Formulation | 50%glycerol/water(v/v) |
Storage | -20°C |
Purity | >95%bySDS-PAGE |
ActivityDetermination | Fibrinogenclottingorchromogenicassay |
ShelfLife(properlystored) | 12months |
Gel | Novex4-12%Bis-Tris |
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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). |
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.
Localization | Plasma | |||||||
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Modeofaction | Serineproteasewhichcleavesfibrinogentoformfibrin;alsoresponsibleforactivationofproteinC,plateletactivationandfeedbackactivationoftheprocofactors,factorVandfactorVIII | |||||||
Molecularweigh | 36,700(3-6) | |||||||
Extinctioncoefficient |
| |||||||
SpecificActivity | approximately3800NIHunits/mg | |||||||
Isoelectricpoint | 7.0-7.6(human)(3) | |||||||
Structure | twosubunits,approximatelyMr=6,000and31,000 | |||||||
Percentcarbohydrate | approximately5% |
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