical Center, Shreveport, Loui-
siana 71130
AALT BAST (28), Department of Pharmaco-
chemistry, Division of 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 of Pharmacochemistry, Divi-

sion of 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 of Molecular Biol-
ogy,
Nuaey,
New Jersey 07110
ENRIQUE CADENAS (9), Department of Mo-
lecular Pharmacology and Toxicology,
School of Pharmacy, University of South-
ern California, Los Angeles, California
90033
ALBERT R. COLLINSON (20), ImmunoGen,
Inc., Cambridge, Massachusetts 02139
JOHN A. COOK (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, UCD Medical Center, Uni-
versify of California, Davis, Sacramento,
California 95817
HEINI W. DIRR (22),
Department of Bio-
chemistry, University of Witwatersrand,
Johannesburg, South Africa
WULF 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
HA6 2JR, United Kingdom
ROBERT C. FAHEY (13),
Department of
Chemistry and Biochemistry, University
of California, San Diego, La Jolla, Cali-
fornia 92093
HEINZ FAULSTICH (34),
Max-Planck Institut
fiir Medizinische Forschung, D-69120
Heidelberg, Germany
THOMAS FISCHBACH (23), Division of Im-
munochemistry, Deutsches Krebsfors-
chungszentrum, 69120 Heidelberg, Ger-

many
ROBERT B. FREEDMAN (38),
Research
School of Biosciences, University of
Kent, Canterbury CT2 7N J, United King-
dom
KAZUKO FUJIWARA (32), The Institute for
Enzyme Research, University of 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 xi
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 of
Membrane Research and Biophysics,
Weizmann Institute of 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 of Biosciences, Biological Labora-
tory, University of Kent, Canterbury CT2
7N J, United Kingdom
ALTON MEISTER (1), Department of Bio-
chemistry, Cornell University Medical
College, New York, New York 10021
DIANA METODIEWA (7), Institute of 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 of Health, Bethesda,
Maryland 20892
JACKOn MOSKOVITZ (45), Roche Research
Center, Roche Institute of 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
of California, San Diego, La Jolla, Cali-
fornia 92093

HANS NOHL (16), Institute of Pharmacology
and Toxicology, Veterinary University of
Vienna, A-I030 Vienna, Austria
KENNETH M. NOEL (46), Department of
Molecular and Cell Biology, University of
Connecticut, Storrs, Connecticut 06269
CHARLES A. O'NEILL (43), Department of
Internal Medicine, UCD Medical Center,
University of California, Davis, Sacra-
mento, California 95817
KAZUKO OKAMURA-IKEDA (32), The Insti-
tute for Enzyme Research, University of
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 of
Molecular and Cell Biology, University of
California at Berkeley, Berkeley, Califor-
nia 94720
RICHARD N. PERHAM (42), Cambridge Cen-
tre for Molecular Recognition, Depart-
ment of Biochemistry, University of Cam-
bridge, Cambridge CB2 1QW, United
Kingdom
L.L. POUESEN (27), Biochemical Institute,
Department of Chemistry and Biochemis-
try, The University of Texas at Austin,
Austin, Texas 78712

WILLIAM B. PRATT (39), Department of
Pharmacology, University of Michigan
Medical School, Ann Arbor, Michigan
48109
MICHAEL PRZYBYLSKI (41), Faculty for
Chemistry, University of Konstanz, 78434
Konstanz, Germany
M. ATIQUR RAHMAN (45), Department of
Internal Medicine, Section of 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 251
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 of 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 of 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 of
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 of 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
[ 1 ] GLUTATHIONE METABOLISM 3
[ 1] Glutathione Metabolism
By ALTON MEISTER
Glutathione (L-y-glutamyl-L-cysteinylglycine; GSH) is widely distrib-
uted in nature and occurs in virtually all animal cells, often in relatively
high (0.1-10 raM) concentrations. 1
HOOCCHNH2(CH2)2CONHCHCONHCH2COOH
I
CHzSH
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.
METHODS IN ENZYMOLOGY, VOL. 251 All rights of reproduction in any form reserved.
4 OVERVIEW [ 1 ]
OXIDATION-REDUCTION ~f"
GSSG~
/i
Transhyd(~rogena/ses / P)oxi~das~es Reduita@
Deoxyribonucleotides ~ I / I I \
Ascorbate "~'~ Flree / / Se Jf NADPH'H +
RSH radicals p +
Disulfides J ~ \
/ / / Q
/=,, ~\
//~
~ _COENZYME
X/~ " GSH - .'.~=~_ Y FUNCTIONS
~/
(7-Glu-CysH-Gly)
~
~ __~ AD p+ p. "" '~ "~' ,,. ,~
V-Glu-Cy~-Gly • 2, f y GLUTAMYL /~ N~ ,~ x
X ~
~
/ " CYC/E G~y ~ *TP 2~ iF~iebdtback
/ ,,
PATHWAY \1 / ,A I~.~.
"
. _ j \ CysH-Gly i~ /,/(-
CY~ -Gly~t'~
\ ~

+H/~ysH /~ ATP
Cys-X ~/-Glu-AA ,~1~ A ~ ~ i
N-Ao-Oyf-P @~ 5"OxOprOlin~ LATP ~
AA
FIG. 1. Metabolism of glutathione.
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. Thompson 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 GSH synthesis; such compounds include

N-acetylcysteine and 2-0xothiazolidine 4-carboxylate. 21 Glutathione levels
may also be increased by administration of y-glutamylcysteine or related
compounds, thus providing substrate for GSH synthetase (reaction 2; Fig.
1). Glutathione esters, such as GSH mono(glycyl) esters and GSH diethyl
ester, provide an efficient way of increasing cellular GSH levels
in vivo
and
in vitro;
these esters have been reviewed in this series. 22'23
HOOCCHNH2CH2CH2CONHCHCONHCHzCOOR
L
CH2SH
Glutathione mono(glycyl) ester
CzH5OOCCHNHzCH2CHzCONHCHCONHCHzCOOC2H5
[
CH2SH
Glutathione diethyl ester
Another approach to the increase in cellular levels of GSH and to the
cellular capacity for GSH synthesis involves the enhancement of synthetase
activities by gene transfer. For example, the genes for the two synthetases
in
Escherichia coli
have been isolated and used to transform the wild strain
to one that overproduces the synthetases. 24 This gene-enriched strain has
a high capacity for GSH synthesis. It also exhibits increased radioresistance,
which is associated with increased capacity to synthesize GSH. Comparable
studies with the mammalian genes are feasible and in progress (see Refs.
6 and 25). Other modulations of GSH metabolism produced by selective
inhibition of various enzymes have also been achieved, 13 and more recent
work on the interactions between GSH and ascorbate have been 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] Thiol/Disulfide Exchange Equilibria and Disulfide
Bond Stability
By HIRAM F. GILBERT
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:
2 RSH ~ RSSR + 2e- + 2H + (1)
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.
METHODS IN ENZYMOLOGY, VOL. 251 All rights of reproduction in any form reserved.
[2] THIOL/DISULFIDE EQUILIBRIA 9
R1-SH + R2SSR2 ~ RIS-SR2 + R2SH (2)
R1S-SR2 + R1-SH ~ RIS-SR1 + RESH (3)
This reaction exchanges redox equivalents between different thiol/disulfide
pairs and serves to oxidize one thiol (RISH) while reducing another disulfide
(R2SSR2). The equilibrium constants for thiol/disulfide exchange reactions
depend on the differences in stability between the two disulfides and the
two thiols. Consequently, disulfide stabilities and redox potentials are most
often determined through thiol/disulfide exchange equilibria with a "stan-
dard" thiol/disulfide pair of known redox potential.
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:
P-SH + GSSG ~ P-S-SG + GSH (4)
The equilibrium constant for this reaction [Eq. (5)]
[PSSG] [GSH]
Kmix
= [PSH] [GSSG] (5)
represents the thermodynamic stability of the mixed disulfide compared to
the stability of GSSG (when PSH is glutathione, the equilibrium constant

for the reaction will be one). The more stable the mixed disulfide compared
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
P(SH)2 + GSSG ~ P(SS) + 2GSH (6)

The equilibrium constant for intramolecular disulfide formation [Eq. (7)]
Kox- [e(ss)] [GSH] 2
[P(SH)2] [GSSG] (7)
represents the stability of the intramolecular disulfide bond relative to the
stability of GSSG. Kox can also be viewed as an oxidation potential of the
dithiol; higher values of Kox indicate a more stable intramolecular disulfide
bond. 12 Because two molecules of GSH are formed in the reaction, Kox
has molar (M) units and represents the ratio [P(SS)]/[P(SH)2] at a [GSH]/
[GSSG] ratio of one and a [GSH] concentration of 1 M. The redox state
of the test system, P(SS)/P(SH)> will depend on the equilibrium constant,
Kox, and the quantity [GSH]2/[GSSG] (which is equivalent to the quan-
tity R[GSH]).
Importance of Thiol / Disulfide Redox Equilibria
The reversible formation of disulfide bonds is involved in a number of
biological processes ranging from protein folding and stability 6 to regulation
of gene expression 13'14 and catalytic activity. 15 Many of these subjects have
been reviewed previously, and several chapters in this volume cover these
topics in more detail. The following discussion, however, emphasizes the
influence of disulfide bond stability and equilibrium behavior.
Structural Stabilization by Disulfide Bonds
The cross-link introduced by the formation of a disulfide bond between
two cysteine residues provides for increased protein stability. The 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).
[2] THIOL/DISULFIDE EQUILIBRIA
11
I,( RED
S SH
H •
Reduced Reduced
Denatured Folded
I
I~ "DEN ~ ~
k" FOLD ¢.0
,-ox ~i o '-ox .
KFOoXD ~Q~f
• b
Oxidized Oxidized
Denatured Folded
FtG. 1. Thermodynamic cycle linking disulfide bond stability in the folded and unfolded
protein with the stability of the reduced and disulfide cross-linked folded structure. Because
the free energies must sum to zero around this closed thermodynamic cycle, the stability
provided to the protein by the disulfide bond,
Kfow,o,,/Kfold,red,
must be equal to the increase
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 number 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
The 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. For example, the Kox of the redox
active disulfide bond in thioredoxin increases from 26 mM 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 The 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
mM in the unfolded state to 0.081 mM 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 more it will contribute to increasing the protein stability.
Regulatory Consequences of Reversible Disulfide Bond Formation
Including Protein S-Thiolation
The concentrations of GSH and GSSG in cells and tissues are not
constant, and cellular levels of GSH and GSSG 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 mM 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 + NADPH + 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 Constants
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
E o , a
Test system Kmix (volts) Ref.
Cysteamine 2.4 -0.251 30, 31
Coenzyme A 1.8 -0.248 30
Cysteine 1.1 -0.241 30
Penicillamine 3.0 - 0.254 30
HMG-CoA reductase treated with mevinolin 27 -0.282 32
Phosphofructokinase 6.1 -0.263 10
Guanidinoacetate methyltransferase 1.7 -0.246 36
YSRCVC mixed disulfide 1.3 -0.243 34
al-Protease inhibitor 0.74 -0.236 35
TNB- 1 × 10 -3 -0.150 33
dsbA mixed disulfide
Native 3.7 × 10 -3 -0.167 19
Denatured 2.1
Glycogen phosphorylase 10 -5 -0.090 31
a E o, values are calculated using Eq. (12) and a 1 M standard state for GSH. The E °'
values reported may differ from previously tabulated values a because more accurate
values for the equilibrium constant for glutathione reductase and the oxidation potential
of dithiothreitol have been used.

Intramolecular Disulfides
A number of factors influence the equilibrium constants for formation
of intramolecular disulfides (Table II). 37-47 In unstructured peptides and
denatured proteins, the sequence distance between the two cysteine residue
is a major factor in determining Kox. Small cysteine-containing loops are not
favorable because of conformational strain, 37 and large ones are entropically
unfavorable. In folded proteins, the tertiary structure may bring two cys-
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
Eopa
Test system Kox (volts)
Ref.
Glutathione
800 M -0.240 26
NADPH 800
M b -0.327 26
Dithiothreitol
260 M -0.312 43, 44
YSRCVC 3 mM -0.164 37
Ac-WCGPCKHI-NH2 16 mM -0.186 38
Ac-GCPYCVRA-NH2 142 mM -0.214 38
NADPH-malate dehydrogenase 4400 M -0.349 45
Bovine trypsin inhibitor
46
C14-C38 1500 M -0.335
C5-C55 1.1 × 107M -0.451
HMG-CoA reductase 0.55 M -0.232 15
With substrates 0.2 M -0.218
Fatty acid synthase
0.015 M -0.185 12
Thioredoxin
10-16 M c -0.269 18, 39
dsbA,
E. coli 81/zM -0.117 19

Protein disulfide isomerase
42/zM-3 mM -0.108 to -0.164 40-42
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
for glutathione reductase and the oxidation potential of dithiothreitol have been
used.
b The
equilibrium constant for oxidation of
NADPH by GSSG
is equivalent to the
Kox
for
NADPH.
c Estimated from
the value of the equilibrium constant for thioredoxin reductase at
pH
7.0 of 4847
and the equilibrium constant for the glutathione reductase reaction at
pH 7.0
of 800 M.
teines into close proximity, and if disulfide formation does not introduce
any strain, the disulfide will be very stable, and Kox will be large. Values
as large as 103-107 M have been observed or estimated for some folded
proteins. Strain in the disulfide bond coupled to strain in the protein can
significantly decrease Kox. As a consequence of the large effects of entropy
and strain on intramolecular disulfide formation in proteins, the oxida-
tion potentials span an extremely large range, with a low of approximately

5x105M.
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,
WCGPCK) has a/Cox of 10-16 M, 18,39 while dsbA (WCXXCK) and protein
disulfide isomerase (PDI) (WCGHCK), 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 mM 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.
Standard 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 NADP +.
P(SS) + 2GSH ~ P(SH)2 + GSSG 1/Kox (8)
NADP + + 2e * NADPH + H + E~ADP (9)
NADPH + GSSG + H + ~ 2GSH + NADP + KGR (10)
P(SS) + 2e- = P(SH)2 E~s (11)
Because the AG ° for each process [Eqs. (8)-(10)] can be summed, the
value of E~s is given by
o RT (12)
E~s = ENADP -~ln(Kox/KG,)
where the standard reduction potential of the disulfide, E ° ss, is in volts,
E~qADP represents the standard reduction potential of NADPH (-0.327
V), 49 R is the gas constant, T is the absolute temperature, n is the number
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
is 800 M at pH 7. The term
(RT/nF)In(Kox/KGR)
is equal to 0.03 log(Kox/
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 Measurements
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-
< Kox >
Kmix Kintra
SH GSSG SSG F tS
I- '1-
"
SH < <
GSH
L SH
GSH
= K
,o mix2
t3
FIG. 2. Redox equilibria involved in the formation of an intramolecular disulfide bond.
The equilibrium constants for formation of mixed disulfides with the redox buffer, Kmix and
Kmix2, are unitless. The thiol/disulfide oxidation potential, Kox, is the equilibrium constant
for formation of the intramolecular disulfide starting from the dithiol (Kox = KmixKintra). Both

Kox and
Kintra
have units of M.
20 OVERVIEW [2]
rium has been reached, measurement of the redox state of the test thiol/
disulfide, and the demonstration that any method 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 (GSH) 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 GSH concentration should be limited to 100-200 mM. Air oxidation
and contamination of GSH with GSSG limits the maximum [GSH]/[GSSG]
ratio to approximately 100. Consequently, the maximum R[GSH] that can
easily be attained for a glutathione redox buffer is approximately 20 M.
Thus, glutathione redox buffers are useful for test systems with Kox values
below 20 M. For more stable disulfides, dithiothreitol (DTT) 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 DTT has been reported, but two careful studies have
determined a value of 260 M at pH 8, 25o. 43'44
DTTSI~ + GSSG ~ DTT s + 2GSH (13)
At a ratio of DTTredDTTox of one, a dithiothreitol redox buffer is equiva-
lent to a glutathione redox buffer with an R[GSH] of 260 M, corresponding
to a GSH concentration of 2.6 M at a [GSH]/[GSSG] ratio of 100. Dithi-
othreitol redox buffers are not likely to form significant concentrations of
mixed disulfide intermediates with the test system because of the intramo-

lecular nature of the reaction. 51
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. The
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.