实验十二转氨作用

1、实验十二转氨作用【实验目的】1 通过实验掌握水平方向滤纸层析原理和技术2了解转氨作用过程。【实验原理】转氨基作用是由转氨酶 （氨基转移酶）催化的，在这个反应中，a -氨基酸的氨基与 a - 酮酸的酮基之间交换，a -氨基酸转变成相应的 a -酮酸，a -酮酸变成新的一种 a -氨基酸。 转氨基作用是一种可逆反应。每个转氨基反应均由专一的转氨酶所催化，在不同的生物有机体中均有转氨酶分布。本实验是将丙氨酸和a -酮戊二酸与肝匀浆一起水浴反应，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 a -酮戊二酸，产生 丙酮酸和谷氨酸。利用圆盘纸层析鉴定谷氨酸的存

2、在， 并且验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，反应方程式如下：COOH1COOHCH2r1CH21CH2CH31CH2CH31谷丙转氨酶I|C= O +1CH NH2 - |CHNH2 +C= O11COOHCOOH1 COOH1COOH-酮戊二酸丙氨酸谷氨酸丙酮酸【实验材料】1. 实验器材培养皿；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 0.01M pH7.4 磷酸缓冲液：0.2MNHPO溶液 81ml, 0.2MNaH 2P0溶液 19ml 混匀，蒸 馏水稀释20倍。0.1M丙氨酸溶液：称取

3、丙氨酸 0.891克先溶于少量磷酸缓冲液中，以 1MNaOH子细调节至pH7.4后，用磷酸盐缓冲液加至100ml。0.01M a -酮戊二酸溶液：称取a -酮戊二酸1.461克先溶于少量0.01M pH7.4磷酸缓 冲液中，用1M Na0H仔细调节至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克先溶于少量 0. 01MpH7.4磷酸缓冲液中，以1MNa0H子细调节至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100ml 95 %乙醇中。（6）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5

4、g，加入20m1预冷0.01M pH7.4磷酸缓冲液，用捣碎机迅速成匀浆（1万转大约30秒）。两人一组进行如下的实验。2. 转氨反应：取干燥大试管二支，分别标明测定管与对照管，按下表进行操作：试剂（ml）测定管对照管肝匀浆0.50.5放入沸水中煮5分钟， 冷却，摇匀0.1M丙氨酸溶液0.50.50.01M a -酮戊二酸溶液0.50.50.01 M pH7.4 磷酸缓冲液1.51.5摇匀，放进37 C水浴保温50分钟沸水浴中煮5分钟，终止反应，取出冷却后摇匀取出冷却后，分别用滤纸过滤或2000rpm离心35分钟，滤液或上清液分别收集到新的干燥小试管中。3. 纸层析： 取直径12cm圆形滤纸一张

5、，通过圆心作两条 2cm相互垂直的线，两个线的末端作点 样点，分别标定“测定”、“对照”、“谷氨酸”、“丙氨酸”。 取4支毛细管，分别吸取测定管溶液、0.1M谷氨酸溶液、对照管溶液、0.1M丙氨酸溶液。在点样处点样，注意斑点不可太大，直径要小于0.3cm。而且每点一滴，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（0.5 x 2.5cm），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培养皿正中，将滤纸放平在培养皿上，灯芯浸入溶剂中，将另一同

6、样大小培养皿反盖 上，溶剂沿灯芯上升到滤纸，再向四周扩展，（层析时间大约 4560分钟）。溶剂前缘距滤纸边缘约1cm时即可取出，用铅笔划出溶剂的边缘，烘箱中干燥之。 显色：将滤纸放在培养皿上，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】用铅笔画出条带的边框，测出表格中的数值，计算R值。测定参数测定样品谷氨酸丙氨酸对照点样点到斑纹中心距离 （cm）点样点到溶剂前沿距离 （cm）R值与已知的标准的氨基酸R进行对比，指出条带所对应的氨基酸，并根据结果解释转氨作用。【注意事项】1 层析滤纸不可用手触摸，以免有手印。2 在滤纸上划线时只需用铅笔，不可用其它笔。3 烘烤时

7、要注意明火。4 .点样时毛细管不能交叉污染。【思考题】1 如果对照管在沸水中煮的时间不够充分，会在层析结果中出现什么现象？2 氨基酸纸上色谱鉴定法操作的关键是什么？Experime nt 12Tran sami natio n【Purpose1. Master the prin ciples and the basic tech no logical operati on of round paper chromatography.2. Learn the process of transamination.【Principle Tran sami natio n react ions are

8、 catalyzed by tran sam in ases (ami notra nsferases). In this process the aamino group is transferred from an aamino acid to an aKeto acid , and the aamino acid forms an aKeto acid. In the mean time, the aKeto acid con verts to a new amino acid. Tran sam in ati on reacti ons are reversible. Every tr

9、an sam in ati on react ion is catalyzed by a specific tran sam in ase. Tran sam in ases are widespread in each orga n of an orga ni sm.In this experiment, liver homogenate is under water bath with L-alanine and pyruvate, while ala nine aminotran sferase (ALT; also called glutamate-pyruvate tran sam

10、in ase,) that are importa nt in the diag no sis of liver damage catalyzes the tran sfer of the ami no group of ala nine to aketoglutarate, thus yield pyruvate and glutamate. Using round paper chromatography can evaluate the existe nee of glutamate and can prove the tran sam in atio n react ion in th

11、e tissue.COOH |COOH1CH2lCH21CH2|CH31谷丙转氨酶1CH2|CH3|c = o +CHNH2 -|CHNH2+1C 二 OCOOHCOOH1COOHCOOHaketoglutarateL-Ala nineL-GlutamatePyruvate【Materials 1. ApparatusPetri dish; Watch-glass; A piece of chromatography filter paper; Homoge ni zer; Test tubes; Test tube rack; Constant temperature water boile

12、r; Several glass capillaries; Pipette ; Sprayer; Scissors; Pen cil; Ruler.2. Reagents 0.01M phosphoric acid buffer of pH 7.4: Prepare 0.2M Na 2HPO4 and 0.2M NaH 2PO4, then mix 81 ml of the former and 19 ml of the latter and dilute 20 times with distilled water. 0.1M ala nine soluti on: Weigh 0.891g

13、ala nine and add trifle 0.01M phosphoric acid buffer of pH 7.4. Adjust pH to 7.4 with 1M NaOH and set the volume at 100ml with 0.01M phosphoric acid buffer. 0.01M aketoglutarate solution: Weigh 1.461ga-ketoglutarate, and add a dollop of 0.01Mphosphoric acid buffer of pH 7.4. Adjust pH to 7.4 with 1M

14、 NaOH and set the volume at 100ml with 0.01M phosphoric acid buffer.(4) 0.1M glutamate solutio n: Weigh 0.735g ala nine, and dissolve it with a dollop of 0.01M phosphoric acid buffer of pH 7.4. Adjust pH to 7.4 with 1M NaOH and set the volume at 100ml with 0.01M phosphoric acid buffer. 0.2% nin hydr

15、in etha nol solve nt: Dissolve 0.2g nin hydrin into 100ml of 95% etha nol.(6) Chromatography solve nt: Phenol saturated by water.【ProceduceS1. The preparation of liver homogenate:Obtain fresh animal liver 5g, add 0.01mol/L (pH7.4) 15ml phosphate buffer in icy bath, and then triturate them to be live

16、r homoge nate using homoge ni zer at about 10000rpm for 30sec on ds.2. Transamination reactions:Get 2 dry tubes, one is determ in ati on tube, the other is con trol tube. Perform accord ing to thefollowi ng table:Additio n (ml)Determ in ati on tubeControl tubeLiver homoge nate0.50.5Bath in boiling w

17、ater for 10minute and cool, mix up0.1M ala nine solutio n0.50.50.01M a-ketoglutarate solution0.50.50.01 M pH7.4 phosphate buffer1.51.5Mix up and bath in 37 C water for 50 minutesBath in boili ng water for 5 minu tes and cool, mix upAfter cooling the tubes, filter with filter paper or 2000 rpm centri

18、fuge for 35 minutes. Tran sfer filtrate or super nata nt to the new tubes marked with the same nu mber.3. Paper chromatography evaluation:(1) Obtain a sheet of 12 cm diameter round filter paper. Draw two 2 cm vertical lines passing its cen ter. Use the termi nal poi nts of the two lines as spot appl

19、icati on and mark “ determ in atio n,“ con trol ” “ glutamate ”，“ ala nine ” ortne edge of the paper corresp onding to each point.(2) Use 4 capillary tubes, absorb one drop of determ in ati on solutio n, 0.1M glutamate soluti on, con trol soluti on, 0.1M ala nine soluti on respectively. Dot the solu

20、ti on at the corresp onding points of the lines. Pay attention to the diameter of the spot less than 0.3 cm. While the spot is dried, dot the solution again, each spot may be dotted for 2 3 times.)Stab a hole (1mm diameter) through the center of the filter paper using a pin, Get another filter paper

21、 strip (0.5 x 2.5cm). Roll it into a cylinder and twist it tightly as a lampwick, insert it into the hole from the reverse side of the dotting spot.(4) Add about 1 ml chromatography solve nt to a 5 cm diameter watch-glass placed in a 10 cm diameter Petri dish. Put filter paper flatly on the Petri di

22、sh in order to soak the lampwick in the chromatography solve nt. Cover the Petri dish with ano ther one of the same size. Solvent rises along the lampwick to the filter paper and diffuses in a circle ( chromatography time is approximately 45 60 minu tes).Allow the solve nt to diffuse to about 1cm di

23、sta nee from the edgeof the filer paper. Remove it from the Petri dish. Draw the edge of the solve nt with a pen cil. Dry it on an electric stove.(5) Developme nt: Put filter paper flatly on the Petri dish. Spray 0.2% nin hydri n etha nol solve nt. Dry it on the electric stove. Purple arc patches then appear on the filter paper.【ResultsDraw the outl ine of the patches with a pen cil. Accord ing to the followi ng table, record releva nt data. Calculate the Rf values.ParametersDeterm in ati onGlutamateAla