高效液相层析法纯化人胎盘谷胱甘肽硫转移酶

足月分娩的新鲜胎盘组织制成匀浆后,经高速离心、超速离心,谷胱甘肽(GSH)Sepharose 6B亲合层析,Amicon pM-10膜超过滤及高效液相层析,最终经SDS-PAGE鉴定,结果呈现单一亚基区带,其亚基分子量为25000。 根据我们现有高效液相设备条件,用ODS柱代替RadulovicL等报道的特异阴离子柱,用磷酸盐洗脱液代替含谷胱甘肽、二硫苏糖醇及氯化钾的梯度洗脱液,从人胎盘组织成功地制备了谷胱甘肽硫转移酶(GST)纯酶,全过程在15min内完成,保留时间及主峰面积的重复性均较理想,7次实验结果的变异系数为0.2％,最终纯化578.9倍。本研究为各种形式GST的纯化制备提供了一个新的、重复性好、分辨率高及回收理想的简易方法。

A Rapid High Performance Liquid Chromatography Method for the Purification of Glutathione S-transferase of Human Placenta

A rapid high performance liquid chromatography (HPLC) procedure is detailed for the purification of glutathione S-transferase of human placenta.The method is based on homogenization and an affinity chromatography column prepared by coupling glutathione to epoxy-activated sepharose and HPLC.The procedure is rapid and resolution of the various forms in 15min.The major form represented about 95.6% of the total recovered activity and exhibited a specific activity of 11.0 Hmol/L/min/mg protein with a purification of 578.9 fold.SDS-PAGE of the major form revealed the presence of a single band.An apparent subunit Mr of 25 000 was calculated.This procedure offers a convenient method for obtaining purified transf-erase in good yields.