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 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. References [1]VeisA.11.eMaeromoleeuhrChemistry0fn.NewYork:AcademicPre,1964 [2】JinGx,PengBX.StudyOllthestructure(Conformation)andpmperti~0fcdlagen/#atin?.Theseparation0fdifferentconformationsinacid- ~lublec0llBn.Phot%cap~Sc/en~andP~try,1993,11(3):204—208(inC~nese) 金桂仙,彭必先.胶原,明胶的结构(构象)与性能研究?.酸溶胶原(SLK)的四种不同 构象的分离.感光科学与光化学,1993,11 (3):204208 118中国科学院研究生院第24卷 JinGX,PengBX.studyonthestructmre(Conformation)andpropertiesofcollagen/gelatin?.Thecharacterizationof4differentconformations 8epedfromcollagen.PhotographicScienceandPhotochemistry,1993,l1(4):302— 306(inCMneso) 金桂仙,彭必先.胶原,明胶的结构(构象)与性能研究?.从酸溶胶原(sLK)中分离出 的四种不同构象的 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 征.感光科学与光化学, 1993,l1(4):302—306 JinGX,Chen13.PengBx.Smdyonthestructure(Conformation)andpmperllesofcollagen/gelatinIV.Ikresearchontheamphotericelectrolyte characterof4differentconformationsofacidsolublecollagen.PhotographicScienceandPhou~hom/sWy,1994,12(1):11—16(inChinese) 金桂仙,陈丽娟,彭必先.胶原,明胶的结构(构象)与性能研究?.酸溶胶原的四种构 象(a,a:,J9和J9)的两性电解质特性研究. 感光科学与光化学,1994,12(1):l1一l6 PengBX.Amicmgelemeterstudyofphysico-mechanicalpropertiesofcollagenfragments. 如urea/of1magi~ScienceandTechnology,1992,36: 544—549 杨永宗.动脉粥样硬化性心血管病基础与I临床.北京:科学出版社,2004 BerryCR,SkillmanDC.DiffractionofX— raysbynascentsilverbromidemicrocrystals.Journ~ofCrystalGrowth,1968,2(3):141— 144 UgwokeMI,KingetR,Influenceofprocessingvariablesonthepropertiesofgelatinmicrosph erespreparedbyemulsificationsolventextraction technique.Journa/ofMicroencapsulation,1998,15:273—281 WangW.Productionofmulti-str~turcsilverbmmolodidec~tallite:[MasterThesis].B喇 ing:InstituteofPhotographicCllemi8,Chinese AcademyofSciences,1996(inChinese) 王巍.多层结构溴碘化银微晶的制备:[硕士学位 论文 政研论文下载论文大学下载论文大学下载关于长拳的论文浙大论文封面下载 ].北京:中国科学院感光化学 所.1996 赵振国.胶体与界面化学实验.北京:北京大学出版社,1993.300304 陈砥.用离子交换法测定明胶的等电点.明胶科学与技术,1985,5(3):130 ChenLJ,PengBX.Theisoelectricpointdistributionofmodifiedgelatins.PhotographicScie nceandPhotochemistry,1984,(4):13—19(in Chinese) 陈丽娟,彭必先.改性明胶的等电点及其分布研究.感光科学与光化学,1984,(4):13—19 郭尧君.蛋白质电泳实验技术.北京:科学出版社,1999 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方法.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… [[;[;[[