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， - 氨基酸的氨基与 - 酮酸的酮基之间交换， - 氨基酸转变成相应的 - 酮酸， - 酮酸变成新的一种 - 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 - 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 - 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在

2、， 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：COOHCOOHCH2CH2CH2CH3CH2CH 3谷丙转氨酶CH NH2C O +CHNH2 +C OCOOHCOOHCOOHCOOH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：0.2MNa2HPO4溶液 81ml , 0.2MNaH 2P04 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 克先溶于少量0

3、.01MpH7.4 磷酸缓冲液中，以1MNa0H仔细调节至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001MpH7.4 磷酸缓冲液中，以1MNa0H仔细调节至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

4、 5g, 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)测定管肝匀浆0.5对照管0.5放入沸水中煮5 分钟，冷却，摇匀0.1M 丙氨酸溶液0.50.50.01M - 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 3 5 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：

5、 取直径 12cm圆形滤纸一张， 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 × 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3 5cm 的干燥表面皿正中， 表面皿置于直

6、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45 60 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Rf 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)Rf 值与已知的标准的氨基酸 Rf 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

7、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？Experiment 12Transamination【 Purpose】1 Master the principles and the basic technological operation of round paper chromatography.2 Learn the process of transamination.【 Principle 】Trans

8、amination reactions are catalyzed by transaminases (aminotransferases). In this processthe -amino group is transferred from an -amino acid to an -Keto acid ， and the -amino acid forms an -Keto acid. In the meantime, the -Keto acid converts to a new amino acid. Transamination reactions are reversible

9、. Every transamination reaction is catalyzed by a specifictransaminase. Transaminases are widespread in each organ of an organism.In this experiment, liver homogenate is under water bath with L-alanine and pyruvate, whilealanine aminotransferase (ALT; also called glutamate-pyruvate transaminase,) th

10、at are importantin the diagnosis of liverdamagecatalyzes the transferof the amino group of alanine to-ketoglutarate, thus yield pyruvate and glutamate. Usinground paperchromatography canevaluate the existence of glutamate and can prove the transamination reaction in the tissue.COOHCOOHCH 2CH 2CH 2CH

11、3GPTCH 2CH3+CHNH2谷丙转氨酶+C OCH NH2C OCOOHCOOHCOOHCOOH-ketoglutarateL-AlanineL-GlutamatePyruvate【 Materials】1. ApparatusPetri dish; Watch-glass; A piece of chromatography filter paper; Homogenizer; Test tubes; Test tube rack; Constant temperature water boiler; Several glass capillaries; Pipette ; Spray

12、er; Scissors; Pencil; 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 alanine solution: Weigh 0.891g alanine and add trifle 0.01M phosphoric acid buff

13、er ofpH 7.4. Adjust pH to 7.4 with 1M NaOH and set the volume at 100ml with 0.01M phosphoric acid buffer. 0.01M -ketoglutarate solution: Weigh 1.461g -ketoglutarate, and add 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 phosp

14、horic acid buffer. 0.1M glutamate solution: Weigh 0.735g alanine, and dissolve it with a dollop of 0.01Mphosphoric 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% ninhydrin ethanol solvent : Dissolve 0.2g ninhydrin into 100ml o

15、f 95% ethanol. Chromatography solvent: Phenol saturated by water.【 Proceduces】1. 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 liver homogenate using homogenizer at about 10000rpm for 30seconds

16、.2. Transamination reactions:Get 2 dry tubes, one is determination tube, the other is control tube. Perform according to thefollowing table:Addition (ml)Determination tubeControl tubeLiver homogenate0.50.5Bath inboiling water for 10minute and cool, mix up0.1M alanine solution0.50.50.01M -ketoglutara

17、te solution0.50.50.01 M pH7.4 phosphate buffer1.51.5Mix up and bath in 37 water for 50 minutesBath in boiling water for 5 minutes and cool, mix upAftercoolingthe tubes, filterwith filterpaper or 2000 rpm centrifuge for 3 5 minutes.Transfer filtrate or supernatant to the new tubes marked with the sam

18、e number.3. Paper chromatography evaluation: Obtain a sheet of 12 cm diameter round filter paper. Draw two 2 cm vertical lines passing itscenter. Use the terminal points of the two lines as spot application and mark “ determination,”“control ”,“ glutamate ”,“alanine ” onhet edge of the paper corresp

19、onding to each point. Use 4 capillary tubes, absorb one drop of determination solution, 0.1M glutamate solution, control solution, 0.1M alanine solution respectively. Dot the solution at the corresponding points of the lines. Pay attention to the diameter of the spot less than 0.3 cm. While the spot

20、 is dried, dotthe 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 anotherfilter paper strip (0.5 × 2.5cm). Roll it into a cylinder and twist it tightly as a lampwick, insert it into the hole from the rever

21、se side of the dotting spot. Add about 1 ml chromatography solvent to a 5 cm diameter watch-glass placed in a 10 cm diameter Petri dish. Put filter paper flatly on the Petri dish in order to soak the lampwick in the chromatography solvent. Cover the Petri dish with another one of the same size. Solv

22、ent risesalong the lampwick to the filter paper and diffuses in a circle ( chromatography time is approximately 45 60 minutes).Allow the solvent to diffuse to about 1cm distance from the edge of the filer paper. Remove it from the Petri dish. Draw the edge of the solvent with a pencil. Dry it on an

23、electric stove. Development: Put filter paper flatly on the Petri dish. Spray 0.2% ninhydrin ethanol solvent.Dry it on the electric stove. Purple arc patches then appear on the filter paper.【 Results】Draw the outline of the patches with a pencil. According to the following table, record relevant data. Calculate the Rf values.ParametersDeterminati