Formulation50%glycerol/water(v/v)Storage-20°CPurity>95%bySDS-PAGEActivityDeterminationClottingassayShelfLife(properlystored)12monthsDomainStructureofProthrombinThedomainstructureofprothrombinisrepresented,where:GLA=regioncontainingγ-carboxyglutamicacidresidues,KRINGLE=regionsofinternalsequencehomology,CATALYTICDOMAIN=regioncontainingtheserineproteasecatalytictriad.ArrowsindicatethesiteswhichareproteolyticallycleavedbyfactorXaduringactivationofthezymogen.SampleGelInformation:GelNovex4-12%Bis-TrisLoad1µgperlane;purifiedhumanprothrombinBufferMOPSStandardSeeBluePlus2;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:LocalizationPlasmaPlasmaconcentration100µg/ml(1)Molecularweight72,000(1,4,5).Fragments:ProthrombinFragment1-21,700ProthrombinFragment2-12,866ProthrombinFragment1.2-34,566PrethrombinFragement1-49,900PrethrombinFragment2-37,580ExtinctioncoefficientE1%1cm,280nm13.8(human)(4)14.4(bovine)(1)Isoelectricpoint4.7-4.9(human)(6)4.4-4.9(bovine)(6)Structuresinglechain,NH2-terminalgladomain,twokringleregionsPercentcarbohydrate8.2%(human)(4)10.0%(bovine)(5)Post-translationalmodificationstenglaresidues(4,5)