胰蛋白酶的提取纯化与活性测定课件

Isolation,PurificationandActivityDeterminationofTrypsin生物技术大实验六SomeKeyWordsYouNeedtoKnowCoenzymeMaximumvelocityMichaelisconstant

RibozymeProstheticgroup

AbzymeSubstrateIsozymeCatalyzeNoncompetitiveinhibitionTransitionstateUncompetitiveinhibitionSteadystateInitialvelocity(Initialrate)KineticsDefinitionofenzymeEnzymesarebiologicalcatalystsACatalystisdefinedas"asubstancethatincreasestherateofachemicalreactionwithoutbeingitselfchangedintheprocess.”Theproteintertiarystructureprovidesadefinedregionoptimizedfortheaccelerationofchemicaltransformations(Activesite)。

CharacterofEnzymesMildreactionconditions-

37℃,physiologicalpH,ambientatmosphericpressure

Substrates

-highlyspecificreactantsforenzymesCatalyst

-speedsupattainmentofreactionequilibriumEnzymaticreactions

-103to1017fasterthanthecorrespondingun-catalyzedreactionsCharacterofEnzymesStereospecificity

-manyenzymesactupononlyonestereoisomerofasubstrateReactionspecificity

-enzymeproductyieldsareessentially100%(thereisnoformationofwastefulbyproducts)Activesite

-whereenzymereactionstakeplaceEnzymaticreactionsareoftenregulated,especiallythoseassociatedwithkeymetabolictransformationsNameofEnzymesEndin–aseIdentifiesareactingsubstance sucrase–reactssucrose lipase-reactslipidDescribesfunctionofenzymeoxidase-catalyzesoxidationhydrolase-catalyzeshydrolysisCommonnamesofdigestionenzymeuse-in:pepsin;trypsin;EffectofsomefactorsonenzymeactivityEffectof[E]onvelocity(V)Fixed,saturating[S]反应速度与酶浓度成正比

[S]>>[E],V∝[E]Effectoftemperatureonvelocity(V)OptimumtemperatureEffectofactivatoronvelocity(V)(ii)OrganicionsReducingagents，suchasGSH,CysEDTA(iii)Proteins:如果酶原一旦被蛋白酶激活，蛋白质可看成是激活剂

14LearningCheckE2

Sucrasehasanoptimumtemperatureof37°CandanoptimumpHof6.2.Determinetheeffectofthefollowingonitsrateofreaction (1)nochange(2)increase(3)decrease

A.Increasingtheconcentrationofsucrose B.ChangingthepHto4 C.Runningthereactionat70°CLearningCheckE3A.Theactivesiteis (1)theenzyme (2)asectionoftheenzyme (3)thesubstrateB.Intheinducedfitmodel,theshapeoftheenzymewhensubstratebinds:Whydoweanalyzeproteins?Proteinsplaycrucialrolesinnearlyallbiologicalprocesses.Thesemanyfunctionsofproteinsarearesultofthefoldingofproteinsintomanydistinct3Dstructures.Proteinanalysistriestoexplorehowaminoacidsequencesspecifythestructureofproteinsandhowtheseproteinsbindtosubstratesandothermoleculestoperformtheirfunctions.Proteinanalysisallowsustounderstandthefunctionoftheproteinbasedonitsstructure.SomeFunctionsofProteins1.Light:theresultofreactioninvolvingtheproteinluciferinandATP,catalyzedbytheenzymeluciferase.2.Oxygentransportfunction:Redbloodcell,hemoglobinEventfulProteinPurification

Growcellsinmedia(vector+tag)Centrifuge,CollectthepelletLysethecells(appropriatebuffer)PurificationStrategyPilotExpressionSDSPAGE,AssaySolubilityAggregationRecombinationCharacterizationMassSpectroscopyX-rayCrystallographyFunctionalAssayNoteinisolationandpurification--Allstepsat0～4℃--Avoidingvigorousstirring--Additionofprotectionreagente.g.EDTAand

少量β-巯基乙醇

--在分离提纯过程中要不断测定酶活力和蛋白质浓度，从而求得比活力，还要计算总活力

2.ProteinExtraction

Involvestheefficientextractionoftheproteinandpeptideinabiologicallyactiveformfromtissue.Extractionofprotein:homogenizeitinaphysiologicalbuffercontainingproteolyticenzymeinhibitors.Extractionofpeptides:boilthetissuewith1MAceticAcidfor5minhomogenizetissueinethanol/0.1MHCl(ratio3:1)at0°C.Thiswillburstopenthevesiclestoreleasethepeptidesinsolution.243.ProteinPurification

Q

Proteinscanbepurifiedaccordingtocertainpropertiestheypossess.Thesepropertiesallowustoemploydifferenttechniquesinpurifyingproteins.ProteinPropertyTechniqueSolubilityDifferentialPrecipitationMolecularSizeGelFiltrationChromatographyMolecularChargeIonExchangeChromatographyHydrophobicityReversedPhaseHPLCBiologicalActivityAffinityChromatographySeparateCellDebrisCentrifugationSupernatePelletFilterStabilizeSampleControlpH:-Useappropriatebuffer;Controltemperature-Keepsamplesoniceorworkincoldroom-PrechillinstrumentsPreventfrothing/foaming-Handlegently;Maintainconcentratedsample

ProteinSolubility[salt]solubility“saltingin”“saltingout”Saltingin:-Ionsshieldchargesandallowproteinstofold.Saltout:-Ionscompetewithwatertointeractwithsidegroups.When[salt]ishighenough,saltwinscausingproteintoprecipitate.-Generallyuseammoniumsulfatetoprecipitateproteinsinthelab.UltravioletAbsorbanceIfyoudon'tknowwhattheproteinconcentrationofanunknownsampleislikelytobe,theultravioletmethodmightbeagoodstartingpoint.Thisisoftenusedtoestimateproteinconcentrationpriortoamoresensitivemethod;Monitorstheabsorbanceofaromaticaminoacids,tyrosineandtryptophanHigherordersofproteinstructure,manyothercellularcomponents,andparticularlynucleicacids,alsomayabsorbUVlightThismethodistheleastsensitiveofthemethodsTherealadvantagesofthismethodarethatthesampleisnotdestroyedandthatitisveryrapid.UltravioletAbsorbanceQuickSamplecanberecoveredUsefulforestimationofproteinbeforeusingamoreaccuratemethodHighlysusceptibletocontaminationbybuffers,biologicalmaterialsandsaltsProteinaminoacidcompositionisextremelyimportant,thusthechoiceofastandardisverydifficult,especiallyforpurifiedproteinsAbsorbanceisheavilyinfluencebypHandionicstrengthofthesolution.UltravioletAbsorbanceZerospectrophotometertowater(orbuffer)Taketheabsorbanceat280nminaquartzcuvetteChangewavelengthto260nmandzerowithwater(orbuffer)Takeabsorptionat260nminaquartzcuvetteUsethefollowingequationtoestimatetheproteinconcentration

[Protein](mg/mL)=1.55*A280–0.76*A260EstimationProcedure

AbsorptionoflightbymoleculesSpectrophotometerWavelengthoflight….Ultrviolet200-350nmVisible400-700Infrared700-MonitoringProgressofPurificationProtocolTotalprotein(mg):-QuantityofproteinpresentinfractionTotalactivity:(unitsofactivity)-Useaportionofsampletodetermineactivity.-Multiplyactivitybytotalvolumetodeterminetotalactivity.Threemeasuresofenzymeactivityarecommonlyused:Turnovernumber

(kcat)-numberofsubstratemoleculesturnedoverperenzymemoleculepersecondInternationalunit(IU)-amountofenzymethatproduces1μMofproductperminute.

i.e.1IU=1μmol/minSpecificactivity-IU/mgproteinDefinitionsofenzymeactivityEnzymeActivityUnitReactiontime

(min)Product[P]010

20

30

40SlopetanS→

P

mmolevo=[P]

/

minUnit=ActivityUnitsProtein(mg)

t

mmole/minyxyx=tanJuangRH(2004)BCbasicsSpecificActivity=MonitoringProgressofPurificationProtocolPurificationlevel:Measureofincreaseinpurityofproteinthroughoutprocedure.

%yield:measureofactivityretainedaftereachstepinprocedure.Purification=SpecificactivityatparticularstepSpecificactivityofinitialextract%yield=TotalactivityatparticularstepTotalactivityofinitialextractMonitoringProgressofPurificationProtocolStepTotalprotein(mg)Totalactivity(units)Specificactivity(units/mg)Yield(%)PurificationlevelInitialextract15,000150,000101001(NH4)2SO4precipitation4,600138,00030923Ion-exchange1,278115,50090779Sizeexclusion68.675,0001,10050110Affinitycolumn1.7552,50030,000353,000(Berg,Tymoczko,&Stryer.(2002)Biochemistry,5thed.W.H.Freeman&Co.,NewYork,NY,p.86)ZymogenorproenzymeZymogensareinactiveenzymeprecursorsDigestiveserineproteasesincludingtrypsin,chymotrypsin,andelastasearesynthesizedandstoredinthepancreasaszymogensStorageofhydrolyticenzymesaszymogenspreventsdamagetocellproteinsZymogensareactivatedbyselectiveproteolysis

Trypsin:-cleavesbasicaminoacidsPepsinogen(inactive)40kD44aminoacidfragmentcutfromN-terminusPepsin+acidpH

Pepsin(active)33kDReactionofPepsinActivationTrypsinogenTrypsin(wt)+-(Autoactivation)ProenzymesEnzymesWhitcombetal,NatureGenetics1996--TrypsinTrypsin--PSTIAutodigestionPancreatitisR122TrypsinH122?PSTIFail-safeTryps