實(shí)驗(yàn)五 細(xì)胞色素C的制備及測(cè)定

實(shí)驗(yàn)五細(xì)胞色素C的制備及測(cè)定【實(shí)驗(yàn)?zāi)康摹?．學(xué)習(xí)細(xì)胞色素C的理化性質(zhì)及其生物學(xué)功能。2．掌握制備細(xì)胞色素C的原理。3．掌握制備細(xì)胞色素C的操作技術(shù)?！緦?shí)驗(yàn)原理】細(xì)胞色素C是呼吸鏈的一個(gè)重要組成成份。是一種含鐵卟啉基團(tuán)的蛋白質(zhì)，在線粒體呼吸鏈上位于細(xì)胞色素b和細(xì)胞色素aa3之間，細(xì)胞色素C的作用是在生物氧化過程中傳遞電子。細(xì)胞色素C分子中含賴氨酸較高，所以等電點(diǎn)偏堿，為pH10.8，分子量為12000～13000道爾頓。它易溶于水及酸性溶液，且較穩(wěn)定，不易變性，組織破碎后，用酸性水溶液即能從細(xì)胞中浸提出來。細(xì)胞色素C分為氧化型和還原型兩種，因?yàn)檫€原型較穩(wěn)定并易于保存，一般都將細(xì)胞色素C制成還原型的，氧化型細(xì)胞色素C在408nm、530nm有最大吸收峰，還原型細(xì)胞色素C的最大吸收峰為415nm、520nm和550nm，這一特性可用于細(xì)胞色素C的含量測(cè)定。由于細(xì)胞色素C在心肌組織和酵母中含量豐富，常以此為材料進(jìn)行分離制備。本實(shí)驗(yàn)以豬心為材料，經(jīng)過酸溶液提取，人造沸石吸附，硫酸銨溶液洗脫，三氯醋酸沉淀等步驟制備細(xì)胞色素C，并測(cè)定其含量?！緦?shí)驗(yàn)材料】1.實(shí)驗(yàn)器材絞肉機(jī)；電磁攪拌器；電動(dòng)攪拌器；離心機(jī)；72l型分光光度計(jì)；玻璃柱(2.5×30cm)；下口瓶；燒杯(2000、1000、500m1)；量筒；移液管；玻璃漏斗和紗布；玻璃棒；透析袋2.實(shí)驗(yàn)試劑⑴2MH2S04溶液；1MNH40H溶液；固體硫酸銨⑵25％硫酸銨溶液:100m1蒸餾水中含25克硫酸銨，約相當(dāng)于25℃時(shí)40％的飽和度⑶0.2％氯化鈉溶液：稱0.2克氯化鈉，用蒸餾水溶解并定容至100ml.⑷BaCl2試劑：稱12克BaCl2,溶于100ml蒸餾水中。⑸20％三氯醋酸溶液(6)人造沸石(60～80目)⑺聯(lián)二亞硫酸鈉(Na2S2O4·2H2O)【實(shí)驗(yàn)操作】1．細(xì)胞色素C的制備⑴材料處理:取新鮮或冰凍豬心，除去脂肪和韌帶，用水洗去積血，將豬心切成小塊，放入絞肉機(jī)絞碎。⑵提取:稱取絞碎豬心肌肉500克，放人2000m1燒杯中，加蒸餾水1000ml，在電動(dòng)攪拌器攪拌下以2MH2S04調(diào)pH至4.0(此時(shí)溶液呈暗紫色)，在室溫下攪拌提取2小時(shí)，在提取過程，使抽提液的pH值保持在4.0左右。在即將提取完畢，停止攪拌之前，以1MNH40H調(diào)pH至6.0，停止攪拌。用八層普通紗布?jí)簲D過濾，收集濾液。濾渣加入750m1蒸餾水，再按上述條件提取1小時(shí)，兩次提取液合并。⑶中和:用1MNH40H調(diào)上述提取液至pH7.2(此時(shí)，等電點(diǎn)接近7.2的一些雜蛋白溶解度小，從溶液中沉淀下來)，靜置30～40分鐘中后過濾，所得濾液準(zhǔn)備通過人造沸石柱進(jìn)行吸附。⑷吸附與洗脫:人造沸石容易吸附細(xì)胞色素C，吸附后能被25％的硫酸銨洗脫下來，利用此特性將細(xì)胞色素C與其它雜蛋白分開。具體操作如下：①人造沸石的預(yù)處理：稱取人造沸石11g，放入500m1燒杯中，加水?dāng)嚢?，用傾瀉法除去12秒鐘內(nèi)不下沉的過細(xì)顆粒。②裝柱：選擇一個(gè)底部帶有濾膜的干凈的玻璃柱(2.5×30cm)，柱下端連接一乳膠管，用夾子夾住，柱中加入蒸餾水至2／3體積，保持柱垂直，然后將己處理好的人造沸石帶水裝填人柱，注意一次裝完，避免柱內(nèi)出現(xiàn)氣泡。③上樣：柱裝好后，打開夾子放水(柱內(nèi)沸石面上應(yīng)保留一薄層水)將準(zhǔn)備好的提取液裝入下口瓶，使其通過人造沸石柱進(jìn)行吸附。柱下端流出液的速度為1.0ml／分鐘。隨著細(xì)胞色素C的被吸附，柱內(nèi)人造沸石逐漸由白色變?yōu)榧t色，流出液應(yīng)為黃色或微紅色。④洗脫：吸附完畢，將紅色人造沸石從柱內(nèi)取出，放入500m1燒杯中，先用自來水，后用蒸餾水?dāng)嚢柘礈熘了?，再?00m10.2％NaCl溶液分三次洗滌沸石，再用蒸餾水洗至水清，按第一次裝柱方法將人造沸石重新裝入柱內(nèi)，用25％硫酸銨溶液洗脫，流速大約2ml/分鐘，收集含有細(xì)胞色素C的紅色洗脫液，當(dāng)洗脫液紅色開始消失時(shí)，即洗脫完畢。人造沸石可再生使用。⑤人造沸石再生：將使用過的沸石，先用自來水洗去硫酸銨，再用0.25M氫氧化鈉和lM氯化鈉混合液洗滌至沸石成白色，前后用蒸餾水反復(fù)洗至pH7～8，即可重新使用。⑸鹽析：為了進(jìn)一步提純細(xì)胞色素C，在上面收集的洗脫液中，加入固體硫酸銨(按每l00m1洗脫液加入20克固體硫酸銨的比例，使溶液硫酸銨的飽和度為45％)邊加邊攪拌，放置30分針后，雜蛋白便從溶液中沉淀析出，而細(xì)胞色素C仍留在溶液中，用濾紙(或離心)除去雜蛋白，即得紅色透亮細(xì)胞色素C溶液。(6)三氯醋酸沉淀：在攪拌情況向所得透亮溶液加入20％三氯醋酸(2.5m1三氯醋酸／100m1細(xì)胞色素C溶液)，細(xì)胞色素C立即沉淀出來（沉淀出來的細(xì)胞色素C屬可逆變性)，立即于3000轉(zhuǎn)／分鐘離心15分鐘，收集沉淀。加入少許蒸餾水，用玻棒攪拌，使沉淀溶解。⑺透析：將沉淀的細(xì)胞色素C溶解于少量的蒸餾水后，裝入透析袋，在500m1燒杯中對(duì)蒸餾水進(jìn)行透析除鹽(電磁攪拌器攪拌)，15分鐘換水—次，換水3至4次后；檢查透析外液SO4是否已被除凈。檢查方法是：取2m1BaCl2溶液于試管中，滴加2至3滴透析外液至試管中，若出現(xiàn)白色沉淀，表示S04未除凈，反之，說明透析完全，將透析液過濾，即得細(xì)胞色素C制品。2.含量測(cè)定所得制品是還原型細(xì)胞色素C水溶液，在波長(zhǎng)520nm處有最大吸收值，根據(jù)這一特性，用721型分光光度計(jì)，先作出一條標(biāo)準(zhǔn)細(xì)胞色素C濃度和對(duì)應(yīng)的光密度值的標(biāo)準(zhǔn)曲線(圖1)，然后根據(jù)測(cè)得的待測(cè)樣品溶液的光密度值就可以由標(biāo)準(zhǔn)曲線的斜率求出待測(cè)樣品的含量。具體操作如下：

⑴標(biāo)準(zhǔn)曲線的繪制：取1毫升標(biāo)準(zhǔn)品(81mg/ml)，稀釋至25ml，從中分別取0.2，0.4，0.6，0.8，1.0ml，分別置于五支試管中，每管補(bǔ)加蒸餾水至4m1，并加少許聯(lián)二亞硫酸鈉作還原劑，然后在520nm處測(cè)得各管的光密度，分別為0.179，0.330，0.520，0.700，0.870。以濃度為橫坐標(biāo)，光密度值為縱坐標(biāo)，作出標(biāo)準(zhǔn)曲線圖，從圖中求得斜率為l／3.71。⑵樣品測(cè)定取1ml樣品，稀釋適當(dāng)倍數(shù)，再加少許聯(lián)二亞硫酸鈉，在波長(zhǎng)520nm處測(cè)定光密度。最后根據(jù)標(biāo)準(zhǔn)曲線的斜率計(jì)算其細(xì)胞色素C的含量?！緦?shí)驗(yàn)結(jié)果】計(jì)算公式：細(xì)胞色素C的含量=3.71×OD×稀釋倍數(shù)×終體積在本實(shí)驗(yàn)中，500克的豬心原料，應(yīng)獲得75mg以上的細(xì)胞色素C的制品。(注：在細(xì)胞色素C的實(shí)際工作中，除了含量測(cè)定以外還要測(cè)定含鐵量即純度的鑒定和活性，后兩項(xiàng)測(cè)定，此處從略)。【注意事項(xiàng)】1．盡可能除掉豬心中的韌帶、脂肪和積血。2．使用離心機(jī)之前，一定要配平。3．透析之前要檢查透析袋。4．在520nm處測(cè)得各管的光密度時(shí)，要加少許聯(lián)二亞硫酸鈉作還原劑?！舅伎碱}】1．制備細(xì)胞色素C通常選取什么動(dòng)物組織?為什么？2．本實(shí)驗(yàn)采用的酸溶液提取，人造沸石吸附，硫酸銨溶液洗脫，三氯醋酸沉淀等步驟制備細(xì)胞色素及含量測(cè)定，各是根據(jù)什么原理?3．請(qǐng)說出其它提取和純化細(xì)胞色素C的方法嗎？請(qǐng)寫出相關(guān)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