实验五 细胞色素C的制备及测定

实验五细胞色素C的制备及测定【实验目的】1．学习细胞色素C的理化性质及其生物学功能。2．掌握制备细胞色素C的原理。3．掌握制备细胞色素C的操作技术。【实验原理】细胞色素C是呼吸链的一个重要组成成份。是一种含铁卟啉基团的蛋白质，在线粒体呼吸链上位于细胞色素b和细胞色素aa3之间，细胞色素C的作用是在生物氧化过程中传递电子。细胞色素C分子中含赖氨酸较高，所以等电点偏碱，为pH10.8，分子量为12000～13000道尔顿。它易溶于水及酸性溶液，且较稳定，不易变性，组织破碎后，用酸性水溶液即能从细胞中浸提出来。细胞色素C分为氧化型和还原型两种，因为还原型较稳定并易于保存，一般都将细胞色素C制成还原型的，氧化型细胞色素C在408nm、530nm有最大吸收峰，还原型细胞色素C的最大吸收峰为415nm、520nm和550nm，这一特性可用于细胞色素C的含量测定。由于细胞色素C在心肌组织和酵母中含量丰富，常以此为材料进行分离制备。本实验以猪心为材料，经过酸溶液提取，人造沸石吸附，硫酸铵溶液洗脱，三氯醋酸沉淀等步骤制备细胞色素C，并测定其含量。【实验材料】1.实验器材绞肉机；电磁搅拌器；电动搅拌器；离心机；72l型分光光度计；玻璃柱(2.5×30cm)；下口瓶；烧杯(2000、1000、500m1)；量筒；移液管；玻璃漏斗和纱布；玻璃棒；透析袋2.实验试剂⑴2MH2S04溶液；1MNH40H溶液；固体硫酸铵⑵25％硫酸铵溶液:100m1蒸馏水中含25克硫酸铵，约相当于25℃时40％的饱和度⑶0.2％氯化钠溶液：称0.2克氯化钠，用蒸馏水溶解并定容至100ml.⑷BaCl2试剂：称12克BaCl2,溶于100ml蒸馏水中。⑸20％三氯醋酸溶液(6)人造沸石(60～80目)⑺联二亚硫酸钠(Na2S2O4·2H2O)【实验操作】1．细胞色素C的制备⑴材料处理:取新鲜或冰冻猪心，除去脂肪和韧带，用水洗去积血，将猪心切成小块，放入绞肉机绞碎。⑵提取:称取绞碎猪心肌肉500克，放人2000m1烧杯中，加蒸馏水1000ml，在电动搅拌器搅拌下以2MH2S04调pH至4.0(此时溶液呈暗紫色)，在室温下搅拌提取2小时，在提取过程，使抽提液的pH值保持在4.0左右。在即将提取完毕，停止搅拌之前，以1MNH40H调pH至6.0，停止搅拌。用八层普通纱布压挤过滤，收集滤液。滤渣加入750m1蒸馏水，再按上述条件提取1小时，两次提取液合并。⑶中和:用1MNH40H调上述提取液至pH7.2(此时，等电点接近7.2的一些杂蛋白溶解度小，从溶液中沉淀下来)，静置30～40分钟中后过滤，所得滤液准备通过人造沸石柱进行吸附。⑷吸附与洗脱:人造沸石容易吸附细胞色素C，吸附后能被25％的硫酸铵洗脱下来，利用此特性将细胞色素C与其它杂蛋白分开。具体操作如下：①人造沸石的预处理：称取人造沸石11g，放入500m1烧杯中，加水搅拌，用倾泻法除去12秒钟内不下沉的过细颗粒。②装柱：选择一个底部带有滤膜的干净的玻璃柱(2.5×30cm)，柱下端连接一乳胶管，用夹子夹住，柱中加入蒸馏水至2／3体积，保持柱垂直，然后将己处理好的人造沸石带水装填人柱，注意一次装完，避免柱内出现气泡。③上样：柱装好后，打开夹子放水(柱内沸石面上应保留一薄层水)将准备好的提取液装入下口瓶，使其通过人造沸石柱进行吸附。柱下端流出液的速度为1.0ml／分钟。随着细胞色素C的被吸附，柱内人造沸石逐渐由白色变为红色，流出液应为黄色或微红色。④洗脱：吸附完毕，将红色人造沸石从柱内取出，放入500m1烧杯中，先用自来水，后用蒸馏水搅拌洗涤至水清，再用100m10.2％NaCl溶液分三次洗涤沸石，再用蒸馏水洗至水清，按第一次装柱方法将人造沸石重新装入柱内，用25％硫酸铵溶液洗脱，流速大约2ml/分钟，收集含有细胞色素C的红色洗脱液，当洗脱液红色开始消失时，即洗脱完毕。人造沸石可再生使用。⑤人造沸石再生：将使用过的沸石，先用自来水洗去硫酸铵，再用0.25M氢氧化钠和lM氯化钠混合液洗涤至沸石成白色，前后用蒸馏水反复洗至pH7～8，即可重新使用。⑸盐析：为了进一步提纯细胞色素C，在上面收集的洗脱液中，加入固体硫酸铵(按每l00m1洗脱液加入20克固体硫酸铵的比例，使溶液硫酸铵的饱和度为45％)边加边搅拌，放置30分针后，杂蛋白便从溶液中沉淀析出，而细胞色素C仍留在溶液中，用滤纸(或离心)除去杂蛋白，即得红色透亮细胞色素C溶液。(6)三氯醋酸沉淀：在搅拌情况向所得透亮溶液加入20％三氯醋酸(2.5m1三氯醋酸／100m1细胞色素C溶液)，细胞色素C立即沉淀出来（沉淀出来的细胞色素C属可逆变性)，立即于3000转／分钟离心15分钟，收集沉淀。加入少许蒸馏水，用玻棒搅拌，使沉淀溶解。⑺透析：将沉淀的细胞色素C溶解于少量的蒸馏水后，装入透析袋，在500m1烧杯中对蒸馏水进行透析除盐(电磁搅拌器搅拌)，15分钟换水—次，换水3至4次后；检查透析外液SO4是否已被除净。检查方法是：取2m1BaCl2溶液于试管中，滴加2至3滴透析外液至试管中，若出现白色沉淀，表示S04未除净，反之，说明透析完全，将透析液过滤，即得细胞色素C制品。2.含量测定所得制品是还原型细胞色素C水溶液，在波长520nm处有最大吸收值，根据这一特性，用721型分光光度计，先作出一条标准细胞色素C浓度和对应的光密度值的标准曲线(图1)，然后根据测得的待测样品溶液的光密度值就可以由标准曲线的斜率求出待测样品的含量。具体操作如下：

⑴标准曲线的绘制：取1毫升标准品(81mg/ml)，稀释至25ml，从中分别取0.2，0.4，0.6，0.8，1.0ml，分别置于五支试管中，每管补加蒸馏水至4m1，并加少许联二亚硫酸钠作还原剂，然后在520nm处测得各管的光密度，分别为0.179，0.330，0.520，0.700，0.870。以浓度为横坐标，光密度值为纵坐标，作出标准曲线图，从图中求得斜率为l／3.71。⑵样品测定取1ml样品，稀释适当倍数，再加少许联二亚硫酸钠，在波长520nm处测定光密度。最后根据标准曲线的斜率计算其细胞色素C的含量。【实验结果】计算公式：细胞色素C的含量=3.71×OD×稀释倍数×终体积在本实验中，500克的猪心原料，应获得75mg以上的细胞色素C的制品。(注：在细胞色素C的实际工作中，除了含量测定以外还要测定含铁量即纯度的鉴定和活性，后两项测定，此处从略)。【注意事项】1．尽可能除掉猪心中的韧带、脂肪和积血。2．使用离心机之前，一定要配平。3．透析之前要检查透析袋。4．在520nm处测得各管的光密度时，要加少许联二亚硫酸钠作还原剂。【思考题】1．制备细胞色素C通常选取什么动物组织?为什么？2．本实验采用的酸溶液提取，人造沸石吸附，硫酸铵溶液洗脱，三氯醋酸沉淀等步骤制备细胞色素及含量测定，各是根据什么原理?3．请说出其它提取和纯化细胞色素C的方法吗？请写出相关的方法及原理。Experiment17PreparationandDeterminationofCytochromec【Purpose】1.LearnphysicalandchemicalpropertiesofCytochromec,anditsbiologyfunctions.2.Mastertheprinciplesofseparation，purification，anddeterminationofCytochromec.3.LearnthebasicexperimentalproceduresfortheproductionofCytochromec.【Principle】Cytochromec,akeycomponentintherespiratorychain,isaproteinthatcontainshemeprostheticgroup.Itissituatedbetweencytochromebandcytochromeaa3inspecificsequenceofcarriersoftherespiratorychainandfunctionsasanelectronshuttle,whichcanbothacceptanddonateelectrons,inthemitochondrialelectrontransportpathandbiologicaloxidationprocess.Thepolypeptidechainsofcytochromeccontainlargeamountsoflysine,whichmakesitspIalkaline(10.8).Themolecularweightofcytochromecisabout12000~13000Da.Itcanbeeasilydissolvedinwateroracidsolutionandquitestable,soitiseasilyisolatedandextractedfromtissueusingacidsolution.Cytochromechastheoxidizedandreducedforms.Theabsorptionpeaksareat550,520,and415nminthereducedform,andat530,408nmintheoxidizedform.Ingeneral,thereducedcytochromecismorestableandeasilyconserved,sopreparationofcytochromecisinthereducedform.Bythisproperty,thecontentofcytochromeccanbedetermined.Cytochromecisrelativelyplentifulintissuessuchasheartmuscleandyeast,whichcanbetherawmaterialforseparatingcytochromec.Inthisexperiment,pigs’heartisselectedastheexperimentalmaterialtoobtaincytochromecthroughtheprocessofextractingatlowpH,bindingwithman-madezeolite,elutingwithammoniumsulfate,precipitatingwithtrichloroaceticacid,andtodeterminateitscontent.【Materials】ApparatusMincer;Electromagneticmixer;Electromotivemixer;Centrifuge;721Spectrophotometer;Glasscolumn(2.5×30cm);Flasks(2000、1000、500m1);Cylinder;Pipette;Glassfunnel;Gauze;Glassstick;Dialyticbag.2.Reagents⑴2MH2SO4solution;1MNH4OHsolution;Solid(NH4)2SO4⑵25%(NH4)2SO4solution:100mldistilledwatercontaining25gammoniumsulfate,whichamountsto40%saturationat25℃.⑶0.2%NaClsolution:Weigh0.2gNaCltodistilledwaterandsetfinalvolumeto100ml.⑷BaCl2solution:Weigh12gBaCl2to100mldistilledwater⑸20%Trichlorocaeticacidsolution⑹Man-madezeolite(60～80screen)⑺SolidNa2S2O4•2H2O【Procedures】1.PreparationDispositionofmaterial:Takefreshorfrozenidealpig’sheart,whichisfreefromfatandligaments,washawaybloodwithwaterandcutintosmallblock.Useminceratthehighestspeedtohomogenizethetissue.⑵Extraction:Weighsamples(500g)ofheartmusclemincemeatandputintoa2000mlflask,thenadd1000mldistilledwater.AdjustthepHto4.0with2MH2SO4(tillthecolorisaboutdarkpurple),inthemeantime,frequentlystirwithanelectricalmixer.Thisstepmaytakeabout2hoursatroomtemperature.BesuretokeepthepHvalueatabout4.0intheextractingprocess.Beforestoppingmixing,adjustpHto6.0with1MNH4OH,andthenstopswirling.Withoutdisturbingthepellet,carefullysqueezethesupernatantoutinapresswitheight-layergauzeandcollectit.Add750mlofdistilledwatertotheresiduetore-suspendthepellet,andre-extractfor1hourasmuch.Combinethesupernatantsolutions.Ifthefilteredsolutionisturbid,filteragain.⑶Neutralization:AdjustthepHoftheextractedsolventto7.2with1MNH4OH(duringthisprocess,someunwantedproteinswillprecipitatebecauseoflowsolubilitywhenthesurroundingpHisequaltotheirpI).After30to40minutesofprecipitation,thesolutionisfiltratedandreadyfornextstep.⑷Bindingandeluting:Theman-madezeolitecaneasilybindwithcytochromec,whichcanbeelutedbysulfateammonium.Accordingtothisproperty,cytochromeccanbeseparatedfromotherunwantedproteins.Thedetailedprocessesarelistedbelow:①Pretreatmentofman-madezeolite:Weigh1gman-madezeolite,putitintoa500mlflask,addwaterandstir,discardthesmallparticlesthatcannotprecipitatein12secondswithpourmethod.②Stuffing:Takeaglasscolumn(2.5×30cm)withfiltermembraneatthebottom,wherealatexpipeisconnected.Shutofftheexitwithaclip,adddistilledwatertillitreaches2/3ofthetotalvolume,andkeepthecolumnupright.Suspendthepretreatedman-madezeoliteinwaterbyswirling,andpouritintothecolumn.Itshouldbeensuredthatthereisnobubble.③Loadingsamples:Afterstuffingthecolumn,openthevalveatthebottomofthecolumnandallowthecolumnmaterialtosettle(besuretoallowthewaterleveltodroptojustabovethetopofthecolumnmaterial).Openthevalveatthebottomofthecolumnandpassthesolutioninstep(3)throughthecolumnattheflowrateof1.0mlperminute.Allowallthesolutiontoflowthroughthecolumn,butdonotletthesolutionleveldropbelowthetopofthecolumnmaterial.Theingredientwantedcanbindwiththeman-madezeoliteinthisstep.Alongwiththebindingofcytochromec,theman-madezeoliteincolumngraduallyturnsredfromwhite,whilethesolventshouldbeyelloworlightred.④Elution:Takeman-madezeoliteoutofthecolumnafterbindingandputitintoa500mlflask.Washitfirstwithwater,thenwithdistilledwatertillthewaterisclear.Washtheman-madezeolite3timeswith100ml0.2%NaClagain.Finallywashitwithwatertillthewaterislucid.Re-stufftheman-madezeoliteintothecolumnandelutewith25%ammoniumsulfateattheflowrateof2mlperminute,andthencollecttheredeluent,whichcontainscytochromec.Whentheeluentturnsredtoclear,itmeanstheendingofelution.Donotdiscardtheman-madezeolitebecauseitcanberegenerated.⑤Regenerationofman-madezeolite:Washawaytheammoniumsulfateontheman-mdezeolitebywater;thenusethemixtureof0.25MNaOHand1MNaCltowashthezeolitetillitbecomeswhite;washitwithwateragainandagainuntilthepHisabout7to8,thentheman-madezeolitecanbereused.⑥Saltprecipitation:InordertofurtherpurifycytochromeC,addsolidammoniumsulfatetotheeluent(add20gsolidammoniumsulfateper100mltomakethefinalconsistencyreach45%ofsaturation)andstirtheeluentwhenadding,thenplaceitstatically.After30minutes,theimpureproteinsprecipitate,whilecytochromecremainsinthesolution.Filtrateitwithfilterpaperstocollecttheredclearfiltratethatcontainscytochromec.⑦Precipitationbytrichloroaceticacid:Add20%trichloroaceticacidtotheclearliquidwithfrequentswirling(2.5mltrichloroaceticacidper100mlliquid),andthenthecytochromecprecipitates(thisprecipitationisreversible).Putthesedimentinacentrifugaltubeimmediatelyandcentrifugefor15minutes(3000rpm),andthencollectthesediment.Addtrifledistilledwaterandstirwithaglasssticktomaketheredcomponentdissolveinthewater.⑧Dialysis:Transferthesuspensionintoadialyticbaganddialysewithdistilledwaterinaflaskusingelectromagneticmixer.Changethewaterevery15minutes,after3to4times,checkoutwhetherSO42-hasbeenclearedaway,theprocessistoput2mlBaCl2intoatesttube,andadd2or3dropsofwaterintheflask,ifprecipitationappears,itmeansthereisstillsomeSO42-inadialyticbag,andviceversa.2.DeterminationofcontentThecytochromecinthesolventisinitsreducedform,whichhasabsorbanceinthewavelengthof520nm.Becauseofthisproperty,using721-ultravioletspectrophotometer,drawabsorbance-cytochromecconcentrationcalibrationcurve(Fig1),thencalculatetheconcentrationofthesamplefromtheslopeofthecurveandtheabsorbance.Theprocessislistedbelow:⑴Drawcalibrationcurve:Take1mlstandardsample(81mg/ml)anddiluteitto25ml.Adddifferentfractionof0.2ml,0.4ml,0.6ml,0.8mland1.0mlofsampleto5testtubesmarkedrespectivelyanddistilledwatertillthefinalvolumeis4mlofeachtesttube.AddlittleNa2S2O4·2H2Oasreducedreagent,andmeasureabsorbancein520nmrespectively.Drawthecalibrationcurvewheretheconcentrationisthex-axis,theabsorbanceisthey-axis.Fromthecalibrationcurve,theslopeis1/3.7