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1、ASSAYMETHODOFNATTOKINASEPRINCIPLENattokinaseactivitycanbeobtainedbythespectrophotometricmeasurementoftheamountofacid-solublelow-molecularproducts,whichisincreasedowingtohydrolysisofthepeptidelinkageswhennattokinaseactsonfibrin.DEFINITIONOFACTIVITYUNITOneunit(1FU)isdefinedastheamountoftheenzymewhichi

2、ncreasestheabsorbanceofthefiltrateat275nmbyperminuteundertheconditionsspecifiedintheprocedure.APPARATUSWaterbath：37Testtubes15X150mmMixer：YAMATOSCIENTIFICMT31Microtesttubes：EPPENDORF,safelockmLHigh-speedmicrocentrifuge(TheRotorwiththeradiusof4cm)Pastuerpipettes：forexampleIWAKIGLASS,9incheslengthSpec

3、trophotometerCuvette：10mminapathlength,4mminapathwidthREAGENTSmolLBoratebuffer(pH,containingNaCl)DissolvegofNa2B4O7?10H2OandgofNaClinabout900mLofdistilledwater.AdjustedpHtowithconcentratedHClandadddistilledwatertomake1000mL.molLTrichloroaceticacid(TCA)solutionDissolvegofTCAindistilledwatertomake1000

4、mL.%FibrinogensolutionPipettethe10mLofmolLBoratebufferintoanErlenmeyerflask,add96mgoffibrinogen(SIGMAFIBRINOGENFractionI)TypeI-S:FromBovinePlasma,PRODUCTNUMBERF8630),andkeepstandingforsometime.Grindtheundisolvedwithglassrodtodissolvecompletely.Filtratethemixturethroughafilterpaper(ADVANTEC,No.6,7cm)

5、,toremoveinsolublematerials.Preparebeforeuse.NoteIThrombinsolutionDissolvethrombin(SIGMA,FromBovinePlasma,PRODUCNTUMBETR6634)inmolLBoratebuffertomakethe1000UmLsolution.Dispenseitlittlebylittleintobottlesandmakethemfreezeforstorage.Diluteoneofthem50-foldwithmolLBoratebufferbeforeuse.1molLAceticacidso

6、lutionDissolvegofCH3COOHindistilledwatertomake1000mL.1molLAcetatebuffer(pH)DissolvegofCH3COONa?3H2Oindistilledwater.AddmLof1mol/LAceticacidsolutionandadddistilledwatertomake1000mL.10%TritonX-100solutionDissolve10gofTritonX-100indistilledwaterbyheatingandadddistilledwatertomake100mL.DiluentDissolveg(

7、finalconc.2mmolL)ofCaSO4?2H2aOndg(finalconc.10mmolL)ofNaClindistilledwater.AddmLof1molLAcetatebuffer(pH),mL(finalconc.%)of10%TritonX-100solution,anddistilledwatertomake1000mL.NoteHPREPARATIONOFSAMPLESOLUTIONDilutethesamplewithdiluenttogivecorrectedabsorbance(A)ofthefiltratebetweenand.Theconcentratio

8、nisnormallytoFUg.PROCEDURE(SAMPLE)DispensethemLofmolLBoratebufferandmLof%Fibrinogensolutionintoatesttube,andpreincubateitina37waterbathfor5minutes.AddmLofThrombinsolutionandmix.Note田Afterexactly10minutes,addmLofthesamplesolution,mixfor5seconds,andincubateat37.After20and40minutesfromthetimethereactio

9、nstarts,mixfor5secondsrespectively.Afterexactly60minutes,add2mLofmolLTCAsolutiontostopthereaction,mix,andincubateat37forfurther20minutes.Transferthemixtureintoamicrotesttubeandcentrifugeat15000rpmfor5minutes.Transferthe1mLofsupernatantcarefullyintoacuvettebyPasteurpipetteandreadtheabsorbance(AT)at27

10、5nm.(BLANK)DispensethemLofmolLBoratebufferandmLof%Fibrinogensolutionintoatesttube,andpreincubateitina37waterbathfor5minutes.AddmLofThrombinsolutionandmix.Afterexactly10minutes,add2mLofmolLTCAsolutionandmixfor5seconds.AddmLofsampleintosolution,mixfor5seconds,andincubateat37for20minutes.Transferthemix

11、tureintoamicrotesttubeandcentrifugeat15000rpmfor5minutes.Transferthe1mLofsupernatantcarefullyintoacuvettebyPasteurpipetteandreadtheabsorbance(AB)at275nm.(4)CALCULATIONNattokinaseactivity(FU/g)=(AT-AB)/x1/60 x1/xD：DilutionrateofsampleNotesI.Themeasuredactivitycanvarywiththelotoffibrinogen.Accordingly,whenfibrinogenslotischanged,correctthemeasuredactivitybymultiplyingthecorrectionfactorifnecessar

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