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). [...]... state changes are comparable in rate to the time required for the assay 15 Suitable control experiments and appropriate selection of assay and preincubation conditions can generally be found so that reasonable estimates of regulatory thiol/disulfide redox potentials can be made using enzyme activity as a reporter of redox state Analyzing Results If the concentrations of all redox species can be measured... 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... (reaction 4) These may be acetylated to form mercapturic acids (reaction 8) Other chemical transformations of the mercapturic acids and their precursors have also been observed TM Glutathione serves as an antioxidant by reacting directly with free radicals (reaction 11) and by providing substrate for the GSH peroxidases and for the GSH transhydrogenases Thus, a variety of reductive reactions that take... 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 o f i n t r a. .. 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 proteinsY Dithiols/Disulfides of Catalytic Importance A number of flavin-dependent reductases, including thioredoxin reductase, glutathione reductase, and lipoyl dehydrogenase (dihydrolipoamide dehydrogenase),26 have vicinal thiols at the active... [2] Exchange Equilibria and Disulfide Bond Stability B y H I R A M 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, disulfide bond stability... 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... consisting of 10 m M RSH and 10 m M RSSR, equilibrium would be expected afte} 8.6 min at p H 7 and 1.0 min at pH 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,... of the incubation time For an accuracy of 5%, this requires that the measurement 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 For an equilibrium constant of one and with a redox buffer... 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 conditions of oxidative stress, [GSH]/[GSSG] ratios may fall to 1-10, and R [GSH] may drop . 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. 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-. (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