实验十二转氨作用(精)

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，α-氨基酸的氨基与α-酮酸的酮基之间交换，α-氨基酸转变成相应的α-酮酸，α-酮酸变成新的一种α-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α-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α-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，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：
CH
COOH CH 2NH 2CH 2COOH +
+
C
O
COOH CH 3CH
CH 3COOH NH 2
C
COOH CH 2O
CH 2COOH 谷氨酸
丙氨酸
丙酮酸α‐酮戊二酸
【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
⑴ 0.01M pH7.4磷酸缓冲液：0.2MNa 2HPO 4溶液81ml , 0.2MNaH 2P04溶液19ml 混匀，蒸馏水稀释20倍。

⑵ 0.1M 丙氨酸溶液：称取丙氨酸0.891克先溶于少量0.01MpH7.4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 。

⑶ 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 。

⑷ 0.1M 谷氨酸溶液：称取谷氨酸0.735克先溶于少量0．01M pH7.4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 。

⑸ 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 对照管 肝匀浆
0.5
0.5
放入沸水中煮5分钟，
冷却，摇匀
0.1M 丙氨酸溶液0.5 0.5
0.01M α-酮戊二酸溶液0.5 0.5
0.01 M pH7.4 磷酸缓冲液 1.5 1.5
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：
⑴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”。

⑵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2～3次。

⑶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。

⑷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。

⑸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 f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 f 值
与已知的标准的氨基酸R f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 12 Transamination
【Purpose 】
1．Master the principles and the basic technological operation of round paper chromatography. 2．Learn the process of transamination. 【Principle 】
Transamination reactions are catalyzed by transaminases (aminotransferases). In this process the α-amino group is transferred from an α-amino acid to an α-Keto acid ，and the α-amino acid forms an α-Keto acid. In the meantime, the α-Keto acid converts to a new amino acid. Transamination reactions are reversible. Every transamination reaction is catalyzed by a specific transaminase. Transaminases are widespread in each organ of an organism.
In this experiment, liver homogenate is under water bath with L-alanine and pyruvate, while alanine aminotransferase (ALT; also called glutamate-pyruvate transaminase,) that are important in the diagnosis of liver damage catalyzes the transfer of the amino group of alanine to α-ketoglutarate, thus yield pyruvate and glutamate. Using round paper chromatography can evaluate the existence of glutamate and can prove the transamination reaction in the tissue.
CH
COOH
CH 2NH 2
CH 2
COOH +
+
C
O
COOH
CH 3
CH
CH 3COOH
NH 2
C
COOH
CH 2O
CH 2COOH L-Glutamate L-Alanine
Pyruvate α-ketoglutarate
【Materials 】
1. Apparatus
Petri dish; Watch-glass; A piece of chromatography filter paper; Homogenizer; Test tubes; Test tube rack; Constant temperature water boiler; Several glass capillaries; Pipette ; Sprayer; Scissors; Pencil; Ruler. 2．Reagents
⑴ 0.01M phosphoric acid buffer of pH 7.4: Prepare 0.2M Na 2HPO 4 and 0.2M NaH 2PO 4, then mix 81 ml of the former and 19 ml of the latter and dilute 20 times with distilled water.
⑵ 0.1M alanine solution: Weigh 0.891g alanine 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.
⑶ 0.01M α-ketoglutarate solution: Weigh 1.461g α-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 phosphoric acid buffer.
⑷ 0.1M glutamate solution: Weigh 0.735g alanine, 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.
⑸ 0.2% ninhydrin ethanol solvent : Dissolve 0.2g ninhydrin into 100ml of 95% ethanol. ⑹ 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. 2. Transamination reactions:
Get 2 dry tubes, one is determination tube, the other is control tube. Perform according to the following table:
Addition (ml) Determination tube Control tube
Liver homogenate 0.5 0.5
Bath in boiling water for 10
minute and cool, mix up
0.1M alanine solution 0.5 0.5
0.01M α-ketoglutarate solution 0.5 0.5
0.01 M pH7.4 phosphate buffer 1.5 1.5
Mix up and bath in 37℃water for 50 minutes
Bath in boiling water for 5 minutes and cool, mix up
After cooling the tubes, filter with filter paper or 2000 rpm centrifuge for 3～5 minutes. Transfer filtrate or supernatant to the new tubes marked with the same number.
3. Paper chromatography evaluation:
⑴Obtain a sheet of 12 cm diameter round filter paper. Draw two 2 cm vertical lines passing its center. Use the terminal points of the two lines as spot application and mark “determination”,“control” ,“glutamate” ,“alanine” on t he edge of the paper corresponding to each point.
⑵Use 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 is dried, dot the solution again, each spot may be dotted for 2～3 times.
⑶Stab a hole (1mm diameter) through the center of the filter paper using a pin, Get another filter 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 reverse side of the dotting spot.
⑷Add 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. Solvent rises along 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 electric stove.
⑸Development: 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 R f values.
Parameters Determination Glutamate Alanine Control
The distance from the spotting point
to the center of the patches (cm)
The distance from the spotting point
to the edge of the solvent (cm)
R f
Contrast the R f values of the patches of “determination” and“control” with the R f values of the known amino acids, infer what amino acid they are. Explain transamination reactions on these grounds.
【Attentions】
1. Do not touch the chromatography filter paper, or else the fingerprints would be kept.
2. Use pencil to draw lines on the filter paper.
3. Pay much attention to fire when roasting
4. Prevent cross pollution when dot solution with capillary tubes.
【Questions】
1. If control tube has not bath in boiling water enough, what is result?
2. What are the key points of amino acid paper chromatography operation?。