Formulation | 50%glycerol/water(v/v) |
Storage | -20°C |
Purity | >95%bySDS-PAGE |
ActivityDetermination | FactorXclottingassayorchromogenicassay |
ShelfLife(properlystored) | 12months |
Gel | Novex4-12%Bis-Tris |
---|---|
Load | HumanFactorXa,1µgperlane |
Buffer | MOPS |
Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
SpecialNotes | Heavychainisadoubletduetothepresenceofupto50%betaform.Theconversionofalpha-Xatobeta-Xaoccursbyautocleavageofalpha-Xabyalpha-XaresultinginthelossofaCOOH-terminalpeptide. |
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.
Localization | Plasma | ||||||||
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Modeofaction | Enzymecomponentoftheprothrombinasecomplex | ||||||||
Molecularweight | 46,000(human)(4) 45,300(bovine)(5) | ||||||||
Extinctioncoefficient |
| ||||||||
SpecificActivity | approximately1000units/mg | ||||||||
Structure | twosubunits,Mr=16,200and29,000(human)(6),Mr=16,500and28,800(bovine)(5),NH2-terminalgladomain,twoEGFdomains | ||||||||
Percentcarbohydrate | 3.0%(human)(8) 2.1%(bovine)(8) | ||||||||
Post-translationalmodifications | elevenglaresidues, oneβ-hydroxyaspartate |
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