实验十二转氨作用

1、实验十二转氨作用(精)实验十二转氨作用(精)5/5实验十二转氨作用(精)实验十二转氨作用【实验目的】1经过实验掌握水平方向滤纸层析原理和技术2认识转氨作用过程。【实验原理】转氨基作用是由转氨酶（氨基转移酶）催化的，在这个反响中，-氨基酸的氨基与-酮酸的酮基之间互换，-氨基酸转变为相应的-酮酸，-酮酸变为新的一种-氨基酸。转氨基作用是一种可逆反响。每个转氨基反响均由专一的转氨酶所催化，在不一样样的生物有机体中均有转氨酶散布。本实验是将丙氨酸和-酮戊二酸与肝匀浆一起水浴反响，肝中的丙氨酸氨基转移酶ALT，又称谷丙转氨酶GPT）含量丰富，该酶可将丙氨酸的氨基转移给-酮戊二酸，产生丙酮酸和谷氨酸。利用

2、圆盘纸层析判断谷氨酸的存在，并且考证组织中的转氨作用。在肝脏谷丙转氨酶(GPT)催化的转氨基作用，反响方程式以下：COOHCOOHCH2CH2CH2CH3CH2CH3谷丙转氨酶CHNH2CO+CHNH2+COCOOHCOOHCOOHCOOH酮戊二酸丙氨酸谷氨酸丙酮酸【实验资料】1实验器械培育皿；表面皿；滤纸；匀浆器；试管；试管架；恒温水浴锅；毛细管；移液管；喷雾器；剪刀；铅笔；格尺。2实验试剂0.01MpH7.4磷酸缓冲液：0.2MNa2HPO4溶液81ml,0.2MNaH2P04溶液19ml混匀，蒸馏水稀释20倍。0.1M丙氨酸溶液：称取丙氨酸0.891克先溶于少许0.01MpH7.4磷酸缓

3、冲液中，以1MNa0H认真调理至pH7.4后，用磷酸盐缓冲液加至100ml。0.01M-酮戊二酸溶液：称取-酮戊二酸1.461克先溶于少许0.01MpH7.4磷酸缓冲液中，用1MNa0H认真调理至pH7.4后，用磷酸盐缓冲液加至100ml。0.1M谷氨酸溶液：称取谷氨酸0.735克先溶于少许001MpH7.4磷酸缓冲液中，以1MNa0H认真调理至pH7.4后，用磷酸缓冲液加至100ml。0.2茚三酮溶液：称取茚三酮0.2克溶于100ml95乙醇中。层析溶剂：水饱和的苯酚。【实验操作】1肝匀浆的制备：取新鲜的猪肝5g,加入20m1预冷0.01MpH7.4磷酸缓冲液，用捣碎机快速成匀浆(1万转大概

4、30秒)。两人一组进行以下的实验。2转氨反响：取干燥大试管二支，分别注明测定管与比较管，按下表进行操作：试剂(ml)测定管肝匀浆0.5比较管0.5放入开水中煮5分钟，冷却，摇匀0.1M丙氨酸溶液1M-酮戊二酸溶液1MpH7.4磷酸缓冲液1.51.5摇匀，放进37水浴保温50分钟开水浴中煮5分钟，停止反响，拿出冷却后摇匀拿出冷却后，分别用滤纸过滤或2000rpm离心35分钟，滤液或上清液分别采集到新的干燥小试管中。3.纸层析：取直径12cm圆形滤纸一张，经过圆心作两条2cm互相垂直的线，两个线的尾端作点样点，分别标定“测定”、“比较”、“谷氨酸”、“丙氨酸”。

5、取4支毛细管，分别汲取测定管溶液、0.1M谷氨酸溶液、比较管溶液、0.1M丙氨酸溶液。在点样处点样，注意斑点不可以太大，直径要小于0.3cm。并且每点一滴，吹干后方可再点第二滴，每个样品可点23次。在滤纸圆心处打一小孔(1mm直径)，另取同类滤纸条(0.52.5cm)，下一半剪成须状，卷成圆筒，如灯芯，从点样相反的一侧插入小孔。将层析溶剂(水饱和酚溶液)放入直径为35cm的干燥表面皿正中，表面皿置于直径10cm培育皿正中，将滤纸放平在培育皿上，灯芯浸入溶剂中，将另一起样大小培育皿反盖上，溶剂沿灯芯上涨到滤纸，再向周围扩展，（层析时间大概4560分钟）。溶剂前缘距滤纸边沿约1cm时即可拿出，用铅

6、笔划出溶剂的边沿，烘箱中干燥之。显色：将滤纸放在培育皿上，喷0.2%的茚三酮乙醇溶液，烘箱中干燥，滤纸上会呈现紫色弧状条带。【实验结果】用铅笔划出条带的边框，测出表格中的数值，计算Rf值。测定参数测定样品谷氨酸丙氨酸比较点样点到花纹中心距离(cm)点样点到溶剂前沿距离(cm)Rf值与已知的标准的氨基酸Rf进行比较，指出条带所对应的氨基酸，并依据结果解说转氨作用。【注意事项】1层析滤纸不可以用手触摸，免得有手印。2在滤纸上划线时只要用铅笔，不可以用其余笔。3烘烤时要注意明火。4点样时毛细管不可以交叉污染。【思虑题】1假如比较管在开水中煮的时间不够充分，会在层析结果中出现什么现象？2氨基酸纸上色谱

7、判断法操作的重点是什么？Experiment12TransaminationPurpose】1Mastertheprinciplesandthebasictechnologicaloperationofroundpaperchromatography.2Learntheprocessoftransamination.Principle】Transaminationreactionsarecatalyzedbytransaminases(aminotransferases).Inthisprocessthe-aminogroupistransferredfroman-aminoacidtoan-

8、Ketoacid，andthe-aminoacidformsan-Ketoacid.Inthemeantime,the-Ketoacidconvertstoanewaminoacid.Transaminationreactionsarereversible.Everytransaminationreactioniscatalyzedbyaspecifictransaminase.Transaminasesarewidespreadineachorganofanorganism.Inthisexperiment,liverhomogenateisunderwaterbathwithL-alani

9、neandpyruvate,whilealanineaminotransferase(ALT;alsocalledglutamate-pyruvatetransaminase,)thatareimportantinthediagnosisofliverdamagecatalyzesthetransferoftheaminogroupofalanineto-ketoglutarate,thusyieldpyruvateandglutamate.Usingroundpaperchromatographycanevaluatetheexistenceofglutamateandcanprovethe

10、transaminationreactioninthetissue.COOHCOOHCH2CH2CH2CH3GPTCH2CH3+CHNH2谷丙转氨酶CHNH2+COCOCOOHCOOHCOOHCOOH-ketoglutarateL-AlanineL-GlutamatePyruvateMaterials】ApparatusPetridish;Watch-glass;Apieceofchromatographyfilterpaper;Homogenizer;Testtubes;Testtuberack;Constanttemperaturewaterboiler;Severalglasscapil

11、laries;Pipette;Sprayer;Scissors;Pencil;Ruler.2Reagents0.01MphosphoricacidbufferofpH7.4:Prepare0.2MNa2HPO4and0.2MNaH2PO4,thenmix81mloftheformerand19mlofthelatteranddilute20timeswithdistilledwater.0.1Malaninesolution:Weigh0.891galanineandaddtrifle0.01MphosphoricacidbufferofpH7.4.AdjustpHto7.4with1MNaO

12、Handsetthevolumeat100mlwith0.01Mphosphoricacidbuffer.0.01M-ketoglutaratesolution:Weigh1.461g-ketoglutarate,andaddadollopof0.01MphosphoricacidbufferofpH7.4.AdjustpHto7.4with1MNaOHandsetthevolumeat100mlwith0.01Mphosphoricacidbuffer.0.1Mglutamatesolution:Weigh0.735galanine,anddissolveitwithadollopof0.0

13、1MphosphoricacidbufferofpH7.4.AdjustpHto7.4with1MNaOHandsetthevolumeat100mlwith0.01Mphosphoricacidbuffer.0.2%ninhydrinethanolsolvent:Dissolve0.2gninhydrininto100mlof95%ethanol.Chromatographysolvent:Phenolsaturatedbywater.Proceduces】Thepreparationofliverhomogenate:Obtainfreshanimalliver5g,add0.01mol/

14、L(pH7.4)15mlphosphatebufferinicybath,andthentrituratethemtobeliverhomogenateusinghomogenizeratabout10000rpmfor30seconds.2.Transaminationreactions:Get2drytubes,oneisdeterminationtube,theotheriscontroltube.Performaccordingtothefollowingtable:Addition(ml)DeterminationtubeControltubeLiverhomogenate0.50.

15、5Bathinboilingwaterfor10minuteandcool,mixup0.1Malaninesolution1M-ketoglutaratesolution1MpH7.4phosphatebuffer1.51.5Mixupandbathin37waterfor50minutesBathinboilingwaterfor5minutesandcool,mixupAftercoolingthetubes,filterwithfilterpaperor2000rpmcentrifugefor35minutes.Transferfiltrateors

16、upernatanttothenewtubesmarkedwiththesamenumber.3.Paperchromatographyevaluation:Obtainasheetof12cmdiameterroundfilterpaper.Drawtwo2cmverticallinespassingitscenter.Usetheterminalpointsofthetwolinesasspotapplicationandmark“determination,”“control”,“glutamate”,“alanine”onhetedgeofthepapercorrespondingto

17、eachpoint.Use4capillarytubes,absorbonedropofdeterminationsolution,0.1Mglutamatesolution,controlsolution,0.1Malaninesolutionrespectively.Dotthesolutionatthecorrespondingpointsofthelines.Payattentiontothediameterofthespotlessthan0.3cm.Whilethespotisdried,dotthesolutionagain,eachspotmaybedottedfor23tim

18、es.Stabahole(1mmdiameter)throughthecenterofthefilterpaperusingapin,Getanotherfilterpaperstrip(0.52.5cm).Rollitintoacylinderandtwistittightlyasalampwick,insertitintotheholefromthereversesideofthedottingspot.Addabout1mlchromatographysolventtoa5cmdiameterwatch-glassplacedina10cmdiameterPetridish.Putfil

19、terpaperflatlyonthePetridishinordertosoakthelampwickinthechromatographysolvent.CoverthePetridishwithanotheroneofthesamesize.Solventrisesalongthelampwicktothefilterpaperanddiffusesinacircle(chromatographytimeisapproximately4560minutes).Allowthesolventtodiffusetoabout1cmdistancefromtheedgeofthefilerpa

20、per.RemoveitfromthePetridish.Drawtheedgeofthesolventwithapencil.Dryitonanelectricstove.Development:PutfilterpaperflatlyonthePetridish.Spray0.2%ninhydrinethanolsolvent.Dryitontheelectricstove.Purplearcpatchesthenappearonthefilterpaper.Results】Drawtheoutlineofthepatcheswithapencil.Accordingtothefollowingtable,recordrelevantdata.CalculatetheRf