| Formulation | 50%glycerol/water(v/v) |
| Storage | -20°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | < 0.5% aPC activity by chromogenic assay |
| ShelfLife(properlystored) | 12months |
ThedomainstructureofproteinCisrepresented,where:GLA=regioncontainingγ-carboxyglutamicacidresidues,EGF=regioncontainingsequenceshomologoustohumanepidermalgrowthfactor,AP=activationpeptidereleaseduponconversionofthezymogentotheactiveserineprotease,CATALYTICDOMAIN=regioncontainingtheserineproteasecatalytictriad.Thearrowindicatesthesitewhichisproteolyticallycleavedbythrombinduringactivationofthezymogen.
SampleGelInformation:
| Load | HumanProteinC,1µgperlane |
|---|---|
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
Overview:
ThevitaminK-dependentzymogen,proteinC,issynthesizedintheliverasasinglechainpolypeptideandissubsequentlyconvertedtoadisulfidelinkedheterodimer,byremovalofadipeptide(Lys-146andArg-147)fromtheprecursormolecule(1,2).Tracequantitiesofthesinglechainformhavebeenobservedinplasma.Thelightchain,whichisresponsIBLeforthecalciumdependentbindingofproteinCtophospholipidvesicles,contains11γ-carboxyglutamicacid(gla)residues,1b-hydroxyasparticacidresidue,and2epidermalgrowthfactor(EGF)homologydomains.Theserineproteasecatalytictriadislocatedintheheavychain.HumanproteinCissusceptibletoproteolyticcleavageofapeptide(Mr=3000)fromtheCOOH-terminalendoftheheavychain,yieldinganalteredformreferredtoasβ-proteinC.Nofunctionaldistinctionbetweenα-andβ-proteinChasbeenobserved.AsinglecleavageatArg-12(Arg-14inbovine)oftheheavychainofhumanproteinCconvertsthezymogenintotheserineprotease,activatedproteinC.Thiscleavageiscatalyzedbyacomplexbetweenα-thrombinandtheendothelialcellsurfaceproteinthrombomodulin.IncontrasttotheothervitaminKdependentcoagulationfactors,activatedproteinCfunctionsasananticoagulantbycatalyzingtheproteolyticinactivationoffactorsVaandVIIIa.APCalsocontributestothefibrinolyticresponsebycomplexformationwithplasminogenactivatorinhibitors.
BovineproteinCispreparedfromfreshcitratedbovineplasmabyamodificationoftheWalkerprocedure(3),asdescribedbyHaleyetal.(4).HumanproteinCispreparedfromfreshfrozencitratedhumanplasmausingacombinationofimmunoaffinitychromatography(5),andconventionaltechniques(4,9).ProteinCisprovidedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20oC.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredusingachromogenicsubstratebasedassay.
Properties:
| Localization | Plasma | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Plasmaconcentration | 4-5µg/ml(human)(6) 5-10µg/ml(bovine)(2) | ||||||||
| Modeofaction | Zymogen;precursortotheserineproteaseactivatedproteinC(APC) | ||||||||
| Molecularweight | 62,000(human)(7) 58,000(bovine)(7) | ||||||||
| Extinctioncoefficient |
| ||||||||
| Isoelectricpoint | 4.4-4.8(human)(8) 4.2-4.5(bovine)(8) | ||||||||
| Structure | twochains,Mr=41,000and21,000,disulfidelinked,NH2-terminalgladomaintwoEGFdomains | ||||||||
| Percentcarbohydrate | 23%(human)(7) 14%(bovine)(7) | ||||||||
| Post-translationalmodifications | elevenglaresidues(bovine),nineglaresidues(human),oneβ-hydroxyaspartate |
