胰彈性蛋白酶的制備與分析

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