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xivContributorsChuang C. ChiuehDivision of Clinical Pharmacy, School of Pharmacy and Taipei MedicalUniversity—Shuang Ho Hospital, Taipei, TaiwanChun-Seok ChoDivision of Life and Pharmaceutical Sciences, Ewha Womans University,Seodaemun-gu, Seoul, KoreaEdward T. ChouchaniMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRCBuilding, Cambridge, UKAndrew G. CoxFree Radical Research Group, Department of Pathology, and National ResearchCentre for Growth and Development, University of Otago, Christchurch,New ZealandVadim N. GladyshevDivision of Genetics, Department of Medicine, Brigham & Women’s Hospital andHarvard Medical School, Boston, Massachusetts, USAYoung-Mi GoDepartment of Medicine, Division of Pulmonary, Allergy and Critical CareMedicine, Emory University, Atlanta, Georgia, USAMark B. HamptonFree Radical Research Group, Department of Pathology, and National ResearchCentre for Growth and Development, University of Otago, Christchurch,New ZealandDolph L. HatfieldMolecular Biology of Selenium Section, Laboratory of Cancer Prevention, Centerfor Cancer Research, National Cancer Institute, National Institutes of Health,Bethesda, Maryland, USAYun HeInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USAQunhua HuangInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USAThomas R. HurdMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRCBuilding, Cambridge, UKHidenori IchijoLaboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences,The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan

ContributorsxvRobert IronsMolecular Biology of Selenium Section, Laboratory of Cancer Prevention, Centerfor Cancer Research, National Cancer Institute, National Institutes of Health,Bethesda, Maryland, USAYvonne M. W. Janssen-HeiningerDepartment of Pathology, University of Vermont College of Medicine,Burlington, Vermont, USADean P. JonesDepartment of Medicine, Division of Pulmonary, Allergy and Critical CareMedicine, Emory University, Atlanta, Georgia, USAP. Andrew KarplusDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis,Oregon, USAKazumi KatagiriLaboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences,The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, JapanYan LuoState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,Sun Yat-Sen University, Guangzhou, ChinaGunther MarscheInstitute of Experimental and Clinical Pharmacology, Medical University of Graz,Universitätsplatz, Graz, AustriaHiroshi MasutaniDepartment of Biological Responses, Institute for Virus Research, KyotoUniversity, Kyoto, JapanYoshiyuki MatsuoDepartment of Biological Responses, Institute for Virus Research, KyotoUniversity, Kyoto, JapanAtsushi MatsuzawaLaboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences,The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, JapanKatja E. MengerMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRCBuilding, Cambridge, UKWang MinInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USA, and State Key Laboratory ofOphthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University,Guangzhou, China

xviContributorsMichael P. MurphyMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRCBuilding, Cambridge, UKKarl OettlInstitute of Physiological Chemistry, Medical University of Graz, Harrachgasse,Graz, AustriaDerek ParsonageDepartment of Biochemistry, Wake Forest University School of Medicine,Winston-Salem, North Carolina, USALeslie B. PooleDepartment of Biochemistry, Wake Forest University School of Medicine,Winston-Salem, North Carolina, USAIrfan RahmanLung Biology and Disease Program, Department of Environmental Medicine,University of Rochester Medical Center, Rochester, New York, USAKota V. RamanaDepartment of Biochemistry and Molecular Biology, University of Texas MedicalBranch, Galveston, Texas, USAStacy A. ReevesDepartment of Biochemistry, Wake Forest University School of Medicine,Winston-Salem, North Carolina, USARaquel RequejoMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRCBuilding, Cambridge, UKNiki L. ReynaertDepartment of Respiratory Medicine, Maastricht University, Maastricht,The NetherlandsSue Goo RheeDivision of Life and Pharmaceutical Sciences, Ewha Womans University,Seodaemun-gu, Seoul, KoreaMaria Pia RigobelloDepartment of Biological Chemistry, University of Padova, Padova, ItalyJolanta SkalskaJames P. Wilmot Cancer Center, University of Rochester Medical Center,Rochester, New York, USASanjay SrivastavaDiabetes and Obesity Center, University of Louisville, Louisville, Kentucky, USA

ContributorsxviiSatish K. SrivastavaDepartment of Biochemistry and Molecular Biology, University of Texas MedicalBranch, Galveston, Texas, USAIsaac K. SundarLung Biology and Disease Program, Department of Environmental Medicine,University of Rochester Medical Center, Rochester, New York, USANicholas K. TonksCold Spring Harbor Laboratory, Cold Spring Harbor, New York, USAPetra TsujiMolecular Biology of Selenium Section, Laboratory of Cancer Prevention, Centerfor Cancer Research, and Cancer Prevention Fellowship Program; NutritionalScience Research Group, Division of Cancer Prevention, National CancerInstitute, National Institutes of Health, Bethesda, Maryland, USAAnton A. TuranovDivision of Genetics, Department of Medicine, Brigham & Women’s Hospital andHarvard Medical School, Boston, Massachusetts, USAChristine C. WinterbournFree Radical Research Group, Department of Pathology, and National ResearchCentre for Growth and Development, University of Otago, Christchurch,New ZealandLuyao (Kevin) XuInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USAHongwei YaoLung Biology and Disease Program, Department of Environmental Medicine,University of Rochester Medical Center, Rochester, New York, USAJunji YodoiDepartment of Biological Responses, Institute for Virus Research, KyotoUniversity, Kyoto, JapanMin-Hyuk YooMolecular Biology of Selenium Section, Laboratory of Cancer Prevention, Centerfor Cancer Research, National Cancer Institute, National Institutes of Health,Bethesda, Maryland, USAEiji YoshiharaDepartment of Biological Responses, Institute for Virus Research, and Division ofSystemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto,Japan

xviiiContributorsHuanjiao ( Jenny) ZhouState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,Sun Yat-Sen University, Guangzhou, ChinaHaifeng ZhangInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USAXu ZheInterdepartmental Program in Vascular Biology and Therapeutics, Yale UniversitySchool of Medicine, New Haven, Connecticut, USA

PREFACESignaling by reactive oxygen- and nitrogen species, mainly non-radicalspecies such as hydrogen peroxide, lipid peroxides, and peroxynitrite—which may be viewed as second messengers—has emerged as a majorregulatory process of cell function. Signaling targets are redox-sensitiveprotein cysteines and the large pool of low-molecular thiols, mainly glutathione. These volumes of Methods in Enzymology on Thiols Redox Transitions in Cell Signaling address two large topics Chemistry and Biochemistryof Low Molecular Weight and Protein Thiols (Part A, Volume 473) andCellular Localization and Signaling (Part B, Volume 474). Both volumesserve to bring together current methods and concepts in the field of cellsignaling driven by thiol redox modifications by techniques such as fluorescence-based proteomics, mass spectrometry approaches, and fluorescencereporters.The editors thank all the contributors, whose thorough and innovativework is the basis of these two Methods in Enzymology volumes. The editorsgive special thanks to Leopold Flohé for providing the introductory chapter‘‘Changing paradigms in thiology: from antioxidant defense toward redoxregulation, whose critical thinking educated us on the major concepts bywhich metabolic regulation and adaptation are transduced via thiolmodifications.ENRIQUE CADENASLESTER PACKERMarch 2010xix

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METHODS IN ENZYMOLOGYVOLUME I. Preparation and Assay of EnzymesEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME II. Preparation and Assay of EnzymesEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME III. Preparation and Assay of SubstratesEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME IV. Special Techniques for the EnzymologistEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME V. Preparation and Assay of EnzymesEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME VI. Preparation and Assay of Enzymes (Continued)Preparation and Assay of SubstratesSpecial TechniquesEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME VII. Cumulative Subject IndexEdited by SIDNEY P. COLOWICK AND NATHAN O. KAPLANVOLUME VIII. Complex CarbohydratesEdited by ELIZABETH F. NEUFELD AND VICTOR GINSBURGVOLUME IX. Carbohydrate MetabolismEdited by WILLIS A. WOODVOLUME X. Oxidation and PhosphorylationEdited by RONALD W. ESTABROOK AND MAYNARD E. PULLMANVOLUME XI. Enzyme StructureEdited by C. H. W. HIRSVOLUME XII. Nucleic Acids (Parts A and B)Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVEVOLUME XIII. Citric Acid CycleEdited by J. M. LOWENSTEINVOLUME XIV. LipidsEdited by J. M. LOWENSTEINVOLUME XV. Steroids and TerpenoidsEdited by RAYMOND B. CLAYTONxxi

xxiiMethods in EnzymologyVOLUME XVI. Fast ReactionsEdited by KENNETH KUSTINVOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)Edited by HERBERT TABOR AND CELIA WHITE TABORVOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHTVOLUME XIX. Proteolytic EnzymesEdited by GERTRUDE E. PERLMANN AND LASZLO LORANDVOLUME XX. Nucleic Acids and Protein Synthesis (Part C)Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMANVOLUME XXI. Nucleic Acids (Part D)Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVEVOLUME XXII. Enzyme Purification and Related TechniquesEdited by WILLIAM B. JAKOBYVOLUME XXIII. Photosynthesis (Part A)Edited by ANTHONY SAN PIETROVOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)Edited by ANTHONY SAN PIETROVOLUME XXV. Enzyme Structure (Part B)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME XXVI. Enzyme Structure (Part C)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME XXVII. Enzyme Structure (Part D)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME XXVIII. Complex Carbohydrates (Part B)Edited by VICTOR GINSBURGVOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVEVOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMANVOLUME XXXI. Biomembranes (Part A)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME XXXII. Biomembranes (Part B)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME XXXIII. Cumulative Subject Index Volumes I-XXXEdited by MARTHA G. DENNIS AND EDWARD A. DENNISVOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK

Methods in EnzymologyVOLUME XXXV. Lipids (Part B)Edited by JOHN M. LOWENSTEINVOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)Edited by BERT W. O’MALLEY AND JOEL G. HARDMANVOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)Edited by BERT W. O’MALLEY AND JOEL G. HARDMANVOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)Edited by JOEL G. HARDMAN AND BERT W. O’MALLEYVOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues,and Organ Systems)Edited by JOEL G. HARDMAN AND BERT W. O’MALLEYVOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)Edited by BERT W. O’MALLEY AND JOEL G. HARDMANVOLUME XLI. Carbohydrate Metabolism (Part B)Edited by W. A. WOODVOLUME XLII. Carbohydrate Metabolism (Part C)Edited by W. A. WOODVOLUME XLIII. AntibioticsEdited by JOHN H. HASHVOLUME XLIV. Immobilized EnzymesEdited by KLAUS MOSBACHVOLUME XLV. Proteolytic Enzymes (Part B)Edited by LASZLO LORANDVOLUME XLVI. Affinity LabelingEdited by WILLIAM B. JAKOBY AND MEIR WILCHEKVOLUME XLVII. Enzyme Structure (Part E)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME XLVIII. Enzyme Structure (Part F)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME XLIX. Enzyme Structure (Part G)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME L. Complex Carbohydrates (Part C)Edited by VICTOR GINSBURGVOLUME LI. Purine and Pyrimidine Nucleotide MetabolismEdited by PATRICIA A. HOFFEE AND MARY ELLEN JONESVOLUME LII. Biomembranes (Part C: Biological Oxidations)Edited by SIDNEY FLEISCHER AND LESTER PACKERxxiii

xxivMethods in EnzymologyVOLUME LIII. Biomembranes (Part D: Biological Oxidations)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME LIV. Biomembranes (Part E: Biological Oxidations)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME LV. Biomembranes (Part F: Bioenergetics)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME LVI. Biomembranes (Part G: Bioenergetics)Edited by SIDNEY FLEISCHER AND LESTER PACKERVOLUME LVII. Bioluminescence and ChemiluminescenceEdited by MARLENE A. DELUCAVOLUME LVIII. Cell CultureEdited by WILLIAM B. JAKOBY AND IRA PASTANVOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMANVOLUME LX. Nucleic Acids and Protein Synthesis (Part H)Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMANVOLUME 61. Enzyme Structure (Part H)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME 62. Vitamins and Coenzymes (Part D)Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHTVOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate andInhibitor Methods)Edited by DANIEL L. PURICHVOLUME 64. Enzyme Kinetics and Mechanism(Part B: Isotopic Probes and Complex Enzyme Systems)Edited by DANIEL L. PURICHVOLUME 65. Nucleic Acids (Part I)Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVEVOLUME 66. Vitamins and Coenzymes (Part E)Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHTVOLUME 67. Vitamins and Coenzymes (Part F)Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHTVOLUME 68. Recombinant DNAEdited by RAY WUVOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)Edited by ANTHONY SAN PIETROVOLUME 70. Immunochemical Techniques (Part A)Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE

Methods in EnzymologyxxvVOLUME 71. Lipids (Part C)Edited by JOHN M. LOWENSTEINVOLUME 72. Lipids (Part D)Edited by JOHN M. LOWENSTEINVOLUME 73. Immunochemical Techniques (Part B)Edited by JOHN J. LANGONE AND HELEN VAN VUNAKISVOLUME 74. Immunochemical Techniques (Part C)Edited by JOHN J. LANGONE AND HELEN VAN VUNAKISVOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LXEdited by EDWARD A. DENNIS AND MARTHA G. DENNISVOLUME 76. HemoglobinsEdited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONEVOLUME 77. Detoxication and Drug MetabolismEdited by WILLIAM B. JAKOBYVOLUME 78. Interferons (Part A)Edited by SIDNEY PESTKAVOLUME 79. Interferons (Part B)Edited by SIDNEY PESTKAVOLUME 80. Proteolytic Enzymes (Part C)Edited by LASZLO LORANDVOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I)Edited by LESTER PACKERVOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix)Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSENVOLUME 83. Complex Carbohydrates (Part D)Edited by VICTOR GINSBURGVOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays)Edited by JOHN J. LANGONE AND HELEN VAN VUNAKISVOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatusand the Cytoskeleton)Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAMVOLUME 86. Prostaglandins and Arachidonate MetabolitesEdited by WILLIAM E. M. LANDS AND WILLIAM L. SMITHVOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates,Stereo-chemistry, and Rate Studies)Edited by DANIEL L. PURICHVOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II)Edited by LESTER PACKER

xxviMethods in EnzymologyVOLUME 89. Carbohydrate Metabolism (Part D)Edited by WILLIS A. WOODVOLUME 90. Carbohydrate Metabolism (Part E)Edited by WILLIS A. WOODVOLUME 91. Enzyme Structure (Part I)Edited by C. H. W. HIRS AND SERGE N. TIMASHEFFVOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies andGeneral Immunoassay Methods)Edited by JOHN J. LANGONE AND HELEN VAN VUNAKISVOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, FcReceptors, and Cytotoxicity)Edited by JOHN J. LANGONE AND HELEN VAN VUNAKISVOLUME 94. PolyaminesEdited by HERBERT TABOR AND CELIA WHITE TABORVOLUME 95. Cumulative Subject Index Volumes 61–74, 76–80Edited by EDWARD A. DENNIS AND MARTHA G. DENNISVOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly andTargeting (General Methods; Eukaryotes)]Edited by SIDNEY FLEISCHER AND BECCA FLEISCHERVOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly andTargeting (Prokaryotes, Mitochondria, and Chloroplasts)]Edited by SIDNEY FLEISCHER AND BECCA FLEISCHERVOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processingand Recycling)Edited by SIDNEY FLEISCHER AND BECCA FLEISCHERVOLUME 99. Hormone Action (Part F: Protein Kinases)Edited by JACKIE D. CORBIN AND JOEL G. HARDMANVOLUME 100. Recombinant DNA (Part B)Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVEVOLUME 101. Recombinant DNA (Part C)Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVEVOLUME 102. Hormone Action (Part G: Calmodulin andCalcium-Binding Proteins)Edited by ANTHONY R. MEANS AND BERT W. O’MALLEYVOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)Edited by P. MICHAEL CONNVOLUME 104. Enzyme Purification and Related Techniques (Part C)Edited by WILLIAM B. JAKOBY

Methods in EnzymologyxxviiVOLUME 105. Oxygen Radicals in Biological SystemsEdited by LESTER PACKERVOLUME 106. Posttranslational Modifications (Part A)Edited by FINN WOLD AND KIVIE MOLDAVEVOLUME 107. Posttranslational Modifications (Part B)Edited by FINN WOLD AND KIVIE MOLDAVEVOLUME 108. Immunochemical Techniques (Part G: Separation andCharacterization of Lymphoid Cells)

Pasadena, California, USA Founding Editors SIDNEY P. COLOWICK AND NATHAN O. KAPLAN. VOLUME FOUR HUNDRED AND SEVENTY-FOUR METHODS IN ENZYMOLOGY Thiol Redox Transitions in Cell Signaling, Part B Cellular Localization and Signaling . Harvard Medical School, Boston, Massachusetts, USA