兔磷酸激酶实验报告英文.docx
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兔磷酸激酶实验报告英文
IsolationandEnzymaticAnalysisofCreatineKinaseofRabbitMuscle
I.Introduction
I.Creatinekinase
Creatinekinaseusuallyexistsincytoplasmandmitochondriaoforganslikeanimalheart,muscle,brainandsoon.Itisanimportantkinasewhichrelatestotheenergyoperationofcells,musclecontractionandATPregeneration.CreatinekinasereversiblycatalyzephosphoryltransferreactionsbetweencreatineandATP.
Therearefourformsofcreatinekinaseisoenzyme:
MM,BB,MB,MiMi.MMmainlyexistsinallkindsofmusclecells,BBmainlyexistsinbraincells,MBmainlyexistsinmyocardialcellswhileMiMiexistsinthemitochondrialinnermembrane.MM,BB,MiMiareplasmacreatinekinase.
Phosphorylationoxidativeoccurinthemitochondrialofcell,whichproduceATP.Butthehigh-energymaterialwhichanimalsusetostoreenergyiscreatinephosphate.Energystorageandtransportationiscompleteviacreatinephosphateshuttle,whichincludeMMandMiMi.ATPproducedbyoxidativephosphorylationcanbedirectlysenttothelocatioinofMiMiintheoutermitochondrialmembrane.MiMitransferthehigh-energyphosphatebondinATPtocreatineintoADPandcreatinephosphateenergymaterial.Creatinephosphatespreadincellandreachmyofibrils.TheM-zoneofmyofibrilscombinesMM,thus,whenmusclecontracts,largeamountsofATParerequiredforenergysupplyandMMcatalystcreatinephosphateintoATP.Thecreatineproducedbythereactionwillgettomitochondriaandgetre-phosphorylated.
Creatinekinasehasimportantphysiologicalfunctionsandmedicalapplications.
Muscle-typecreatinekinasemoleculeiscomposedoftwoidenticalsubunitsdimer.Incatalyticprocessthesmallestfunctionalunitoftheenzymeissubunitwhichhavecatalysisfunctionindependently.Accordingtothecreatinekinaseprimarystructuremeasuredinrabbits,human,chicken,mouserecently,M-typesubunitconsistof387aminoacidresiduesandhasthemolecularweightaround43000ug.Thereare8sulfhydrylbuthasnodisulfidebond. Naturalcreatinekinasemoleculeisacompactglobularstructure.Accordingtothestudyofopticalrotatorydispersion,thesubunitofthisenzymehasabout25%-30%oftheα-helix,15%oftheβ-sheet.
2.Purificationofcreatinekinase
Therearemanywaysofpurifyingcreatinekinase,andthepurificationmethodusedinthisstudyisimprovedKubymethod.
3.Measurementofcreatinekinaseactivities
Therearemanywaysofmeasuringtheactivitiesofcreatinekinase,andweusepH-colorimetryinthisstudy.
pH-colorimetry
Whencreatinekinaseiscatalyzeforwardreaction,withthetransferofPhosphorylfromATPtocreatine,theequimolarH+isproduced,whoseoptimumpHis7.5~9.0.Withinthiscontext,thegenerationrateofH+measuredcanbeusedasindicatorsofactivity.Inthisstudy,thymolblueisusedasapHindicator.TheactivitiesofcreatinekinaseismeasuredbypH-colorimetryonspectrophotometer,whichismonitoringchangesinabsorbanceunderthewavelengthof597nm.Inthecuvette,H+producedbytheenzyme-catalyzedreactionlowerthepHvalueofsolution.ThecolorofsubstratesolutiongraduallychangefromdeeppurpleredyellowgreenandlowertheAbsorbancevalueofA597.InthepH-colorimetry,thesubstratesolutionrequirestobenew.
4.Measurementofcreatinekinasesolution
280nmlightabsorption
Methodforproteinmeasurement
Sensitivity
Time
Principle
Interference
Comments
Spectrophotometric
(A280)
Moderate
50-1000g
Rapid
5-10min
Absorptionof280nmlightbyaromaticresidues
Purines,pyrimidines,nucleicacids
Usefulformonitoringcolumneluents.Nucleicacidabsorptioncanbecorrected.Nondestructivetoproteinsamples.Varieswithproteins.
II.Experimental
1.ReagentsandEquipments
1)Reagents
1.Preparationofcrudeextract:
0.01MKCl,NH4Cl,5MNH4OH,anhydrous ethanol,pH8.5-9.02MMgSO4
a.KCl:
0.01mol/L,200ml.(0.15gKCladdwaterto200ml)
b.NH4OH:
1.7mmol/L,1000ml. (take0.12mlNH3H2O,addH2Oto1000ml)
c.NH4Cl
d.Anhydrousethanol
e.MgSO4:
2.0mol/L,pH8.5,20ml
f.MgAC2:
0.07mol/L,pH9.0,100ml
g.Tris-HCl:
0.1mol/L,pH8.0,1000ml(Tris12.1g,HCl23ml,pH为8.0,addwaterto1000ml)
h.Salt
2.Chromatography
a.48mmol/Lcreatinekinasesolution
b.0.1mol/LMgAC2
c.0.1%Thymolblue(100mgThymolblue,dissolvewith20mlethanoland60mlddwater).
d.0.1mol/LpH9.0Gly-NaOHbuffer
e.ATP
f.0.1mol/LNaOH
g.0.07M.MgAC2pH9.0
h.0.01MTris-HClpH8.0
2)Equipments
1.Preparationofcrudeextract:
Knife,meatchopper,plasticbasin,1/100balance,High-Speeddiffuser,refrigerator;
100mlbeaker,25mlbeaker,100mlmeasuringcylinder,250mlmeasuringcylinder;
Pippet,burette,glassstick;
0.5mlEptube,1.5mlEptube;
centrifugalmachine,50mlcentrifugaltube,stainlesssteelscoop,mortar;
drinkingpaper,accuratepHtestpaper
2.Measurementofenzymesactivities:
SPECORD200Spectrophotometer,Pippete,50mlbeaker,burette.
3.Measurementofproteinsolutions:
SPECORD200Spectrophotometer.
2.Procedure
1)Preparationofcrudeextract
1.Withasharpknife,cutthemuscleintocubes4to5cmwidefromtheice-coldrabbitmuscle.
2.Quicklyweighabout50gofthemusclecubesandsuspendthecubesin140mLof0.01mol/LKClat0C.
3.Homogenize140mLofthesuspensioninaglassbeakerwithablender(turnontheblenderathighspeedfor20seconds)andstirthemwithaglassstirringrodat0Cfor15minutes.
4.Centrifugethehomogenatefor10minutesat10000r/minat0C.
5.Carefullydecantthesupernatantandmeasurethevolumeofthesupernatant(V1)(keep0.6mLforproteinassayat–20C).Discardthepellet.
6.Addgroundammoniumchloride(NH4Cl)tomake0.1mol/LsolutionandthenadjustpHto9.0with5mol/Lammoniumhydroxide(NH4OH),keepstirringthesolutionat0Cfor30minutes.
7.Add1-foldV1ofcold95%ethanolandstirthesolutionat20Cfor30minutes.
8.Centrifugethesolutionfor10minutesat10000r/minat-8C.Discardthepelletandmeasurethevolumeofthesupernatant(V2)(keep0.6mLforproteinassayat–20C).
9.Takethesupernatant(V2)andaddVA(mL)of2mol/LMgSO4(pH8.5)toafinalconcentrationof0.03mol/L.
10.Add1-foldVAofcold95%ethanolandstirthesolutionat0Cfor30minutes.
11.Centrifugethesolutionfor10minutesat10000r/minat-8C.Pouroffanddiscardthesupernatant.
12.Suspendthepelletwith1/10V10.07mol/LMgAC2(pH9.0).
13.Stirthesuspensionat0Cfor30minutes.Centrifugethesuspensionfor10minutesat12000r/minat0C.Pouroffanddiscardthepellet.
14.Measurethevolumeofthesupernatant(V3)(keep0.6mLforproteinassayat–20C).
15.AdjustthepHofthesupernatantto8.0with1mol/LNaOHandaddVBofcold95%ethanoltoafinalconcentrationof36%inanice-saltbath.CalculatetheVBusing:
[(V3-thevolumeofMgAC2)x0.5+VB]/(V3+VB)=0.36.
16.Stirthesolutionat0Cfor30minutes.Centrifugefor10minutesat12000r/minat-8C.Pouroffanddiscardthepellet.Measurethevolumeofthesupernatant(V4)(keep0.6mLforproteinassayat–20C).
17.AddVCcold95%ethanoltoafinalconcentrationof50%inanice-saltbath.CalculatetheVCusing:
(V4x0.36+VC)/(V4+VC)=0.6.
18.Stirthesolutionat0Cfor30minutes.Centrifugefor10minutesat12000r/minat-8C.Pouroffanddiscardthesupernatant.Suspendthepelletinabout5mLof0.01mol/LTris-HCl(pH7.5).Centrifugefor10minutesat12000r/minat0C.Measurethevolumeofthesupernatant(V5)(keep3-4mLforproteinassayat–20C).
19.StoretheV5at–20Covernight.
2)Chromatography
1.QSepharoseHighPerformancecolumnhasbeenpreparedforyouruse.Beforestartingarun,thecolumnhastobeequilibrated.Thisisdonebypumping5columnvolumesofstartbuffer(bufferA:
0.01mol/LTris-HCl,pH7.5)throughthecolumn.
2.Measureout4~5mLofV5forapplicationtothecolumn,thenwashthecolumnwith2columnvolumesofbufferAandelutethesamplewithbufferB(0.01mol/LTris-HCl,0.3mol/LNaCl,pH7.5)inalineargradient,flowrateofthecolumnisabout1mL/min.
3.Collect1mLfractionsintesttubesinafractioncollector.Transferthecontentsofeveryotherfractiontoaquartzcuvetteandmeasuretheabsorbanceat280nmoruseaUVmonitor.RecordtheA280valuesinyournotebook.Whenyoubegintodetectproteinelutingfromthecolumn(A280>0),measuretheA280ofeveryfraction.
3)MeasurementofEnzymeActivities
1.ThedetectionofthespecificactivityofthecreatinekinaseisbasedonthepH-colorimetry.ItisrathersensitivefortheadvancedspectroscopytomonitorthealterationofthecolorofThymolblue.
2.Thewaytocalculatethespecificactivityisaccordingtothefollowingequation:
1.3isaconvertingcoefficient.Ustandsforspecificactivity.VAstandsforthevolumeofsubstrate(VA=1.0mL).VBstandsforthevolumeofenzymesolution(VB=0.01mL).Cistheconcentrationofenzymesolution,anditisdeterminedbytheabsorbanceofthesolutionat280nm.
3.Fillthespecificactivityofthecreatinekinaseineachfractionintheformbellow.
Fraction
ProteinConcentration(mg/mL)
SpecificActivity
FoldPurification
Recovery(%)
4)SDS-PAGE
Thesamplesobtainedineachstepofcrudeextractionandthefinalproductsfromion-exchangechromatographyareloadedontosodiumdodecylsulfate-poly-acrylamidegelelectrophoresis(SDS-PAGE).Thestackinggelwasmadeof3%acrylamide,andtheseparatinggelwasmadeof12.5%acrylamide.
III.Experimentalrecord
1.Thevolumeineachpurificationstage
Chart1.Volumeoftheprotein.
V1
V2
V3
V4
V5
46.8ml
83.1ml
4.98ml
5