胎儿肝脏谷胱甘肽转移酶的纯化和部分性质的研究

Purification and characterization of acidic glutathion S-transferase from human fetal liver

本文采用S_已基谷胱甘肽-sephsrose 6B亲和层析和DE-52纤维素离子交换层析法,从胎儿肝脏中,分离纯化了酸性谷胱甘肤转移酶(GST)。纯化后的GST的比活力为41.39μmol/(min·mg)。纯化倍数为112,回收率为22.2％。它在SDS-聚丙烯酰胺凝胶电泳上,为一条区带,其亚基分子量为23000,在等电聚焦电泳上,也为一条区带,等电点为4.8。以谷胱甘肽(GSH)和1-氟-2,4-二硝基苯(CDNB)为底物,测定了其Km值,对底物GSH和CDNB的Km值分别为0.637mmol/L和1.43mmol/L。

The acidic glutathione S-transferase from human foetal liver was purified approximately 40 fold to homogenity from 105000g by affinity chromatography on S-hexyl glutathine sepharose 6B. This purification procedure resulted in 59％ yield of the original GSTs activity toward 1-chloro-2, 4donitrobezene. Further purification by DE-52 cellulose column chromatography, 112 fold apparently purified enzyme acidic GST was obtained. For the characterization of the GST, SDS-PAGE, kinetic studies and isoelectrofocusing were performed. The Km of the enzyme for reduced GSH was 0.637 mmol/L at fixed concentration of CDNB (1mmol/L), while its Km of CDNB was 1.43 mmol/L at the fixed concentration of 1 mmol/L GSH. The molecular weight of subunit is 23 000. Isoelectrofocusing of the enzyme showed only one band, whose pl was 4.8.