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1、ASSAY METHOD OF NATTOKINASEPRINCIPLENattokinaseactivitycanbeobtainedbythespectrophotometricmeasurementoftheamountofacid-solublelow-molecularproducts,whichisincreasedowingtohydrolysisofthepeptidelinkageswhennattokinaseactsonfibrin.DEFINITIONOFACTIVITYUNITOneunit
2、(1FU)isdefinedastheamountoftheenzymewhichincreasestheabsorbanceofthefiltrateat275nmby0.01perminuteundertheconditionsspecifiedintheprocedure.APPARATUS(1)Waterbath：37±0.3℃(2)Testtubes：15×150mm(3)Mixer：YAMATOSCIENTIFICMT－31(4)Microtesttubes：EPPENDORF,safelock2.0mL
3、(5)High-speedmicrocentrifuge(TheRotorwiththeradiusof4cm)(6)Pastuerpipettes：forexampleIWAKIGLASS,9incheslength(7)Spectrophotometer(8)Cuvette：10mminapathlength,4mminapathwidthREAGENTS(1)0.05mol／LBoratebuffer(pH8.5,containingNaCl)Dissolve19.07gofNa2B4O7?10H2Oand9.
4、0gofNaClinabout900mLofdistilledwater.AdjustedpHto8.5withconcentratedHClandadddistilledwatertomake1000mL.(2)0.2mol／LTrichloroaceticacid(TCA)solutionDissolve32.68gofTCAindistilledwatertomake1000mL.(3)0.72%FibrinogensolutionPipettethe10mLof0.05mol／LBoratebufferint
5、oanErlenmeyerflask,add96mgoffibrinogen(SIGMA,FIBRINOGEN(FractionⅠ)TypeⅠ-S:FromBovinePlasma,PRODUCTNUMBERF8630),andkeepstandingforsometime.Grindtheundisolvedwithglassrodtodissolvecompletely.Filtratethemixturethroughafilterpaper(ADVANTEC,No.6,7cm),toremoveinsolub
6、lematerials.Preparebeforeuse.〔NoteⅠ〕(4)ThrombinsolutionDissolvethrombin(SIGMA,FromBovinePlasma,PRODUCTNUMBERT6634)in0.05mol／LBoratebuffertomakethe1000U／mLsolution.Dispenseitlittlebylittleintobottlesandmakethemfreezeforstorage.Diluteoneofthem50-foldwith0.05mol／L
7、Boratebufferbeforeuse.(5)1mol／LAceticacidsolutionDissolve60.1gofCH3COOHindistilledwatertomake1000mL.(6)1mol／LAcetatebuffer(pH6.0)Dissolve129.6gofCH3COONa?3H2Oindistilledwater.Add47.6mLof1mol/LAceticacidsolutionandadddistilledwatertomake1000mL.(7)10%TritonX-100s
8、olutionDissolve10gofTritonX-100indistilledwaterbyheatingandadddistilledwatertomake100mL.(8)DiluentDissolve0.334g(finalconc.2mmol／L)ofCaSO4?2H2Oand0.585g(finalconc.10mmol／L)o
