浊度/分光光度法与毛细管等电点聚焦法测定明胶等电点及其分布的比较研究
浊度,分光光度法与毛细管等电点聚焦法
测定明胶等电点及其分布的比较研究
第24卷第1期
2007年1月
中国科学院研究生院
JournaloftheGraduateSchooloftheChhe~AcademyofSciences
Vo1.24No.1
January2007
ArfldeliD:10o2.1175(2Oo7)01.0114-05
ComparativestudyofdeterminingIEPandIEP
distributionofgelatinwithturbidimetry/
spectrometryandcapillaryIEPfocusingmethods
LUYang'PENGBi.Xian
(1TechnicalInstitutePhysicsandChemistry,ChineseAcademy&/ences,lg100080,China;
2GraduateUn~rsity,C轴?eAcademySc,驷100049,China)
(Received20Jmuary2006;Revised3Ap,412006)
LuY.PangBX.ComparativestudyofdeterminingIEPandIEPdistribution0fgelatinwlflaturbidlmetry/spectrometryand
eapmaryIEPfocusingmethods.JournaloftheGraduateSchooloftheChOteseAcademyofSc/ences,20O6,/,4(1):114—118
Abstract.nleisoelectricpoint(IEP)isoneofthemostimportantphysicochemicalpropertiesof
proteins.TherearemanydifferentmethodsforthedeterminationofIEP.Inthispaper,acomparative
studybetweenthesimplestturbidimetry/spectrometrymethodandthemostadvancedcapillaryIEP
focusing(CalPF)methodwasmade.Henancattlehidegelatin'SIEPwagdeterminedwiththetwo
methodsandtheadvantagesoftheCaIPFmethodovertheothermethodsweredemonstratedand
discussed.
Keywordsisoelectricpoint,gelatin,turbidimetry/spectrometry,capillaryIEPfocusing CI.CO64
Asatypeofampholytes,proteinshavemanycharacteristicpropertiesthathelpthemexerttheirbiochemical
functions.Isoelectricpointsandtheirdistributionsareoneofthemostimportantphysicochemicalpropertiesof
proteinmacromolecules.Inthemacromoleculesofcollagenproteins,differentcomponentssuchasland口2chains
ofunripeTypeIcollagenarefoundtohavedifferentIEPs(pIvalues)afterbeingseparatedanddetermined卜.The
differencesaretheresultsofthedistinctionsoftheirprimarystructures(thejointsequencesofaminoacidresidues).
Componentscontainingmorehydrophilicacidicgroups(Glutamicacid,asparticacid,etc.)havelowerpIvalues
whilecomponentscontainingmorehydrophobicbasicgroups(Lysine,arginine,etc.)havehigherpIvalues.Thepl
vahlesandtheirdistributionsdecidethehydrophilicityandhydrophobicityofproteinmacromoleculestoagreatextent
andconsequentlyinfluencetheirbiochemicalactivity.Inthecomponentsofcollagenfoundincartilages(thejoint
partsofal'throses),口
chaincontainsplentyofhydrophilicaminoacidresiduesanddemonstratesniceflexibility. ~fcortespondingauthor.E-mail:pengbx@mail.ipc.?.cn
第1期
LUYang,PENGBi-Xian:Comparativestudyofdetermining1EPandIEPdistributionofgelatinwith……115
I11eirIEPsarealsolowert.
Theabove-mentionedinstanceisaboutthecollagenproteins.Anotherkind,lipopioteins,whichcanbedivided
intothreetypes,high-densitylipopmteins(HDL),low-densi~lipopmteins(LDL)andverylow—densitylipopmteins
(VLDL),haveverygreatdisparitiesintheproteinprimarystructures,molecularweights,ampholytecharacteristics
(IEP)andlipidcomponents(Triglycerides,phospholipidsandcholesterol,etc.).Forexample,inordertoconvey
HDLfrombloodvesselstolivereasily,proteinsconnectedwithHDLshouldcontainfeweraminoacids(Dozensonly)
andtherebyhavepIvaluesnearthepHofbloodplasma.Therefore,thedeterminationofdifferentlipopmteins'
IEPcanhelpustoknowthetransportandmetabolismoflipopmteins.
Gelatinsarethehydrolyzedproductsofcollagens.Theyhaveimportantdispersingandwrappingeffectsinthe
preparationofphotosensitiveemulsionmicmcrystals,microspheresandmicmcapsules.ThemediumpH,thepI
ofgelatinandthepXofemulsion(X=C1,Br,I)areconsideredtobethreeimportantparametersthatcontrolthe
formationofphotosensitivesilverhalidemicrocrystals.Duringthepreparationofmicrospheres,themediumpHand
gelatin'sIEParealsothekeyparametersthatinfluencethedispersivityofmicmspheres.Therefore,theprecise
determinationofthepIvaluesofproteinmacromolecules(includingtheirconformationunits:al,a2,etc.)hasnot
onlyacademicsignificancebutalsosomepotentialforapplicationincertainfields.
Inthiswork,wemadeacomparativestudyontheIEPdeterminationwithturbidimetry/spectrometryand
capillaryIEPfocusing(CaIPF)methods,usingagelatinsampleastheresearchobject. 2Experimentalprocedure
2.1Reagentsandapparatus
Gelatin(Henancattlehide21220);HAc,NaAc(anhydions)andanhydionsalcoholareallA.R.andproduced
bytheBeijingChemicalPlant;HI8314membranepHmeter(HannaInstruments(AsiaPacific)E.Ltd.);U-
2001UV-Visspectrometer(Hitachi).
2.2Determinationofgelatin'sIEP
Therearemanymethodstodeterminegelatins'IEPsuchastheturbidimetrymethod…,theion
exchange
method….
theIEPfocusingmethod【J2_andtheCaIPFmethod
【13J.Thefirsttwomethodsaresimpleandeasyto
handle,buttheirprecisionsareconsiderednottobeverynice.Thelasttwomethodsarethebasicmeansthattoday'
sbiochemicalresearchersuseandshowveryhighprecisions.BoththevaluesanddistributionrangesofIEPcanbe
detected.ButtheampholineneededtodetermineIEPisverydifficulttosynthesize?,andtheirexperimental
operationsarequitetime-consumingandcosily.
2.2.1Turbidimetry/spectrometrymethod
Inthetraditionalturbidimetrymethod,theturbidityvalueofgelatinsolutionafterflocculationisdetectedby
eyesorwithaturbidimeter.Weusedaspectrometerinsteadtoenhancethedetectionsensitivity.Themanipulation
stepswereasfollows:HAcandNaAcsolutionswiththeconcentrationof0.1mol/Landgelatinsolution(1%w/v)
wereprepared.BuffersolutionswithdifferentpHsof4.5,4.7,4.8,4.9,5.0,5.4,5.7,5.8,5.9,6.0and6.3were
thenpreparedwiththeHAcandNaAcsolutions.3mLofbuffersolutionandlmLofgelatinsolutionwereaddedtoa
seriesoftesttubesandshakenup,respectively.Thesamevolumeofanhydrous-alcoholwasaddedtoeachtubewith
anacidicburetuntilalltubesturnedturbid.andthetubeswereshakenduringadding.Thelightabsorbcneiesof
eachsampleatthewavelengthsof400nmand60Ontoweredeterminedwithaspectrometer.ThepHofthesolution
withthelargestabsorbencyisjustthegelatin'spIvalue.
2.2.2Ca?.Fmethod
TheprinciplesandoperationsofIEPfocusingmethodscanbereferredtoinRef.[12].Inprinciple,the
differencebetweenconventionalIEPfocusingandtheCaIPFmethodsis:Liquidelectrolytewithdifferentdensity
l16中国科学院研究生院第24卷
gradientstopreventturbulentflow(usingsucro8eascarrier)isusedinthefirstmethod;butinthelattermethod,
polymerisappliedasthefixedphaseandwiththeampholineandstandardsintegratedtobeapHlineargradient(e.
g.pH3—
10).ThiscarrierCanbepurchasedeasily.ThedetailedmanipulationofthismethodCanbereferredtoin
themonograph[.
Thegelatinweusedisakindofhidegelatinmadefromthecollagenproteinofcattlehide.Therelateddataof
pIvaluedetectedwithtnrbidimetry/spectrometrymethodarelistedinTable1andalsoshowninFig.1.The
correspondingresultsdetectedwithCaIPFmethodalegiveninTable2andFig.2.
0.130
0125
0120
喜0.115
0.110
0l05
0lo0
TaMe1LightabsorbenciesofHenancattlehidegelatinsolution atdmerentpHsafteraddingthe懿I皿amount{120drops)ofalcohol
4.4464.85052545+65860626.4
pH
Hg.1Relationshipbetweentheu#tabsorbencies and#IsofHew~cattlehidegelatinsolutionafter 8ddingthegmltu~mountofalcohol
O00
2000
0o0
Absorbanc~
0.00500.0100
0+00500.0100
Absorbance
1pI9.45
Fig.2SpectrumofdeterminingHemmcattle
hldegelatin'SIEPwiththeCalPFmethod
Table2DataofdeterminingHenancattlehldegelatin'sIEPwiththeCalPFmethod
3.1Evaluationofturbidimetry/spectrometrymethod Theturbidimetrymethodissimpleandeasytohandle.Sofar,itisstillwidelyusedintheanalyti
callabofa
gelatinfactory.ThepeakvalueofpICanbejudgedbyeyes.Butit'8notpreciseenoughtoconfir
mthedistribution
rangeofpI.Afterthismethodisreplacedbytheturbidimetry/spectrometrymethod,thedistributionrangeCanbe
第1期
LUYang,PENGBi.Xian:ComparativestudyofdeterminingIEPandIEPdistribution0fgelatinwith……117
confirmed.Inthisexperiment,fromTable1andFig.1wecangeethatthepIvalueofthisgelatinis5.81andits
distributionrangeispH5.60—5.95.
3.2Judgmentofminorcomponents'IEPwithturbidimetry/spectrometrymethod Gelatinisakindofbiopo1)metwithpolydispersemolecularweight.Itsdifferentcomponentshavedifferent
IEPs.InFig.1,tllepH(5.81)correspondingtothelargestabsorbencyisjustthepIvalueofthisgelatin(the
influenceofinorganicmetallicionisignored.)Butthereisanothersmall,bun-shapedpeal【atlowerpHexceptfor
thismajorpeal
【.ItscorrespondingpHis4.75,whichistheaverageIEPoftheminorcomponents.Andits distributionrangeis4.50—
5.00.Butitcan'tbejudgedwhethertherearestillsomedifferentcomponentsexisting inthisgelatin.
3.3Determinationofmajorcomponents'IEP,订thCalPFmethod
Inthismethod,3standardmattersareapplied.Theyareribonuclease(RNAase,pI9.45),-lactoglobulin(pI
5.10)andSflankingpeptide(pI2.75).Basedonthelinearfunctionbetweentheabsorbenciesatthewavelengthof
400nmandelectmphoreticmovingdistances(orretentiontime),theunknownmatter'8IEPiscalculatedwithspecial
software.ThepeakareaandpIvaluesarealsoprintedinthemeantime.ThepIvalueofthisgelati
ndetectedwith
thismethodis5.90.It'sthefirsttimetodeterminegelatin'sIEPwiththeCaIPFmethod.Thisresultissocloseto
thepIvalue(5.81)detectedwiththeturbidimetry/spectmmetrymethod.AnditsdistributionrangeispH5.25—
6.55.
3.4Determinationofminorcomponents'IEPwithCalPFmethod
FromFig.2wecanseethat,exceptforthemajorcomponent(pI5.90),therearestilltwominorcomponents
withsharpandsmallpeaks.Onecomponent'spIis4.92whiletheother'sis4.71.Theyares0closetoeachother
butstillwithgooddifferentialratio.TheirdistributionrangearepH4.60—4.75andpH4.70,
5.15.respectively.
Therefore,theCaIPFmethoddisplaysahigherdifferentialratiothantheturbidimetry/spectrometrymethoddoes.
IEP'sdistributionrangeisaveryimportantparametertoevaluatethepurityofaproteinsample.Inthisregard,
CaIPFcancontributeagreateraction.
3.5ComparisonofcolumnIEPfocusing(CoIPF)andCaIPFmethods
FromRef.[12]andRef.[13]wecanseethat,InCoIPF,thenecessarystandardampholineisquitedifficultto
synthesize.Anditisverytime-consumingtoestablishthedensitygradientsandtofocus(thetotalmanipulativetime
isaslongas3—
4d).Andsometimesinappropriatedyeingandfadingmayoccur.ButinCaIPF,thefixedphase,the
standardmattersandunknownmatterareplacedtogether.Sotheabove-mentionedproblemswon'tappear.In
general,thewholeworktodetectasampleonlyneeds2—
3h.Theoperationaltimecanbesho~enedgreatlyandits
differentialratioismuchhigherthanCoIPF's.Therefore,theapplicationofCaIPFshouldbeextended.
AcknowledgementsTheauthorsalehighlygratefultoProfessorCHENLi-Juanforhervaluableadvicesand
discussionsonthispaper.AndwealsothankMr.ZHANGXin—
ZhuangforprovidinguswiththeUV-Vis
spectrometer.
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浊度/分光光度法与毛细管等电点聚焦法测定明胶等电点
及其分布的比较研究
陆扬彭必先
(1中国科学院理化技术研究所,北京100080;2中国科学院研究生院,北京100049)
摘要等电点是蛋白质最重要的物理化学性质之一.目前有多种测定等电点的
.本文利用最简单
的浊度/分光光度法和最先进的毛细管等电点聚焦法分别测定了河南牛皮明胶的等电点,对两种方法进
行比较并论证了后一种方法的优势.
关键词等电点,明胶,浊度/分光光度法,毛细管等电点聚焦法
]];]】i]???引…
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