Formulation	20mMHepes,150mMNaCl,pH7.4
Storage	-80°C
Purity	>95%bySDS-PAGE
ActivityDetermination	FactorXclottingassayorchromogenicassay
ShelfLife(properlystored)	12months

ParticipationofFactorXainProthrombinase
TheparticipationoftheserineproteasefactorXaintheprothrombinasecomplexisillustrated.MembraneboundfactorXabindstomembraneboundfactorVatoformtheprothrombinasecomplex.Thiscomplexeffectivelyconvertsthezymogenprothrombin(II)totheactiveserineproteasethrombin(IIa)byproteolyticremovalofthefragment1.2(F1.2)portionofprothrombin.

SampleGelInformation:

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.

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:

Localization	Plasma
Modeofaction	Enzymecomponentoftheprothrombinasecomplex
Molecularweight	46,000(human)(4) 45,300(bovine)(5)
Extinctioncoefficient	E 1% 1cm,280nm =11.6(human)(9)     =12.4(bovine)(7)
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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