| Formulation | 20mMHepes,150mMNaCl,pH7.4 |
| Storage | -80°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | FactorIXclottingassay |
| ShelfLife(properlystored) | 12months |
TheactivationoffactorXbytheintrinsicfactorXasecomplexisshown.TheintrinsicfactorXasecomplexconsistsofanenzyme,factorIXa,andacofactor,factorVIIIa,assembledonaphospholipidsurfaceinthepresenceofcalciumions.Theenzymecomplexproteolyticallycleavesa10,000molecularweightactivationpeptidefromtheNH2-terminaloftheheavychainoffactorX,thusexpressingtheactive-siteoffactorXa.
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | HumanFactorIXa,1µgperlane |
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
Overview:
FactorIXaisproducedfromitsinactiveprecursor,factorIX,viaproteolyticcleavagebyfactorXIaorthetissuefactor/factorVIIa/phospholipidcomplex.TheactivationresultsfromthecleavageoftwopeptidebondsinthefactorIXmolecule,releasinganactivationglycopeptidewithanapparentmolecularweightof10,000.TheheavychainoffactorIXa(Mr=28,000)containstheserineproteasecatalyticdomain,whilethelightchain(Mr=17,000)containsthemembranebindingdomain.
FactorIXafunctionsasaserineproteaseinvolvedintheactivationofthezymogen,factorX,toformtheenzyme,factorXa.ThefactorIXaenzymaticactivityisgreatlyenhancedbyinclusionofitscofactor,factorVIIIa,inthepresenceofcalciumionsonaphospholipidsurface.FactorIXaisreADIlyinhibitedbyantithrombinIII,andthisinhibitionisgreatlyacceleratedbythepresenceofheparin.FactorIXaisnotinhibitedbyDFP.
FactorIXaispreparedfromhighlypurifiedfactorIXbyactivationwithfactorXIa,asdescribedbyLindquistetal.(5).ThefactorIXaisfurtherpurifiedbygelfiltration,followedbyimmunoaffinitypurification.FactorIXaisalsoavailablewiththeactivesiteirreversIBLyblockedbythetripeptidechloromethylketone,EGRck,orbythefluorescentinhibitor,Dansyl-EGRck.Itissuppliedin50%(vol/vol)glycerol/H2Oforstorageat-20°C.PurityisassessedbySDS-PAGEanalysis.Activityisdeterminedinaone-stageclottingassay.
Properties:
| Localization | Plasma | |||||
|---|---|---|---|---|---|---|
| Modeofaction | EnzymecomponentoftheFactorXasecomplex | |||||
| Molecularweight | 45,000(7) | |||||
| Extinctioncoefficient |
| |||||
| Structure | 2subunits,Mr=28,000and17,000(5),NH2-terminalgla-domain,twoEGFdomains | |||||
| Post-translationalmodifications | oneb-hydroxyaspartate(3),twelveglaresidues(4) |
