Formulation	100mMNaPO4,0.1%PEG,pH6.5
Storage	-80°C
Purity	>95%bySDS-PAGE
ActivityDetermination	Fibrinogenclottingassay
ShelfLife(properlystored)	12months

ProteolyzedFormsofα-Thrombin
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	E 1% 1cm,280nm =18.3
Structure	b-thrombin:threechains(A,B1,B2),disulfidelinkbetweentheAandtheB2chains.g-thrombin:fourchains(A,B1,B5,B4)withadisulfidelinkbetweentheApeptideandtheB5peptide.

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