镉诱导兔肝金属硫蛋白的分离纯化

Isolation and Purification of Hepatic Metallothionein from Rabbit

家兔经皮下连续注射CdCl_2四周,停药两天后处死动物,取兔肝匀奖,经热处理、离心后除去较多杂蛋白。上清经SephadexG75柱层析。收集液在6.8～8.5％柱体积时出现250nm高吸收峰,ε250/280为3.0。金属硫蛋白得率为1.0mg/g肝。Cd、Zn含量与250nm紫外吸收峰一致。Cd∶Zn=4.5∶1,Pr∶Cd(Mol)=1∶4.4。样品经SephadexG50层析分离,分别于3.1～5.7/10和6.2～7.9/10柱体积时出现250nm高吸收峰,Cd含量与吸收峰一致。峰Iε250/280为1.5,Pr∶Cd(Mol)=1∶5.0;峰Ⅱε250/280为12.0,Pr∶Cd(Mol)=1∶2.6。Cd回收率为82％,蛋白回收率为80％。聚丙烯酰胺凝胶电泳结果与Sigma公司产品一致。中毒剂量的Cd-ZnMT皮下注射大鼠有明显肾毒性。

Rabbits were given CdCl_2 subcutaneously for 4 weeks and then untreated for 2 weeks. Upon sacrifice, the livers were removed and homogenized. After centrifugation the supernatant was heated,the precipitate was removed by centrifugation again. The second supernatant was applied to a Sephadex G75 column for the primary purification. The metallothionein fractions, which had an 250/280 ratio of only 3 and Cd/Zn ratio of 4. 5,Pr/Cd ratio (Mol) of 1/4. 4, were collected and concentrated. After it was applied to a Sephadex G50 column,MT-Ⅰ and MT-Ⅱ fractions were finally purificated. Both MT-Ⅰ and MT-Ⅱ had an 250/280 ratio of 1. 5 and 12. 0,Pr/Cd(Mol)was 1/5. 0 and 1/2. 6 respectively. The recoveries of protein and Cd were 82% and 80% respectively. The result of polyacrylamid gel electrophoresis was showed no difference with metallothionein suppplied by Sigma. It tprovided an evidence that acute nephrotoxicity was caused by injection of renal toxic dose of Cd、ZnMT subcutaneously in rats.