A simplified method for the preparation of 14 C-labelled branched-chain -oxo acids.
A simplified method for the preparation of 14 C-labelled branched-chain -oxo acids.
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一种制备 14 C 标记的支链含氧酸的简化方法。
DOI：
10.1042/bj1260445
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发表时间：
1972
期刊：
影响因子：
--
通讯作者：
H W Goedde
中科院分区：
文献类型：
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作者：
H W Rüdiger;U. Langenbeck;H W Goedde
材料和方法L-[U-14 C]亮氨酸、L-[U-14 C]异亮氨酸、L-[U-14 C]-缬氨酸、L-[1- 14 C]亮氨酸和DL-[1 - 14 C]缬氨酸得自放射化学中心（阿默舍姆，巴克斯，UK），L-[1- 14 C]异亮氨酸来自Calbiochem（洛杉矶，CA，美国）。L-氨基酸氧化酶（EC 1.4. 3.2)和过氧化氢酶（EC 1.11. 1.6)购自Boehringer（曼海姆，德国联邦共和国）。Dowex 50 W（X8; 200-400目）购自Serva（Heidelberg，德国联邦共和国）。所有其它物质，包括硅胶tlc板，均来自Merck（达姆施塔特，德国联邦共和国）。将50 μ Ci部分的冻干氨基酸（比放射性通常为10- 20 mCi/mmol）溶解于两份500 μ l部分的蒸馏水中，并移液至14 ml瓦尔堡容器中。然后加入500 μ l 1 M-tris-HCl缓冲液，pH 7。依次加入7.50 μ l过氧化氢酶溶液和200 μ l L-氨基酸氧化酶溶液，得到总体积为1750.1 μ l。用O2对容器充气1分钟，紧密封闭，并在37 ℃下连续振荡孵育3小时. 1小时后，重复用O2充气。为了从剩余的氨基酸中分离α-含氧酸，将Dowex 50 W（X8）依次用1 M-NaOH、水、1 M-HCl和水洗涤，并填充在10 mm × 70 mm柱中。将整个反应混合物轻轻地施加到柱上，并用蒸馏水进行洗脱，
Materials and methods L-[U-14C] Leucine, L-[U-14C] isoleucine, L-[U-14C]-valine, L-[1-14C] leucine and DL-[l _14C] valine were obtained from The Radiochemical Centre (Amersham, Bucks., UK), and L-[1-14C] isoleucine was from Calbiochem (Los Angeles, Calif., USA). L-Amino acid oxidase (EC 1.4. 3.2) and catalase (EC 1.11. 1.6) were purchased from Boehringer (Mannheim, German Federal Republic). Dowex 50W (X8; 200-400 mesh) was purchased from Serva (Heidelberg, German Federal Republic). All other substances, including the silica-gel tlc plates, were from Merck (Darmstadt, German Federal Republic).A 50, uCi portion of freeze-dried amino acid (specific radioactivity usually 10-2OmCi/mmol) was dissolved in two 500, u1 portions of distilled water and pipetted into a 14ml Warburg vessel. Then 500, ul of 1 M-tris-HCl buffer, pH7. 7, 50, ul of catalase solution and 200, Iu of L-amino acid oxidase solution were added in that sequence, yielding a total volume of 1750, 1. The vessel was gassed for1 min with 02, closed tightly and incubated with continuous shaking for 3 h at 37 C. After 1 h the gassing with 02 was repeated. To separate the a-oxo acid from the remaining amino acid, Dowex 50W (X8) waswashedsuccessively with 1 M-NaOH, water, 1 M-HCl and water, and was packed in a 10mmx 70mm column. The whole reaction mixture was gently applied to the column, and elution was carried out with distilled water, the