Formulation	50%glycerol/water(v/v)
Storage	-20°C
Purity	>95%bySDS-PAGE
ActivityDetermination	N/A
ShelfLife(properlystored)	12months

DomainStructureofProthrombin
Thedomainstructureofprothrombinisrepresented,where:GLA=regioncontainingγ-carboxyglutamicacidresidues,KRINGLE=regionsofinternalsequencehomology,CATALYTICDOMAIN=regioncontainingtheserineproteasecatalytictriad.ArrowsindicatethesiteswhichareproteolyticallycleavedbyfactorXaduringactivationofthezymogen.

SampleGelInformation:

Gel	Novex4-12%Bis-Tris
Load	1µgperlane;purifiedhumanprothrombin
Buffer	MOPS
Standard	SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)

Overview:

ProthrombinisavitaminK-dependentplasmaproteinwhichissynthesizedintheliver(1).Priortosecretionintoplasma,prothrombinundergoespost-translationalmodificationbyavitaminK-dependentcarboxylasewhichconvertstenspecificglutamicacidresiduestoγ-carboxyglutamicacid(gla).Thetenglaresiduesarelocatedwithinthefirst40aminoacidsofthematureproteinandcontributetotheABIlityofprothrombintobindtonegativelychargedphospholipidmembranes.Prothrombincontainstworegionsofinternalhomologywhicharereferredtoas"kringle"structures.Theseregionsofconspicuoussecondarystructurearelocatedbetweenresidues40and270ofthematureplasmaproteinandreplacethegrowthfactordomainsfoundinseveralotherplasmaserineproteases.Thusfar,nofunctionhasbeenascribedtotheseregions,butthereissUSPicionthattheymayplayaroleinoneofseveralbinaryproteininteractionsinvolvingprothrombin.Thematuresinglechainproteincirculatesinplasmaasazymogenand,duringcoagulation,isproteolyticallyactivatedtothepotentserineproteaseα-thrombin.Thisproteolysisiscatalyzedbytheprothrombinaseenzymecomplex.Duringactivation,prothrombiniscleavedatArg271-Thr272(human)/Arg273-Thr274(bovine)andatArg320-Ser321(human)/Arg323-Ser324(bovine)toa"pro"fragment(fragment1.2)andthrombin,thelatterofwhichiscomposedoftwochainscovalentlylinkedbyadisulfidebond.Inthecaseofhumanprothrombin/thrombin,thereisanadditionalthrombinfeed-backcleavageatArg284-Thr285resultinginanadditional13aminoacidsbeingremovedfromthematurethrombin“A”chain.

HumanprothrombinispreparedfromfreshfrozenhumanplasmaasdescribedbyBajajandcoworkers(2).BovineprothrombinispreparedfromfreshbovineplasmausingamodificationoftheproceduredescribedbyOwenandcoworkers(3).Purifiedprothrombinissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20oC.PurityisdeterminedbySDS-PAGEanalysis,andactivityismeasuredbyclottingand/orchromogenicsubstrateassay,followingconversionofprothrombintothrombin.

Properties:

Localization	Plasma
Plasmaconcentration	100µg/ml(1)
Molecularweight	72,000(1,4,5). Fragments: ProthrombinFragment1-21,700 ProthrombinFragment2-12,866 ProthrombinFragment1.2-34,566 PrethrombinFragement1-49,900 PrethrombinFragment2-37,580
Extinctioncoefficient	E 1% 1cm,280nm 13.8(human)(4) 14.4(bovine)(1)
Isoelectricpoint	4.7-4.9(human)(6) 4.4-4.9(bovine)(6)
Structure	singlechain,NH2-terminalgladomain,twokringleregions
Percentcarbohydrate	8.2%(human)(4) 10.0%(bovine)(5)
Post-translationalmodifications	tenglaresidues(4,5)

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