Formulation50%glycerol/water(v/v)Storage-20°CPurity>95%bySDS-PAGEActivityDeterminationClottingassayShelfLife(properlystored)6monthsActivationofFactorVbyAlpha-ThrombinTheactivationofsinglechainfactorV(330,000)byalpha-thrombinisrepresented.ArrowsindicatethesiteatwhichfactorViscleavedbythrombintoyieldtwoactivationpeptides(71,000and120,000),inadditiontotheheavy(94,000)andlight(74,000)chainswhichformVa.TheheavyandlightchainsoffactorVaremainnon-covalentlyassociatedinthepresenceofcalciumions.SampleGelInformation:GelNovex4-12%Bis-TrisLoadHumanFactorV,1µgperlaneBufferMOPSStandardSeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)SpecialNotesFactorVisaverylABIleprotein.Somedegradedfragmentsareusuallynoticeableinpreparations.Overview:FactorVisalarge,singlechain,plasmaglycoproteinwhichisanessentialcomponentinthebloodcoagulationcascade(1).Duringcoagulation,theprocofactor,factorV,isconvertedtotheactivecofactor,factorVa,vialimitedproteolysisbytheserineproteasealpha-thrombin(illustratedabove),andlessefficientlybyfactorXa.TheactivecofactoriscomposedofanNH2-terminalderivedheavychain(Mr=94,000)andaCOOH-terminalderivedlightchain(Mr=74,000)whichremainnon-covalentlyassociatedinthepresenceofcalciumions.FactorVaservesasacofactorfortheserineproteasefactorXa.FactorVaandXaassembleonaphospholipidsurfaceinanon-covalentandcalciumion-dependentmanner,toformtheprothrombinasecomplex.TheprothrombinasecomplexisresponsIBLefortherapidconversionofthezymogenprothrombintotheactiveserineprotease,alpha-thrombin.Assemblyoftheprothrombinasecomplexincreasestherateatwhichprothrombinisconvertedtothrombinbynearly300,000-foldrelativetotheratewithfactorXaalone.DownregulationoftheprothrombinasecomplexisaccomplishedpartlythroughtheinactivationoffactorVabyactivatedproteinC.HumanfactorVispreparedfromfreshfrozenhumanplasmausingimmunoaffinitychromatographyasdescribedbyKatzmannandcoworkers(2).BovinefactorVispreparedfromfreshbovineplasmausingconventionalchromatographictechniquesasdescribedbyNesheimandcoworkers(3).PurifiedfactorVissuppliedin50%glycerol(vol/vol),andshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinafactorVclottingassay.Properties:LocalizationPlasmaandplateletsPlasmaconcentration7.0µg/ml(human)(4,5)35µg/ml(bovine)(5)ModeofactionProcofactor;activatedbythrombintoformtheactivecofactor,factorVaMolecularweight330,000(3)-(determinedforbovinefactorV)ExtinctioncoefficientE1%1cm,280nm=9.6(3)-(determinedforbovinefactorV)Structureonesubunit,2196aminoacids(1,6)-(determinedforhumanfactorV)Percentcarbohydrateapproximately25%(baseduponthecalculatedmolecularweightofhumanfactorV)