Purificationofthecreatinekinase.docx

Purificationofthecreatinekinase.docx

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Purificationofthecreatinekinase

Isolationandenzymaticanalysisofcreatinekinase

ofrabbitmuscle

●Background

1Creatinkinase:

Creatinkinase（CK,EC2.7.3.2）isakeyenzymeintheenergymetabolismoftissuewithhighenergydemandlikecardiacandskeletalmuscle,brain,retinaandspermatozoa.Itcatalyzesthemagnesium-dependentreversibletransphosphorylationbetweenphosphocreatine（PCr）andATPaccordingtothefollowingbiochemicalreaction:

Inrabbittissues,fourdifferentsubunitisoformsofCKareexpressed:

MK（Mstandingformuscle）,B-CK（Bstandingforbrain）,Mis-andMib-CK（Mistandingformitochondria）.Whereasonasubcellularleveltheformertwoarelocalizedinthecytosol,thelattertwoarefoundwithinthemitochondria.

Theexpressionofthesemultiplecreatinkinaseisoenzymeswithinceraincelltypesprovidesasystemfortheprocessofthemetabolicenergytransferknownasthecreatinephosphateshuttle（Bessman,1972;BessmanandCarpenter,1985）.Itisthissystemthatusescreatinephosphateandcreatinetointerconnectsitesofenergyproductionandutilizationwithincellsfortherapidreplenishmentofmetabolicenergy.AccordingtothePCrcircuitmodel,mitochondriacreatinkinaseontheouteersurfaceoftheinnermitochondriamembranemetabolizesATP,generatedbyoxidativephosphorylationinthemitochondriamatrix,andCrtoyieldPCrandADP.PCristhoughttobetheactual“highenergycompound”diffusing,withinthecellsofthetissuesmentionedabove,toplaceswhereenergy-consumingreactionstakeplace.Atthesesites,thecytosolicCKisoenzymesregenerateATP,andtheCrtherebyproduceddiffusesbacktothemitochondria.M-CKandthetwoMi-CKsareinvolvedinthismechanism.

Besidesitsroleinenergytransfer,oneofthekeyfunctionsofcreatinkinaseinthetissuesdescribedaboveisitscapacitytomaintaincytosolicATPlevelsrelativelyconstantduringsteadystateactivityandduringmodestworktransitionsdespitedecreaseinPCr.Itperformsthisbufferfunctioninvivobymaintainingnear-equilibriumbetweenallreactioncomponets.Therexplanationare（a）theenzymebeingpresentatactivitiesintissuesfarinexcessoftherateofATPutilization,and（b）thatitisnotkineticallylimitedbyeitherofitssubstratessincetheKmvaluesareintheapproximaterangeofconcentrationreportedinthetissyue.

CKicadimericenzyme,composedoftwoidenticalsubunits.Accordingtotheprimarystructuredetermined,thenaturallyoccuringM-CKfromchicken,human,andrataswellasfromrabbitisconsistingof387aminoacidresidues,eachwithamolecularweightabout43000.Thereareeightthioylgroupsinasinglemolecule,butnodisufarbondsareformedbetweenthem.X-raydiffractionshowsthatthe3dimensionalstructureofCKmoleculeisacondensedspherewithabout25percentα-helixand15percentβ-sheet.

2Measuringtheactivityofcreatinkinase（pH--colorimetricmethod）:

WhenCKcatalysestheforwardreaction,morephosphorylasesaretransferredandthesamemolecularamountofH+isgenerated.TheoptimalpHis7.5-9.0.Inthisrange,theH+generatingratecanbeusedtomeasuretheactivityofCK.Inthisexperiment,thymolblueservesaspHindicator.AspHdecreaseswithincreasingamountofgeneratingH+,thecolorofsubstratesolutionwillchangefromdarkmagentatoolivine.Whenthewavelengthis597nm,theabsorbanceA597willconstantlydecrease.

TheactivityofCKcanbecalculatedbythefollowingformula

dilutionratio（μmol／（minmg））

VA=1.0ml（Thevolumeofsubstratesolution），

VB=0.01ml（Thevolumeofenzymesolutionadded）

C：

theconcentrationoenzymesolutionadded

ΔA597:

ThechangeofA597inaminute

3Measureproteinconcentrationbyspectrophotometricassay:

Mostproteinshaverelativelyintenseultravioletlightabsorptioncenteredat280nm.Thisisduetothepresenceoftyrosineandtryptophanresiduesintheprotein.However,theamountoftheseaminoacidresiduesvariesindifferentproteins,aswaspointedoutearlier.Ifcertainprecautionsaretaken,theA280valueofproteinsolutionisproportionaltotheproteinconcentration.Theprocedureissimpleandrapid.AproteinsolutionistransferredtoaquartzcuvetteandtheA280isreadagainstareferencecuvettecontainingtheproteinsolventonly（buffer,water,etc.）.

●Materialsandsupplies

1Chemicalreagents:

Form1Thechemicalreagentinvolvedinthisexperiment

Chemical

Concentration

Function

KCl

0.01mol/L

Solutetheproteinbysalt

NH4OH

1.7mol/L

ControlpH=8.0

ammoniumcitrate

0.05mol/L

Tris-HCl

0.1mol/L（pH8.0）

ControlthepH=8.0

NH4Cl

Saltoutimpurities

NH4OH

5mol/L

ControlthepH,discardtheimpurities

MgSO4

2.0mol/L（pH8.5）

SaltoutCK

MgAC2

0.07mol/L（pH9.0）

SoluteCKremaininginthepellet

NaOH

0.2，0.5，1.0，2.0，5.0mol/L

AdjustthepH

HAC

0.2mol/L

AdjustthepH

CH3CH2OH

95%

Denaturetheimpurities

Crudesalt

Maintainlowtemperature

Form2Theprotocolofsubstratesolution

Thevolumeofsubstratesolution

10ml

15ml

20ml

Creation（48mmol/L）

5ml

7.5ml

10ml

MgAC2（0.1mol/L）

0．5ml

0.75ml

1ml

thymolblue（0.1％）

1ml

1.5ml

2ml

Gly-NaOH（0.1mol/L）pH9.0

0．5ml

0．75ml

1ml

ATP

24mg

36mg

48mg

Add0.1～0.2mol/LNaOHuntilthecolorofsolutionturnspurple（A5971.8～2.0）

A5971.6～1.8

PH=9.0

A5971.6～1.8

PH=9.0

A5971.6～1.8

PH=9.0

AddH2Oto

10ml

15ml

20ml

2Instruments

（1）Specord200spectrometerforkineticspectroscopy（Germany）.

（2）Speckol1200spectrometer（Germany）.

（3）Constant-flowPump（China）.

（4）FractionCollector（China）.

（5）Bio-RADElectrophoresisSet（USA）.

（6）EupaBlender（China）.

●Procedure

1Preparationofcrudeextract

（1）.Withasharpknife,cutthemuscleintocubes4to5cmwidefromtheice-coldrabbitmuscle.Thewhiteconnectivetissueshouldbesetaside.

（2）.Quicklyweighabout50gofthemusclecubesandsuspendthecubesin150mLof0.01mol/LKClat00C.

Thethreadlikeconnectivetissueshouldbediscardedascompletelyaspossibleinthisstep.Otherwise,itmayinterferewiththestirring.

（3）Homogenize150mLofthesuspensioninaglassbeakerwithablenderfor3times（turnontheblenderathighspeedfor30secondseachtime.Theintervalbetweeneachtimeis10s）andstirthemwithaglassstirringrodat00Cfor3times.（20seachtime,withanintervalof10s）.

Thestirshouldnottouchthesidewallofthebreaker.

（4）Centrifugethehomogenatefor10minutesat8000r/minat00C.Usetheelectronicbalancetobalancethetwocentrifugetube（Don'tforgetthelid）andensurethedifferencebetweentheirmassislessthan0.1g.

Ensurethatnowaterdropadheredtotheoutersidewallofthecentrifugetube.Otherwise,theadheredwatermayfreezeinthecentrifugalmachine.

（5）Carefullydecantthesupernatantandmeasurethevolumeofthesupernatant（V1）（keep0.6mLforproteinassayinthreeEPtubesat–20oC）.Discardthepellet.

（Usesteelspoontodigitout）OurV1=45mL）

（6）.Addgroundammoniumchloride（NH4Cl）tomake0.1mol/Lsolution

TheNH4Clweaddedis0.2354g.NH4Clshouldbeaddedslowly.Otherwiselocalconcentrationwillbesohighthattheenzymemayloseitsactivity.

Wemadeamistakeinthisstep,weaddedNH4Clrapidlyandthismayleadtotheinactivationofenzyme.

（7）AdjustpHto9.0with5mol/Lammoniumhydroxide（NH4OH）,keepstirringthesolutionat00Cfor30minutes.

TheNH4OHshouldbeaddedhalfadroponetimeandmeasurethepHeachtime.

（8）AddV1ofcold95%ethanolandstirthesolutionat200Cfor30minutes.

（9）Centrifugethesolutionfor10minutesat8000r/minat-80C.Discardthepelletandmeasurethevolumeofthesupernatant（V2）（keep0.6mLforproteinassayat–200C）.

V2=68mL

（10）Takethesupernatant（V2）andaddVA（mL）of2mol/LMgSO4（pH8.5）toafinalconcentrationof0.03mol/L.

VA=1.020mL

（10）AddVAofcold95%ethanolandstirthesolutionat00Cfor30minutes.

（11）Centrifugethesolutionfor10minutesat8000r/minat-80C.Pouroffanddiscardthesupernatantandassemblethepelletinatube.

Inprinciple,thesolutionshouldbedividedin4centrifugetubes,butouramountisrelativelylittle,todecreasetheloss,wedividedthesolutionin3tubesandfillwaterintheforthtubetobalance.

（12）.Suspendthepelletwith1/10V10.07mol/LMgAC2（pH9.0）.

Actually,wedidn'tdothisstepinthefirstexperiment.

（13）Storethepelletat–200Covernight.

（14）.SolvethepelletinMgAc2.Stirthesuspensionat0oCfor30minutes.Centrifugethesuspensionfor10minutesat12000r/minat00C.Pouroffanddiscardthepellet.

ThevolumeofMgAc2=1/10thevolumeofV1,soweadded4.5mLMgAc2（aq）

（15）.Measurethevolumeofthesupernatant（V3）.Keep0.6mLforproteinassayinEPtubesat–200C（0.2mLineachtube）.

OurV3=6.4mL

（16）AdjustthepHofthesupernatantto8.0with0.5mol/LNaOHandaddVBofcold95%ethanoltoafinalconcentrationof36%inanice-saltbath.CalculatetheVBusing:

（V3-thevolumeofMgAC2）x0.5+VB/（V3+VB）=0.36

OurVB=2.1156mL

Beforeadding,wepreparedtheicesaltbath.Whatisimportantisthatthesaltshouldbedistributearoundthebreakerbutavoiddistributingitintothebreaker.

Otherwise,theenzymewillbesaltoutorinactivated.

Unfortunately,inthisstep,wedidn'tgetanysediment.Perhaps,ourenzymeispureenoughormaybewedidn'tsetthesysteminaconditionthatfavorstheformationofproteinsediment.

（17）Stirthesolutionat00Cfor30minutes.Centrifugefor10minutesat12000r/minat

-80C.Pouroffanddiscardthepelletandkeep0.6mLforproteinassayin3EPtubesat–200C（02mLineachtube）.Measurethevolumeofthesupernatant（V4）.