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Experiment1DepartmentofBiologicalPharmacy,ChinaPharmaceuticalUniversity,2011PreparationofElasticProteasefromPancreasandAnalysisofEnzymeActivityCONTENTSPartI:PreparationofElasticProtease2PartII:AnalysisofEnzymeActivity3Purposes1PurposesLearntheprincipleofpreparationofelasticproteasefrompancreas.Mastertheanalysismethodofenzymeactivity.12PartIPreparationof
ElasticProteaseProceduresIFrozenPancreas75gPancreatic
Plasma(1)Activatetheenzymeat24℃for24h(2)removethefat,cutintopieces(3)add50mlHAc-buff,mashedFiltrate(1)add250mLHAc-buff,adjustpHto4.5
(2)
25℃
stirfor1hours,(3)
Filter
Continuedon
nextpage…ProceduresIIContinuedfrom
previouspage…FiltrateResin(toabsorbprotein)(1)add40gresinforadsorption(2)
25℃stirfor1.5h(3)washwithwaterEluate(1)
add50ml1MNH4Cl-buff,adjustpHto9.0(2)
stirfor1h(3)
adjustpHto5.2~6.0(4)
filterContinuedon
nextpage…ProceduresIIIContinuedfrom
previouspage…EluatePrecipitationAdd3-time-volumncoldacetoneElastasepowder(1)
washtwicewithacetone(2)
vacuumdryingEndProductObtainedPartIIAnalysisof
EnzymeActivityDefinitionDefinitionofEnzymeActivityTheamountofenzymehydrolyzing1.0mgCongoredelastinwithin20minatpH8.8and37℃isdefinedasanactivityunit.PreparationofReferenceSubstanceI30mgCongoredelastin
(takenaccurately)1220mLstandardelasticenzymesolution
(mustbesufficient)100mL
volumetricflask3Keeptheflaskat37℃inawaterincubator4Shakethebottlegentlyuntilallsubstratesdissolves(about60min)PreparationofReferenceSubstanceII6addboricacidbufftothescale3Keeptheflaskat37℃inawaterincubator4Shakethebottlegentlyuntilallsubstratesdissolves(about60min)50mLphosphatebuffer(pH6.0)5100mL
volumetricflaskSamplePreparation5mgenzymesample
(takenaccurately)1mortar25mLpH8.8boricacidbuff
(addasmallamountatfirst)43grinduntileverythingdessolvesDiluteenzymesolutionwithboricacidbuffto2~3unit/mL1mLboricacidbuffStandardCurveTakeseriesamountsofreferencesubstancesolution,addingmixtureofboricacidbufferandphosphateacidbuffer(1:1),asindicatedinthetablebelow.Measuretheabsorptionat495nm,usingtheabsorptionoftube0ascontrol.Plotastandardcurve.TubeNo012345Referencesubstance(mL)02.04.06.08.010.0Mixtureofbuffer(mL)10.08.06.04.02.00A495value12SamplePreparationTake3tubestooperateaccordingtothetablebelow:TubeNo.012Congoredelastin(mg)~1.2~3~3pH8.8boricacidbuff(mL)544Awaitmeasuringenzymeliquid(mL)-11Hydrolyzethemixureat37℃for20min(Theintermittenceisstirred20ormoretimes)pH6.6phosphoricacidbuff(mL)555Takethesupernatantaftercentrifugation(3000rpm×10min)A495valueCaculationTaketheaverageabsorptionandcheckthecorrespondingenzymeactivityaccordingtothestandardcurve.Calculatetherelativeenzymeactivity(enzymeactivity/enzymeamount)bytakingi
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