Formulation50%glycerol/water(v/v)Storage-20°CPurity>95%bySDS-PAGEActivityDeterminationFactorXclottingassayorchromogenicassayShelfLife(properlystored)12monthsParticipationofFactorXainProthrombinaseTheparticipationoftheserineproteasefactorXaintheprothrombinasecomplexisillustrated.MembraneboundfactorXabindstomembraneboundfactorVatoformtheprothrombinasecomplex.Thiscomplexeffectivelyconvertsthezymogenprothrombin(II)totheactiveserineproteasethrombin(IIa)byproteolyticremovalofthefragment1.2(F1.2)portionofprothrombin.SampleGelInformation:GelNovex4-12%Bis-TrisLoadHumanFactorXa,1µgperlaneBufferMOPSStandardSeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)SpecialNotesHeavychainisadoubletduetothepresenceofupto50%betaform.Theconversionofalpha-Xatobeta-Xaoccursbyautocleavageofalpha-Xabyalpha-XaresultinginthelossofaCOOH-terminalpeptide.Overview:Activationofthezymogen,factorX,byeithertheintrinsicorextrinsicfactorXasecomplexesproducestheactiveserineproteasefactorXa(1,2).TheactivationoffactorXrequiresproteolyticcleavageoftheheavychain,resultinginthereleaseofanactivationglycopeptide.TheheavychainregioninfactorXacontainstheserineproteasecatalyticdomain,whilethelightchain,asinthezymogen,containsthemembranebindingdomain.FactorXaparticipatesintheprothrombinasecomplex,whichcatalyzestherapidconversionofprothrombintothrombin.ProthrombinaseisanenzymecomplexcomposedoffactorXa(enzyme)andfactorVa(cofactor)assembledonacellularsurfaceinthepresenceofcalciumions.AlthoughfactorXacanindependentlycatalyzetheactivationofprothrombin,therateatwhichthisreactionoccursisincreasednearly300,000-foldwithcompleteassemblyoftheprothrombinasecomplex.TheclottingactivityoffactorXainvivoisterminatedbyeitherinactivationofthecofactor,factorVa,orbydirectinhibitionoffactorXabyinhibitors,suchasATIII,afterdisassemblyoftheprothrombinasecomplex.Inrecentyears,molecularBIOLOGistshaveutilizedfactorXaforsitespecificcleavageoffusionproteinsexpressedinbacteria(9-12).AfactorXa-sensitivesiteisincorporatedbetweentherecombinantproteinofinterestandpeptidesorproteinswhichfacilitatepurificationand/orexpression.ThetargetproteinisreleasedfromtheexpressedhybridbycleavagewithfactorXa.ThefactorXacanthenbeeasilyremovedbyaffinitychromatography.FactorXaispreparedbyactivatingpurifiedfactorXwiththefactorXactivatorisolatedfromRussell"svipervenom.FactorXaispurifiedfromtheactivationmixturebychromatographyoverbenzamidine-Sepharosefollowedbygelfiltration(1,3).SeveralmodifiedformsoffactorXaarealsoavailableincluding:A)active-siteblockedfactorXacontainingeitherthetripeptidechloromethylketoneinhibitorEGRck,orthefluorescentinhibitorDansyl-EGRck;andB)humanGla-domainlessβ-factorXa.Theenzymeissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinafactorXaclottingassayand/orchromogenicsubstrateassay.InadditiontoitsbroadapplicationincoagulationresearchfactorXacanbeusedforsitespecificcleavageoffusionproteins.AfactorXasensitivesiteisincorporatedbetweentherecombinantproteinofinterestandpeptidesorproteinswhichfacilitatepurificationand/orexpression.ThetargetproteinisreleasedfromtheexpressedhybridbycleavagewithfactorXa.FactorXacanthenbeeasilyremovedbyaffinitychromatography.LottolotconsistencyensuresreproducIBLeresultseverytime.Forexperimentsinvolvingcellcultures,pleasecontactustodiscusscustom,lowendotoxinlotsdesignatedforcellcultureuse.Properties:LocalizationPlasmaModeofactionEnzymecomponentoftheprothrombinasecomplexMolecularweight46,000(human)(4)45,300(bovine)(5)ExtinctioncoefficientE1%1cm,280nm=11.6(human)(9)  =12.4(bovine)(7)SpecificActivityapproximately1000units/mgStructuretwosubunits,Mr=16,200and29,000(human)(6),Mr=16,500and28,800(bovine)(5),NH2-terminalgladomain,twoEGFdomainsPercentcarbohydrate3.0%(human)(8)2.1%(bovine)(8)Post-translationalmodificationselevenglaresidues,oneβ-hydroxyaspartate