| Formulation | 50%glycerol/water(v/v) |
| Storage | -20°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | Clottingassay |
| ShelfLife(properlystored) | 12months |
ThedomainstructureoffactorIXisrepresented,where:GLA=regioncontainingγ-carboxyglutamicacidresidues,EGF=regioncontainingsequenceshomologoustohumanepidermalgrowthfactor,AP=activationpeptidereleaseduponconversionofthezymogentotheactiveserineprotease,andCATALYTICDOMAIN=regioncontainingtheserineproteasecatalytictriad.ArrowsindicatethesiteswhichareproteolyticallycleavedbyfactorXIaduringactivationofthezymogen,witharrowAbeingcleavedfirst.
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | HumanFactorIX,1µgperlane |
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
Overview:
ThezymogenfactorIXisasinglechainvitaminK-dependentglycoproteinwhichissynthesizedintheliver(1-3).ThedomainstructureoffactorIXissimilartothatoftheothervitaminKdependentcoagulationfactors.TheNH2-terminalregioncontains12γ-carboxyglutamicacid(gla)residueswhichfacilitatethecalciumdependentbindingoffactorIXtonegativelychargedphospholipidsurfaces.Twodomainswhicharehomologoustoepidermalgrowthfactor(EGF)spantheregionbetweentheNH2-terminalgladomainandtheactivationpeptide(Ala-146toArg-180).
FactorIXisactivatedbyeitherfactorXIaorthefactorVIIa/tissuefactor/phospholipidcomplex.CleavageatsiteA(seefigure)yieldstheintermediateIXawhichissubsequentlyconvertedtothefullyactiveformIXaβbycleavageatsiteB.TheNH2-terminallightchain(GLAandEGFdomains)remainscovalentlyattachedtotheCOOH-terminalheavychainbyadisulfidebond.Theserineproteasecatalytictriad(Ser-365,His221,Asp-269)islocatedintheheavychain.FactorIXaβisthecatalyticcomponentofthe"intrinsicfactorXasecomplex"(factorVIIIa/IXa/Ca2+/phospholipid)whichproteolyticallyactivatesfactorXtofactorXa.
HumanfactorIXispreparedfromfreshfrozenplasmabyacombinationofconventionalprocedures(4)andimmunoaffinitychromatography(5).BovinefactorIXispreparedfromfreshcitratedbovineplasmabyamodificationofthemethoddescribedbyFujikawaetal.(6).Thepurifiedproteinsaresuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20oC.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredusingafactorIXclottingassay.
Properties:
| Localization | Plasma | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Plasmaconcentration | 4-5ug/ml(human)(1) | ||||||||
| Modeofaction | Zymogen;precursortotheserineproteasefactorIXa | ||||||||
| Molecularweight | 55,000(human)(8) 55,400(bovine)(6) | ||||||||
| Extinctioncoefficient |
| ||||||||
| Isoelectricpoint | 4.2-4.5(human)(7) 3.7(bovine)(6) | ||||||||
| Structure | singlechain,NH2-terminalgla-domain,twoEGFdomains | ||||||||
| Percentcarbohydrate | 17%(human)(7) 26%(bovine)(6) | ||||||||
| Post-translationalmodifications | oneβ-hydroxyaspartate(9),twelveglaresidues(7) |
