METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N. ABELSON MELVIN I. SIMON AND Division of Biology California Institute of Technology Pasadena, California Founding Editors SIDNEY P. COLOWICK NATHAN O. KAPLAN AND AcademicPressisanimprintofElsevier 525BStreet,Suite1900,SanDiego,CA92101-4495,USA 30CorporateDrive,Suite400,Burlington,MA01803,USA 32JamestownRoad,LondonNW17BY,UK Firstedition2011 Copyright#2011,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:978-0-12-381268-1 ISSN:0076-6879 PrintedandboundinUnitedStatesofAmerica 11 12 13 10 9 8 7 6 5 4 3 2 1 C ONTRIBUTORS AndrewJ.Andrews Department of Biochemistry and Molecular Biology, Colorado State University, FortCollins,Colorado,USA StefanT.Arold Department of Biochemistry and Molecular Biology, University of Texas MD AndersonCancerCenter,Houston,Texas,USA ZeljkoBajzer Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester,Minnesota,USA NicoleA.Becker Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester,Minnesota,USA DorothyBeckett Department of Chemistry and Biochemistry, Center for Biological Structure and Organization,UniversityofMaryland,Maryland,USA WlodzimierzBujalowski DepartmentofBiochemistryandMolecularBiology,andDepartmentofObstetrics andGynecology,TheSealyCenterforStructuralBiology,SealyCenterforCancer Cell Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas,USA JamesL.Cole Department of Molecular and Cell Biology, and Department of Chemistry, UniversityofConnecticut,Storrs,Connecticut,USA DeborahG.Conrady Department of Molecular Genetics, Biochemistry & Microbiology, University of CincinnatiCollegeofMedicine,Cincinnati,Ohio,USA ErnestoJ.Fuentes Department of Biochemistry,Roy J. and LucilleA.Carver College of Medicine, UniversityofIowa,andHoldenComprehensiveCancerCenter,IowaCity,Iowa, USA PetrHerman InstituteofPhysics,CharlesUniversity,KeKarlovu,Prague,CzechRepublic xi xii Contributors AndrewB.Herr Department of Molecular Genetics, Biochemistry & Microbiology, University of CincinnatiCollegeofMedicine,Cincinnati,Ohio,USA NicholasG.Housden DepartmentofBiology(Area10),UniversityofYork,York,UnitedKingdom MariaJ.Jezewska DepartmentofBiochemistryandMolecularBiology,TheSealyCenterforStruc- tural Biology, Sealy Center for Cancer Cell Biology, The University of Texas MedicalBranchatGalveston,Galveston,Texas,USA JasonD.Kahn Department of Chemistry and Biochemistry, University of Maryland, College Park,Maryland,USA ColinKleanthous DepartmentofBiology(Area10),UniversityofYork,York,UnitedKingdom JohnE.Ladbury Department of Biochemistry and Molecular Biology, University of Texas MD AndersonCancerCenter,Houston,Texas,USA KatherineLauner-Felty Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut,USA J.ChingLee Department of Biochemistry and Molecular Biology, The University of Texas MedicalBranchatGalveston,Galveston,Texas,USA VinceJ.LiCata Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana,USA Chin-ChiLiu Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana,USA AaronL.Lucius Department of Chemistry, The University of Alabama at Birmingham, Birmingham,Alabama,USA KarolinLuger Department of Biochemistry and Molecular Biology, Colorado State University, FortCollins,Colorado,andHowardHughesMedicalInstitute,USA L.JamesMaherIII Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester,Minnesota,USA Contributors xiii JustinM.Miller Department of Chemistry, The University of Alabama at Birmingham, Birmingham,Alabama,USA JustinP.Peters Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester,Minnesota,USA BurkiRajendar Department of Chemistry, The University of Alabama at Birmingham, Birmingham,Alabama,USA EmilyM.Rueter Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester,Minnesota,USA TysonR.Shepherd Department of Biochemistry,Roy J. and LucilleA.Carver College of Medicine, UniversityofIowa,IowaCity,Iowa,USA C.JasonWong Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut,USA P REFACE This volume is the continuation in a series of Methods in Enzymology volumes which promotes thermodynamics as an important tool for the study of biological systems. One of the many examples of biological ther- modynamics is the cooperative binding of oxygen by hemoglobin. Hemoglobin is the quintessential example of a ligand-binding protein. Mostbiochemistrytextbooksexplainthatthehemoglobintetramerexistsin two structural states, a low-affinity structure without oxygen bound and a high-affinity structure with oxygen bound. This is the classic two-state allosteric model as presented by Monod, Wyman, and Changeux (1965, J. Mol. Biol., 12, 88–118) and extended by Ackers and Johnson (1981, J. Mol. Biol. 147, 559–582). Unfortunately, this model tells us nothing about the specific molecular interactions that are altered by the binding of oxygen, which force the hemoglobin to shift to the alternative structural state. Investigating hemoglobin at this level is analogous to viewing only the first and last act of a tragedy by William Shakespeare (e.g., King Lear or Macbeth).Thefirstactbeingacelebrationandinthelastact,thestageisfull of dead bodies. While only the first and last acts are provocative, the truly wonderful part of a Shakespearean play is the character interactions in the interveningstepsbetweentheinitialandfinalstates,whichforcethelastact to follow from the first act. Thermodynamics provides a conceptual and mathematical framework, thatis,a“logictool,”whichallowstheinvestigationofthespecificmolec- ular interactions and the concomitant energetics such as those that are altered by the binding of oxygen, which force the hemoglobin to shift to the alternative structural and/or association states. MICHAELL JOHNSON, JO HOLT AND GARY K ACKERS xv 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 xvii xviii MethodsinEnzymology VOLUMEXVI.FastReactions EditedbyKENNETHKUSTIN VOLUMEXVII.MetabolismofAminoAcidsandAmines(PartsAandB) EditedbyHERBERTTABORANDCELIAWHITETABOR VOLUMEXVIII.VitaminsandCoenzymes(PartsA,B,andC) EditedbyDONALDB.MCCORMICKANDLEMUELD.WRIGHT VOLUMEXIX.ProteolyticEnzymes EditedbyGERTRUDEE.PERLMANNANDLASZLOLORAND VOLUMEXX.NucleicAcidsandProteinSynthesis(PartC) EditedbyKIVIEMOLDAVEANDLAWRENCEGROSSMAN VOLUMEXXI.NucleicAcids(PartD) EditedbyLAWRENCEGROSSMANANDKIVIEMOLDAVE VOLUMEXXII.EnzymePurificationandRelatedTechniques EditedbyWILLIAMB.JAKOBY VOLUMEXXIII.Photosynthesis(PartA) EditedbyANTHONYSANPIETRO VOLUMEXXIV.PhotosynthesisandNitrogenFixation(PartB) EditedbyANTHONYSANPIETRO VOLUMEXXV.EnzymeStructure(PartB) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEXXVI.EnzymeStructure(PartC) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEXXVII.EnzymeStructure(PartD) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEXXVIII.ComplexCarbohydrates(PartB) EditedbyVICTORGINSBURG VOLUMEXXIX.NucleicAcidsandProteinSynthesis(PartE) EditedbyLAWRENCEGROSSMANANDKIVIEMOLDAVE VOLUMEXXX.NucleicAcidsandProteinSynthesis(PartF) EditedbyKIVIEMOLDAVEANDLAWRENCEGROSSMAN VOLUMEXXXI.Biomembranes(PartA) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMEXXXII.Biomembranes(PartB) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMEXXXIII.CumulativeSubjectIndexVolumesI-XXX EditedbyMARTHAG.DENNISANDEDWARDA.DENNIS VOLUMEXXXIV.AffinityTechniques(EnzymePurification:PartB) EditedbyWILLIAMB.JAKOBYANDMEIRWILCHEK MethodsinEnzymology xix VOLUMEXXXV.Lipids(PartB) EditedbyJOHNM.LOWENSTEIN VOLUMEXXXVI.HormoneAction(PartA:SteroidHormones) EditedbyBERTW.O’MALLEYANDJOELG.HARDMAN VOLUMEXXXVII.HormoneAction(PartB:PeptideHormones) EditedbyBERTW.O’MALLEYANDJOELG.HARDMAN VOLUMEXXXVIII.HormoneAction(PartC:CyclicNucleotides) EditedbyJOELG.HARDMANANDBERTW.O’MALLEY VOLUMEXXXIX.HormoneAction(PartD:IsolatedCells,Tissues, andOrganSystems) EditedbyJOELG.HARDMANANDBERTW.O’MALLEY VOLUMEXL.HormoneAction(PartE:NuclearStructureandFunction) EditedbyBERTW.O’MALLEYANDJOELG.HARDMAN VOLUMEXLI.CarbohydrateMetabolism(PartB) EditedbyW.A.WOOD VOLUMEXLII.CarbohydrateMetabolism(PartC) EditedbyW.A.WOOD VOLUMEXLIII.Antibiotics EditedbyJOHNH.HASH VOLUMEXLIV.ImmobilizedEnzymes EditedbyKLAUSMOSBACH VOLUMEXLV.ProteolyticEnzymes(PartB) EditedbyLASZLOLORAND VOLUMEXLVI.AffinityLabeling EditedbyWILLIAMB.JAKOBYANDMEIRWILCHEK VOLUMEXLVII.EnzymeStructure(PartE) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEXLVIII.EnzymeStructure(PartF) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEXLIX.EnzymeStructure(PartG) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUMEL.ComplexCarbohydrates(PartC) EditedbyVICTORGINSBURG VOLUMELI.PurineandPyrimidineNucleotideMetabolism EditedbyPATRICIAA.HOFFEEANDMARYELLENJONES VOLUMELII.Biomembranes(PartC:BiologicalOxidations) EditedbySIDNEYFLEISCHERANDLESTERPACKER xx MethodsinEnzymology VOLUMELIII.Biomembranes(PartD:BiologicalOxidations) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMELIV.Biomembranes(PartE:BiologicalOxidations) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMELV.Biomembranes(PartF:Bioenergetics) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMELVI.Biomembranes(PartG:Bioenergetics) EditedbySIDNEYFLEISCHERANDLESTERPACKER VOLUMELVII.BioluminescenceandChemiluminescence EditedbyMARLENEA.DELUCA VOLUMELVIII.CellCulture EditedbyWILLIAMB.JAKOBYANDIRAPASTAN VOLUMELIX.NucleicAcidsandProteinSynthesis(PartG) EditedbyKIVIEMOLDAVEANDLAWRENCEGROSSMAN VOLUMELX.NucleicAcidsandProteinSynthesis(PartH) EditedbyKIVIEMOLDAVEANDLAWRENCEGROSSMAN VOLUME61.EnzymeStructure(PartH) EditedbyC.H.W.HIRSANDSERGEN.TIMASHEFF VOLUME62.VitaminsandCoenzymes(PartD) EditedbyDONALDB.MCCORMICKANDLEMUELD.WRIGHT VOLUME63.EnzymeKineticsandMechanism(PartA:InitialRateand InhibitorMethods) EditedbyDANIELL.PURICH VOLUME64.EnzymeKineticsandMechanism (PartB:IsotopicProbesandComplexEnzymeSystems) EditedbyDANIELL.PURICH VOLUME65.NucleicAcids(PartI) EditedbyLAWRENCEGROSSMANANDKIVIEMOLDAVE VOLUME66.VitaminsandCoenzymes(PartE) EditedbyDONALDB.MCCORMICKANDLEMUELD.WRIGHT VOLUME67.VitaminsandCoenzymes(PartF) EditedbyDONALDB.MCCORMICKANDLEMUELD.WRIGHT VOLUME68.RecombinantDNA EditedbyRAYWU VOLUME69.PhotosynthesisandNitrogenFixation(PartC) EditedbyANTHONYSANPIETRO VOLUME70.ImmunochemicalTechniques(PartA) EditedbyHELENVANVUNAKISANDJOHNJ.LANGONE