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

ActivationofFactorXbyFactorIXa
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	E 1% 1cm,280nm =14.0(5)
Structure	2subunits,Mr=28,000and17,000(5),NH2-terminalgla-domain,twoEGFdomains
Post-translationalmodifications	oneb-hydroxyaspartate(3),twelveglaresidues(4)

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