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

ProteolyticActivationofProthrombin
Theserineproteaseα-thrombinisproducedbyproteolyticactivationofthezymogen,prothrombin.Theenzymecomplex,prothrombinase,catalyzestheproteolysisoftwopeptidebondsinprothrombin,whichgivesrisetoanNH2-terminalderivedF1.2regionandtheheterodimer,α-thrombin.Alpha-thrombiniscomposedofan"A"chain(Mr=6000)whichiscovalentlylinkedtoa"B"chain(Mr=31,000)throughasingledisulfidebond.

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).

Overview:

Alpha-thrombinisahighlyspecificserineproteasegeneratedbyproteolyticactivationofthezymogenprothrombin(1).Duringcoagulation,thrombincleavesfibrinogentoformfibrin,leADIngtotheultimatestepincoagulation,theformationofafibrinclot.ThrombinisalsoresponsIBLeforfeedbackactivationoftheprocofactorsfactorVandfactorVIII.ThrombinhasalsobeenreportedtoactivatefactorXIIIandplatelets,andalsofunctionsasavasoconstrictorprotein.Theprocoagulantactivityofthrombinisarrestedintwoways:1)inhibitionbyeitherheparincofactorIIortheantithrombinIII/heparincomplex;or2)complexformationwiththrombomodulin.Formationofthethrombin/thrombomodulincomplexresultsintheinABIlityofthrombintocleavefibrinogenandactivatefactorsVandVIII,butincreasestheefficiencyofthrombinforactivationoftheanticoagulant,proteinC.

ThrombinisatwochainenzymecomposedofanNH2-terminal"A"chain(Mr=6,000)andaCOOH-terminal"B"chain(Mr=31,000)whichremaincovalentlyassociatedthroughasingledisulfidebond.Humanthrombinis13aminoacidsshorterthanthebovinethrombinduetoathrombincleavagesiteonthehumanproteinthatisnotpresentinthebovineprotein.

Thrombinisalsoutilizedforsitespecificcleavageoffusionproteinsexpressedinbacteria(9-11).Athrombinsensitivesiteisincorporatedbetweentherecombinantproteinofinterestandpeptidesorproteinswhichfacilitatepurificationand/orexpression.Thetargetproteinisreleasedfromtheexpressedhybridbycleavagewiththrombin.Thrombincanthenbeeasilyremovedbyaffinitychromatography.

Human,bovineandmousethrombinarepreparedfrompurifiedprothrombinusingamodificationoftheLundbladprocedure(1)asdescribedbyNesheimetal.(2).Thrombinissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20oC.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinathrombinspecificclottingassay,andcomparedtostandardizedNIHthrombin.ThrombinisalsoavailablewiththeactivesiteblockedwitheitherDFP,FPRck,orbiotinlyatedFPRck.

CleavageofFusionProteins
Inadditiontoitsbroadapplicationincoagulationresearchthrombincanbeusedforsitespecificcleavageoffusionproteins.Athrombinsensitivesiteisincorporatedbetweentherecombinantproteinofinterestandpeptidesorproteinswhichfacilitatepurificationand/orexpression.Thetargetproteinisreleasedfromtheexpressedhybridbycleavagewiththrombin.Thrombincanthenbeeasilyremovedbyaffinitychromatography.Lottolotconsistencyensuresreproducibleresultseverytime.Forexperimentsinvolvingcellcultures,pleasecontactustodiscusscustom,lowendotoxinlotsdesignatedforcellcultureuse.

Properties:

Localization	Plasma
Modeofaction	Serineproteasewhichcleavesfibrinogentoformfibrin;alsoresponsibleforactivationofproteinC,plateletactivationandfeedbackactivationoftheprocofactors,factorVandfactorVIII
Molecularweigh	36,700(3-6)
Extinctioncoefficient	E 1% 1cm,280nm =18.3(human)(6) =19.5(bovine)(7)
SpecificActivity	approximately3800NIHunits/mg
Isoelectricpoint	7.0-7.6(human)(3)
Structure	twosubunits,approximatelyMr=6,000and31,000
Percentcarbohydrate	approximately5%

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