Measurement-of-Protein-Using-Bicinchoninic-Acid

ANALYTICALBIOCHEMISTRY19,76-85(1985)MeasurementofProteinUsingBicinchoninicAcid’P.K.SMITH,*R.I.KROHN,G.T.HERMANSON,A.K.MALLIA,F.H.GARTNER,M.D.FROVENZANO,E.K.FUJIMOTO,N.M.GOEKE,B.J.OLSON,ANDD.C.KLENKBiochemicalResearchDivision,PierceChemicalCompany,P.O.BoxI17,Rockford,Illinois61IO5ReceivedApril30,1985Bicinchoninicacid,sodiumsalt,isastable,water-solublecompoundcapableofforminganintensepurplecomplexwithcuprousion(Cu’+)inanalkalineenvironment.ThisreagentformsthebasisofananalyticalmethodcapableofmonitoringcuprousionproducedinthereactionofproteinwithalkalineCu2+(biuretreaction).Thecolorproducedfromthisreactionisstableandincreasesinaproportionalfashionoverabroadrangeofincreasingproteinconcentrations.WhencomparedtothemethodofLowryetal.,theresultsreportedheredemonstrateagreatertoleranceofthebicinchoninatereagenttowardsuchcommonlyencounteredinterferencesasnonionicde-tergentsandsimplebuffersalts.Thestabilityofthereagentandresultingchromophorealsoallowsforasimplified,one-stepanalysisandanenhancedflexibilityinprotocolselection.Thisnewmethodmaintainsthehighsensitivityandlowprotein-to-proteinvariationassociatedwiththeLowrytechnique.01985AcademicFPLS,hc.KEYWORDS:proteindetermination;bicinchoninicacid.ThewidelyusedmethodofLowryetal.(1)forproteindeterminationreliesontheFolin-Ciocalteaureagenttoenhancethesensitivityofthebiuretreaction.Theinstabilityofthisreagentinanalkalinemediumdemandsthatexactingtechniquebeexercisedinthetimingofbothreagentadditionandmixingwithsampleinordertoobtainaccurateresults.Thetwo-stepnatureoftheLowrymethodisme-chanicallycumbersomeandtedious,andaddsconsiderablecomplexitytoeffortstoattempttoutilizeitinanautomatedorpost-column-detectionformat(2).Additionally,nonionicdetergentsaswellassomebuffersaltsusedatlevelsusefulforproteinsolubilizationcanin-terferebyforminginsolubleprecipitateswiththeFolin-Ciocalteaureagent.Itseemsappar-entthatmostofthedifficultiesassociatedwiththeLowrymethodareduetothepeculiaritiesofthedetectionreagentused.Inthispaperwedescribeanewproteinmeasurementmethodbaseduponanalter-nativedetectionreagent,namelybicinchon-inicacid(BCA).3Intheformofitswater-sol-ublesodiumsalt,BCAisasensitive,stable,andhighlyspecificreagentforCu’+(3).Pre-viously,thisattributehasbeenutilizedtomonitorthelevelsofothersubstancescapableofreducingCu*+toCu’+suchasuricacid(4)andglucose(5).Toourknowledge,noonehasexploitedaBCAindicatorsystemtomon-itorCu’+producedduringthebiuretreaction.MATERIALSANDMETHODSChemicals.Bicinchoninicacid,sodiumsalt,wassynthesizedbythePfitzingerreactionofisatinandacetoin(Aldrich),substitutingso-diumhydroxideforpotassiumhydroxideinthemethodofLesesneandHenze(6).The’ThispaperisdedicatedtothememoryofDr.E.Melvin3Abbreviationsused:BCA,bicinchoninicacid;BCA-Gindler,along-timefriendandcolleague.WithoutMel’sNa2,bicinchoninicacid,disodiumsalt;RIBO,ribonucleaseknowledgeandloveofchemistrywhichhesogenerouslyA;CHYMO,chymotrypsinogen;IgG,humanimmuno-shared,thisworkwouldnothavebeenpossible.globulinG,BSA,bovineserumalbumin;S-WR,Standard’Towhomcorrespondenceshouldbedirected.WorkingReagent.0003-2697185$3.00Copyright01985byAcademicPress.Inc.Allrightsofreproductioninanyform-ed.76PROTEINASSAYUSINGBIClNCHONINICACID77crudeproductobtainedwassubjectedtore-crystallizationfromaminimumamountof75°Cwateruntiltheabsorbanceblankofthefreshlypreparedworkingreagent(describedbelow)waslessthan0.065at562nm.Threerecrystallizationswereusuallyrequired.Thehydrated,nearlycolorlessneedlesisolatedre-verttoamorphous,cream-coloredanhydrouspowderupondryingat60°C.Technical-gradeBCAcurrentlyavailablefromvarioussources(Sigma,Hach,Pierce)givesasomewhathigherblankreadingandhasnotbeenfurthereval-uated.Inorganicsaltsusedinthereagentfor-mulationswereofreagent-gradequality(J.T.BakerorFisher).Allpreparedsolutionswerepassedthroughl-pmfilterstoremoveinsol-ubledebrisassociatedwiththesalts.Folin-Ciocalteaureagent(2N)waspurchasedfromFisheranddilutedto1Npriortouse.Com-poundsusedintheinterferingsubstancesstudieswereofthehighestqualitycommer-ciallyavailable,whilenonionicdetergentswerefurtherpurifiedbydescribedmethods(7,8).Allwaterusedwasdeionized,butcaremustbetakentoavoidobtainingwaterfromsys-temscontainingcopperlinesandfittings,be-causeenoughdissolvedcopperasCu’+maybepresenttocontributeappreciablytoblankreadings.Wefoundthatdeionizedwater(18Mohm-cm)deliveredfromallplasticcar-tridgedunits(MilliporeorBamsted)wassat-isfactory.Proteins.BSA(crystallized)andIgG(hu-man)wereobtainedfromMiles.Ribonuclease,chymotrypsinogen,andhumaninsulinwerefromBoehringer-Mannheim,andavidin(eggwhite)wasofaffinity-purifiedgradeobtainedfromPierce.Standardproteinsolutionswerepreparedineitherisotonicsalineor,inthecaseoftheinterferingsubstancesstudies,inasolutioncontainingtheparticularcompoundunderstudy.Lowryreagentandprotocol.TheLowryre-agentformulationsandprotocolusedinthisstudywereasdescribed(1).StandardBCAreagentandprotocol.Re-agentAconsistsofanaqueoussolutionof1%BCA-Na2,2%Na2C03-HZO,0.16%Naztar-trate,0.4%NaOH,and0.95%NaHC03.Ifneeded,appropriateadditionofNaOH(50%)orsolidNaHC03ismadetoreage