METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N. ABELSON MELVIN I. SIMON AND Division of Biology California Institute of Technology Pasadena, California, USA Founding Editors SIDNEY P. COLOWICK NATHAN O. KAPLAN AND AcademicPressisanimprintofElsevier 525BStreet,Suite1900,SanDiego,CA92101-4495,USA 30CorporateDrive,Suite400,Burlington,MA01803,USA 32JamestownRoad,LondonNW17BY,UK Firstedition2010 Copyright#2010,ElsevierInc.AllRightsReserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinany formorbyanymeanselectronic,mechanical,photocopying,recordingorotherwisewithoutthe priorwrittenpermissionofthepublisher PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRightsDepartment inOxford,UK:phone(+44)(0)1865843830;fax(+44)(0)1865853333;email:permissions@ elsevier.com.AlternativelyyoucansubmityourrequestonlinebyvisitingtheElsevierwebsiteat http://elsevier.com/locate/permissions,andselectingObtainingpermissiontouseElseviermaterial Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or propertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseoroperation ofanymethods,products,instructionsorideascontainedinthematerialherein.Becauseofrapid advancesinthemedicalsciences,inparticular,independentverificationofdiagnosesanddrug dosagesshouldbemade ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com ISBN:978-0-12-381003-8 ISSN:0076-6879 PrintedandboundinUnitedStatesofAmerica 10 11 12 10 9 8 7 6 5 4 3 2 1 C ONTRIBUTORS ScottW.Aesif Department of Pathology, University of Vermont College of Medicine, Burlington,Vermont,USA KellyK.Andringa Department ofEnvironmentalHealthSciences,CenterforFreeRadicalBiology, UniversityofAlabamaatBirmingham,Birmingham,Alabama,USA ShannonM.Bailey Department ofEnvironmentalHealthSciences,CenterforFreeRadicalBiology, UniversityofAlabamaatBirmingham,Birmingham,Alabama,USA StevenBernstein James P. Wilmot Cancer Center, University of Rochester Medical Center, Rochester,NewYork,USA AruniBhatnagar DiabetesandObesityCenter,UniversityofLouisville,Louisville,Kentucky,USA AlbertoBindoli Institute of Neuroscience (CNR), Section of Padova, c/o Department of BiologicalChemistry,Padova,Italy BenoitBoivin ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork,USA PaulBrookes Department of Anesthesiology, University of Rochester Medical Center, Rochester,NewYork,USA SamuelCaito Lung Biology and Disease Program, Department of Environmental Medicine, UniversityofRochesterMedicalCenter,Rochester,NewYork,USA BradleyA.Carlson MolecularBiologyofSeleniumSection,LaboratoryofCancerPrevention,Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,Maryland,USA ZheChen Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto,Japan xiii xiv Contributors ChuangC.Chiueh Division of Clinical Pharmacy, School of Pharmacy and Taipei Medical University—ShuangHoHospital,Taipei,Taiwan Chun-SeokCho Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu,Seoul,Korea EdwardT.Chouchani Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building,Cambridge,UK AndrewG.Cox Free Radical Research Group, Department of Pathology, and National Research Centre for Growth and Development, University of Otago, Christchurch, NewZealand VadimN.Gladyshev DivisionofGenetics,DepartmentofMedicine,Brigham&Women’sHospitaland HarvardMedicalSchool,Boston,Massachusetts,USA Young-MiGo Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine,EmoryUniversity,Atlanta,Georgia,USA MarkB.Hampton Free Radical Research Group, Department of Pathology, and National Research Centre for Growth and Development, University of Otago, Christchurch, NewZealand DolphL.Hatfield MolecularBiologyofSeleniumSection,LaboratoryofCancerPrevention,Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,Maryland,USA YunHe InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA QunhuaHuang InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA ThomasR.Hurd Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building,Cambridge,UK HidenoriIchijo Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, TheUniversityofTokyo,Hongo,Bunkyo-ku,Tokyo,Japan Contributors xv RobertIrons MolecularBiologyofSeleniumSection,LaboratoryofCancerPrevention,Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,Maryland,USA YvonneM.W.Janssen-Heininger Department of Pathology, University of Vermont College of Medicine, Burlington,Vermont,USA DeanP.Jones Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine,EmoryUniversity,Atlanta,Georgia,USA P.AndrewKarplus Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon,USA KazumiKatagiri Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, TheUniversityofTokyo,Hongo,Bunkyo-ku,Tokyo,Japan YanLuo State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, SunYat-SenUniversity,Guangzhou,China GuntherMarsche InstituteofExperimentalandClinicalPharmacology,MedicalUniversityofGraz, Universita¨tsplatz,Graz,Austria HiroshiMasutani Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto,Japan YoshiyukiMatsuo Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto,Japan AtsushiMatsuzawa Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, TheUniversityofTokyo,Hongo,Bunkyo-ku,Tokyo,Japan KatjaE.Menger Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building,Cambridge,UK WangMin InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA,andStateKeyLaboratoryof Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou,China xvi Contributors MichaelP.Murphy Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building,Cambridge,UK KarlOettl Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse, Graz,Austria DerekParsonage Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem,NorthCarolina,USA LeslieB.Poole Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem,NorthCarolina,USA IrfanRahman Lung Biology and Disease Program, Department of Environmental Medicine, UniversityofRochesterMedicalCenter,Rochester,NewYork,USA KotaV.Ramana DepartmentofBiochemistryandMolecularBiology,UniversityofTexasMedical Branch,Galveston,Texas,USA StacyA.Reeves Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem,NorthCarolina,USA RaquelRequejo Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building,Cambridge,UK NikiL.Reynaert Department of Respiratory Medicine, Maastricht University, Maastricht, TheNetherlands SueGooRhee Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu,Seoul,Korea MariaPiaRigobello DepartmentofBiologicalChemistry,UniversityofPadova,Padova,Italy JolantaSkalska James P. Wilmot Cancer Center, University of Rochester Medical Center, Rochester,NewYork,USA SanjaySrivastava DiabetesandObesityCenter,UniversityofLouisville,Louisville,Kentucky,USA Contributors xvii SatishK.Srivastava DepartmentofBiochemistryandMolecularBiology,UniversityofTexasMedical Branch,Galveston,Texas,USA IsaacK.Sundar Lung Biology and Disease Program, Department of Environmental Medicine, UniversityofRochesterMedicalCenter,Rochester,NewYork,USA NicholasK.Tonks ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork,USA PetraTsuji MolecularBiologyofSeleniumSection,LaboratoryofCancerPrevention,Center for Cancer Research, and Cancer Prevention Fellowship Program; Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute,NationalInstitutesofHealth,Bethesda,Maryland,USA AntonA.Turanov DivisionofGenetics,DepartmentofMedicine,Brigham&Women’sHospitaland HarvardMedicalSchool,Boston,Massachusetts,USA ChristineC.Winterbourn Free Radical Research Group, Department of Pathology, and National Research Centre for Growth and Development, University of Otago, Christchurch, NewZealand Luyao(Kevin)Xu InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA HongweiYao Lung Biology and Disease Program, Department of Environmental Medicine, UniversityofRochesterMedicalCenter,Rochester,NewYork,USA JunjiYodoi Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto,Japan Min-HyukYoo MolecularBiologyofSeleniumSection,LaboratoryofCancerPrevention,Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,Maryland,USA EijiYoshihara DepartmentofBiologicalResponses,InstituteforVirusResearch,andDivisionof Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan xviii Contributors Huanjiao(Jenny)Zhou State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, SunYat-SenUniversity,Guangzhou,China HaifengZhang InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA XuZhe InterdepartmentalPrograminVascularBiologyandTherapeutics,YaleUniversity SchoolofMedicine,NewHaven,Connecticut,USA P REFACE Signaling by reactive oxygen- and nitrogen species, mainly non-radical species such as hydrogen peroxide, lipid peroxides, and peroxynitrite— which may be viewed as second messengers—has emerged as a major regulatory process of cell function. Signaling targets are redox-sensitive protein cysteines and the large pool of low-molecular thiols, mainly gluta- thione. ThesevolumesofMethodsinEnzymologyon ThiolsRedoxTransi- tionsinCellSignalingaddresstwolargetopicsChemistryandBiochemistry of Low Molecular Weight and Protein Thiols (Part A, Volume 473) and Cellular Localization and Signaling (Part B, Volume 474). Both volumes serve to bring together current methods and concepts in the field of cell signalingdrivenbythiolredoxmodificationsbytechniquessuchasfluores- cence-based proteomics, mass spectrometry approaches, and fluorescence reporters. The editors thank all the contributors, whose thorough and innovative work is the basis of these two Methods in Enzymology volumes. The editors givespecialthankstoLeopoldFlohe´ forprovidingtheintroductorychapter ‘‘Changing paradigms in thiology: from antioxidant defense toward redox regulation, whose critical thinking educated us on the major concepts by which metabolic regulation and adaptation are transduced via thiol modifications. ENRIQUE CADENAS LESTER PACKER March 2010 xix M E ETHODS IN NZYMOLOGY VOLUMEI.PreparationandAssayofEnzymes EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEII.PreparationandAssayofEnzymes EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEIII.PreparationandAssayofSubstrates EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEIV.SpecialTechniquesfortheEnzymologist EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEV.PreparationandAssayofEnzymes EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEVI.PreparationandAssayofEnzymes(Continued) PreparationandAssayofSubstrates SpecialTechniques EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEVII.CumulativeSubjectIndex EditedbySIDNEYP.COLOWICKANDNATHANO.KAPLAN VOLUMEVIII.ComplexCarbohydrates EditedbyELIZABETHF.NEUFELDANDVICTORGINSBURG VOLUMEIX.CarbohydrateMetabolism EditedbyWILLISA.WOOD VOLUMEX.OxidationandPhosphorylation EditedbyRONALDW.ESTABROOKANDMAYNARDE.PULLMAN VOLUMEXI.EnzymeStructure EditedbyC.H.W.HIRS VOLUMEXII.NucleicAcids(PartsAandB) EditedbyLAWRENCEGROSSMANANDKIVIEMOLDAVE VOLUMEXIII.CitricAcidCycle EditedbyJ.M.LOWENSTEIN VOLUMEXIV.Lipids EditedbyJ.M.LOWENSTEIN VOLUMEXV.SteroidsandTerpenoids EditedbyRAYMONDB.CLAYTON xxi