實(shí)驗(yàn)十二轉(zhuǎn)氨作用

1、實(shí)驗(yàn)十二轉(zhuǎn)氨作用【實(shí)驗(yàn)?zāi)康摹? 通過實(shí)驗(yàn)掌握水平方向?yàn)V紙層析原理和技術(shù)2了解轉(zhuǎn)氨作用過程。【實(shí)驗(yàn)原理】轉(zhuǎn)氨基作用是由轉(zhuǎn)氨酶 （氨基轉(zhuǎn)移酶）催化的，在這個(gè)反應(yīng)中，a -氨基酸的氨基與 a - 酮酸的酮基之間交換，a -氨基酸轉(zhuǎn)變成相應(yīng)的 a -酮酸，a -酮酸變成新的一種 a -氨基酸。 轉(zhuǎn)氨基作用是一種可逆反應(yīng)。每個(gè)轉(zhuǎn)氨基反應(yīng)均由專一的轉(zhuǎn)氨酶所催化，在不同的生物有機(jī)體中均有轉(zhuǎn)氨酶分布。本實(shí)驗(yàn)是將丙氨酸和a -酮戊二酸與肝勻漿一起水浴反應(yīng)，肝中的丙氨酸氨基轉(zhuǎn)移酶（ALT,又稱谷丙轉(zhuǎn)氨酶 GPT含量豐富，該酶可將丙氨酸的氨基轉(zhuǎn)移給 a -酮戊二酸，產(chǎn)生 丙酮酸和谷氨酸。利用圓盤紙層析鑒定谷氨酸的存

2、在， 并且驗(yàn)證組織中的轉(zhuǎn)氨作用。 在肝臟 谷丙轉(zhuǎn)氨酶（GPT）催化的轉(zhuǎn)氨基作用，反應(yīng)方程式如下：COOH1COOHCH2r1CH21CH2CH31CH2CH31谷丙轉(zhuǎn)氨酶I|C= O +1CH NH2 - |CHNH2 +C= O11COOHCOOH1 COOH1COOH-酮戊二酸丙氨酸谷氨酸丙酮酸【實(shí)驗(yàn)材料】1. 實(shí)驗(yàn)器材培養(yǎng)皿；表面皿；濾紙；勻漿器；試管；試管架；恒溫水浴鍋；毛細(xì)管；移液管；噴霧 器；剪刀；鉛筆；格尺。2. 實(shí)驗(yàn)試劑 0.01M pH7.4 磷酸緩沖液：0.2MNHPO溶液 81ml, 0.2MNaH 2P0溶液 19ml 混勻，蒸 餾水稀釋20倍。0.1M丙氨酸溶液：稱取

3、丙氨酸 0.891克先溶于少量磷酸緩沖液中，以 1MNaOH子細(xì)調(diào)節(jié)至pH7.4后，用磷酸鹽緩沖液加至100ml。0.01M a -酮戊二酸溶液：稱取a -酮戊二酸1.461克先溶于少量0.01M pH7.4磷酸緩 沖液中，用1M Na0H仔細(xì)調(diào)節(jié)至pH7.4后，用磷酸鹽緩沖液加至100ml。0.1M谷氨酸溶液：稱取谷氨酸0.735克先溶于少量 0. 01MpH7.4磷酸緩沖液中，以1MNa0H子細(xì)調(diào)節(jié)至pH7.4后，用磷酸緩沖液加至 100ml。0.2 %茚三酮溶液：稱取茚三酮0.2克溶于100ml 95 %乙醇中。（6）層析溶劑：水飽和的苯酚?！緦?shí)驗(yàn)操作】1. 肝勻漿的制備：取新鮮的豬肝5

4、g，加入20m1預(yù)冷0.01M pH7.4磷酸緩沖液，用搗碎機(jī)迅速成勻漿（1萬轉(zhuǎn)大約30秒）。兩人一組進(jìn)行如下的實(shí)驗(yàn)。2. 轉(zhuǎn)氨反應(yīng)：取干燥大試管二支，分別標(biāo)明測(cè)定管與對(duì)照管，按下表進(jìn)行操作：試劑（ml）測(cè)定管對(duì)照管肝勻漿0.50.5放入沸水中煮5分鐘， 冷卻，搖勻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 磷酸緩沖液1.51.5搖勻，放進(jìn)37 C水浴保溫50分鐘沸水浴中煮5分鐘，終止反應(yīng)，取出冷卻后搖勻取出冷卻后，分別用濾紙過濾或2000rpm離心35分鐘，濾液或上清液分別收集到新的干燥小試管中。3. 紙層析： 取直徑12cm圓形濾紙一張

5、，通過圓心作兩條 2cm相互垂直的線，兩個(gè)線的末端作點(diǎn) 樣點(diǎn)，分別標(biāo)定“測(cè)定”、“對(duì)照”、“谷氨酸”、“丙氨酸”。 取4支毛細(xì)管，分別吸取測(cè)定管溶液、0.1M谷氨酸溶液、對(duì)照管溶液、0.1M丙氨酸溶液。在點(diǎn)樣處點(diǎn)樣，注意斑點(diǎn)不可太大，直徑要小于0.3cm。而且每點(diǎn)一滴，吹干后方可再點(diǎn)第二滴，每個(gè)樣品可點(diǎn)23次。在濾紙圓心處打一小孔 （1mm直徑），另取同類濾紙條（0.5 x 2.5cm），下一半剪成須 狀，卷成圓筒，如燈芯，從點(diǎn)樣相反的一側(cè)插入小孔。 將層析溶劑（水飽和酚溶液）放入直徑為35cm的干燥表面皿正中，表面皿置于直徑 10cm培養(yǎng)皿正中，將濾紙放平在培養(yǎng)皿上，燈芯浸入溶劑中，將另一同

6、樣大小培養(yǎng)皿反蓋 上，溶劑沿?zé)粜旧仙綖V紙，再向四周擴(kuò)展，（層析時(shí)間大約 4560分鐘）。溶劑前緣距濾紙邊緣約1cm時(shí)即可取出，用鉛筆劃出溶劑的邊緣，烘箱中干燥之。 顯色：將濾紙放在培養(yǎng)皿上，噴0.2%的茚三酮乙醇溶液，烘箱中干燥，濾紙上會(huì)呈現(xiàn)紫色弧狀條帶?！緦?shí)驗(yàn)結(jié)果】用鉛筆畫出條帶的邊框，測(cè)出表格中的數(shù)值，計(jì)算R值。測(cè)定參數(shù)測(cè)定樣品谷氨酸丙氨酸對(duì)照點(diǎn)樣點(diǎn)到斑紋中心距離 （cm）點(diǎn)樣點(diǎn)到溶劑前沿距離 （cm）R值與已知的標(biāo)準(zhǔn)的氨基酸R進(jìn)行對(duì)比，指出條帶所對(duì)應(yīng)的氨基酸，并根據(jù)結(jié)果解釋轉(zhuǎn)氨作用。【注意事項(xiàng)】1 層析濾紙不可用手觸摸，以免有手印。2 在濾紙上劃線時(shí)只需用鉛筆，不可用其它筆。3 烘烤時(shí)

7、要注意明火。4 .點(diǎn)樣時(shí)毛細(xì)管不能交叉污染?！舅伎碱}】1 如果對(duì)照管在沸水中煮的時(shí)間不夠充分，會(huì)在層析結(jié)果中出現(xiàn)什么現(xiàn)象？2 氨基酸紙上色譜鑒定法操作的關(guān)鍵是什么？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谷丙轉(zhuǎn)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