| Formulation | 10mMNaPO4,0.3MNaCl,pH6.5 |
| Storage | -80°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | Fibrinogenclottingassay |
| ShelfLife(properlystored) | 12months |
Thedegradationpatternofhumanα-thrombintolessactiveformsisillustratedabove.β-thrombinisgeneratedbycleavageoftheintactB-chainattheArg106-Tyr107bondtoyieldtheB1andB2fragments.γ-thrombinisgeneratedbyfurthercleavageoftheβ-thrombinB2-chainattheLys190-Gly191bondtoyieldtheB4andB5fragments.Althoughnotillustrated,theB3fragmentisapotentialintermediatethatisderivedbycleavageoftheintactB-chainattheLys190-Gly191bond,resultinginaproductthatisoftenreferredtoasβ"-thrombin.(TheaminoacidnumberingusedherebeginsattheNH2-terminalendoftheA-chainandcontinuesthroughtheB-chain).
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | HumanBeta&GammaThrombin,1µgperlane |
| Buffer | MES |
| Standard | SeeBluePlus2;Myosin(188kDa),PhosphorylaseB(98kDa),BSA(62kDa),GlutamicDehydrogenase(49kDa),AlcoholDehydrogenase(38kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(17kDa),Lysozyme(14kDa),Aprotinin(6kDa),Insulin,Bchain(3kDa). |
| Note | Othercontaminantsareofthrombinorigin,andmaymakeupto~5%oftheadditionalbandmass. |
Overview:
Alpha-thrombinisahighlyspecificserineproteasegeneratedbyproteolyticactivationofthezymogenprothrombin(1).Purifiedformsofα-thrombinhavebeenshown(2-4)toundergoautolysisuponlongtermstoragetolessactiveforms.Similarinactivationwasobserveduponlimiteddigestionofa-thrombinwithtrypsin(5).Theseproteolyzedformsofα-thrombinhavebeentermedβ-thrombinandγ-thrombin.Humanβ-thrombinisgeneratedbycleavageoftheB-chainattheArg106-Tyr107bond.γ-thrombinisgeneratedbyfurthercleavageoftheβ-thrombinB2-chainattheLys190-Gly191bond.Anotherformofproteolyzedthrombin,termedβ"-thrombinisformedbythesinglecleavageofthrombinatArg-154.γ-thrombinisproducedbyproteolyticcleavageatbothofthesesites(Arg70/73andArg-154)intheB-chain.Thesecleavagescausereleaseofpeptidesthatarenolongercovalentlyattachedtothethrombinmolecule,butremainassociatedthroughionexchangeandgelfiltrationchromatography.TheseproteolyzedformsofthrombinretaintheirABIlitytocleavesmallsyntheticsubstrates(6,7)andsomeproteinsubstratessuchasfactorXIII(8),antithrombinIII(9)andprothrombin(10).Theirabilitytoclotfibrinogen(11),cleavethrombospondin(12)oractivateproteinC(9)havebeenmarkedlydecreased.
Humanβ-thrombinandγ-thrombinarepreparedfrompurifieda-thrombinbylimitedproteolysiswithTPCK-treatedtrypsin,essentiallybythemethodofBraunetal.(5).Theproteolyzedformsofa-thrombinaresuppliedin100mMNaH2PO4,pH6.5bufferandshouldbestoredat-20ºC.PurityisassessedbySDS-PAGEandactivityisassessedusingafibrinogenclottingassay.
Properties:
| Modeofaction | Proteolyzedformsof-thrombinwhichretainactivitytowardsmallsubstrates,factorXIIIandprothrombin,buthavereducedactivitytowardfibrinogen,proteinCactivationandantithrombinIIIbinding. | |||||
|---|---|---|---|---|---|---|
| Molecularweight | 36,700(b-thrombin) 36,700(g-thrombin) 4,000(Achain)* 34,300(Bchain)* 6,000(B1chain)* 10,400(B5chain)* 11,800(B4chain)* *ApparentmolecularweightasdeterminedbySDS-PAGEanalysis | |||||
| Extinctioncoefficient |
| |||||
| Structure | b-thrombin:threechains(A,B1,B2),disulfidelinkbetweentheAandtheB2chains.g-thrombin:fourchains(A,B1,B5,B4)withadisulfidelinkbetweentheApeptideandtheB5peptide. |
