Formulation50%(vol/vol)glycerol/5mMCaCl2Storage-20°CPurity>95%bySDS-PAGEActivityDeterminationClottingassayShelfLife(properlystored)6monthsParticipationofFactorVainProthrombinaseAssemblyandexpressionoftheprothrombinasecomplexisillustrated.FactorVa,composedofaheavy(VaH)andlight(VaL)chain,bindstonegativelychargedphospholipidmembranesandeffectivelyservesasareceptorformembraneboundfactorXa(Xa).Theenzymecomplexservestoconvertthezymogenprothrombin(II)totheactiveserineproteasethrombin(IIa),inaproteolyticeventwhichremovesthefragment1.2(F1.2)portionofprothrombin.SampleGelInformation:GelNovex4-12%Bis-TrisLoadHumanFactorVa,1µgperlaneBufferMOPSStandardSeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)SpecialNotesFactorVisaverylABIleprotein.Somedegradedfragmentsareusuallynoticeableinpreparations.Overview:FactorVaisacofactorfortheserineproteasefactorXa,andinthepresenceofcalciumionstheycollectivelyassembleonaphospholipidsurfacetoformtheprothrombinasecomplex(1).TheprothrombinasecomplexisresponsIBLefortherapidconversionofprothrombintothrombin.FactorVaisderivedfromthepro-cofactor,factorV,uponlimitedproteolysisbyalpha-thrombin.ThethrombincleavageoffactorVliberatestwoheavilyglycosylatedactivationpeptidesfromthecentralportionofthemoleculewhichhavenocofactorfunction.FactorVaiscomprisedofanNH2-terminalderivedheavychain(Mr=94,000)andaCOOH-terminalderivedlightchain(Mr=74,000)whichremainassociatedinthepresenceofcalciumions.Thecofactorbindstophospholipid(cellmembrane)surfacesandeffectivelyservesasareceptorformembraneboundfactorXa.Completeassemblyoftheprothrombinasecomplex(factorXa,factorVa,phospholipid,andcalcium)resultsina300,000-foldincreaseintherateofprothrombinconversionrelativetotherateobservedwithfactorXaalone.TheinteractionbetweenfactorVaandfactorXaismediatedbyboththeheavyandlightchainoffactorVa,whilethebindingofprothrombintofactorVaismediatedsolelybytheheavychain.FactorVaispreparedbyactivatingpurifiedfactorVwiththrombinandissubsequentlypurifiedbyimmunoaffinitychromatography(2).Thisprocessresultsincofactorpreparationswhicharefreeofbothactivationpeptidesandthrombin.PurifiedfactorVaissuppliedin50%glycerol(vol/vol),5.0mMCaCl2,andshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinafactorVaclottingassay.Properties:LocalizationPlasmaandmembranesurfacesModeofactioncofactorforfactorXaintheprothrombinasecomplexMolecularweight168,000(baseduponthecombinedmolecularweightofsubunits)ExtinctioncoefficientE1%1cm,280nm=17.4(3)Structuretwosubunits,Mr~94,000(heavychain)and74,000(lightchain)(4)Percentcarbohydrateapproximately8%(baseduponthecalculatedmolecularweightofhumanfactorVa)