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

DomainStructureofProteinS
ThedomainstructureofproteinSisrepresented,where:GLA=regioncontainingγ-carboxyglutamicacidresidues,EGF=regioncontainingsequenceshomologoustohumanepidermalgrowthfactor,TSR=thrombinsensitiveregion,?=regionofunknownfunctionwhichreplacesthecatalytictriadfoundinvitaminK-dependentserineproteases.

SampleGelInformation:

Load	HumanProteinS,1µgperlane
Buffer	MOPS
Standard	SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)

Overview:

ProteinSisasinglechainvitaminK-dependentproteinwhichisthoughttofunctioninboththecoagulationandcomplementcascades(1,2).Approximately60%ofproteinScirculatinginplasmaiscomplexedtoC4bbindingprotein(C4BP).Ithasbeensuggestedthatg-carboxyglutamicacid(gla)dependentbindingofproteinStonegativelychargedphospholipidsmayfunctiontoconcentrateC4BPatcellsurfacesfollowinginjury.

Inthecoagulationsystem,proteinSfunctionsasananticoagulantcofactorprotein.ActivatedproteinC(APC)formsa1:1stoichiometriccomplexwithproteinSinthepresenceofCa2+andphospholipidvesicles(Kd=6x10-9M)(3).InthepresenceofproteinS,amoderateincrease(3-10fold)intherateoffactorVaandfactorVIIIainactivationbyAPCisobservedinplasmaandonthesurfaceofunstimulatedplatelets.ProteinSboundtoC4BPdoesnotpossessAPCcofactoractivity.Recently,anadditionalbindingproteinwhichenhancestheactivityofproteinShasbeendescribed(4).ProteolyticinactivationofproteinSbythrombinhasbeenproposedasaregulatorymechanisminthissystem.AsinglecleavagebythrombinabolishesproteinScofactoractivitybyremovinganNH2-terminalpeptide(Mr=8000)whichcontainsthegladomain.

ThedomainstructureofproteinSissimilartothatoftheothervitaminK-dependantcoagulationfactorswiththeexceptionthatproteinSdoesnotpossessthecatalytictriad.ProteinSisasinglechainproteincontaining10glaresiduesintheNH2-terminaldomainand4epidermalgrowthfactor(EGF)domains.

HumanproteinSisisolatedfromfreshfrozenplasmabyacombinationofconventionalmethods(9)andimmunoaffinitychromatographyasdescribedbyJennyetal.(5).PurifiedproteinSissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysis.

Properties:

Localization	Plasma,freeandcomplexedtoC4BP
Plasmaconcentration	10µg/ml(free)(6)
Modeofaction	CofactorforactivatedproteinC
Molecularweight	69,000(7)
Extinctioncoefficient	E 1% 1cm,280nm =9.5(7)
Isoelectricpoint	5.0-5.5(7)
Structure	singlechain,NH2-terminalgladomain,fourEGFdomains
Percentcarbohydrate	7%(7)
Post-translationalmodifications	oneβ-hydroxyaspartate(8)tenglaresidues(7),threeβ-hydroxyasparagine(8)

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