昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ，ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨，经１００００ｇ和１０００００ｇ分级离心；取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白；然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４），四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４），铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质，通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明，采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰，在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带，分子量为２４ｋＤ，２４．５ｋＤ左右；Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰，呈现出一条带，分子量为２４ｋＤ左右；ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰，呈现出一条带，分子量为２３ｋＤ左右

A New Method of Separating Glutathione S Transferses from Insects

The glutahione S transferases (GST)are the isoenzymes of multiple physiological role.It is introducted that glutathione S transferases were separated from Galleria mellonella extract solution.First step,the freezing Glleria mellonella was homogenated in the phosphate buffer(pH7 4) and centrifuged at 10 000 g and 100 000 g.A part of proteins and impurities in the supernatant were removed by QAE Sephadex A 25 chromatography.Second step,GST was separated by GSH QT 4 and BSP QT 4 affinity chromatography,Cu 2+ QT 6 metal chelatting chromatography and PBE 94 Sepharose chromatofocusing.The biological activity of purified GST was assayed by CDNB and DCNB as substrate,and the molecular weight were determined by SDS PAGE.The electrophoresis results showed that molecular weight were 24 kD,24 5 kD by GSH QT 4 affinity chromatography,24 kD by Cu 2+ QT 6 metal chelatting chromatography,23 kD in the peakⅠ,23 5 kD,23 kD in the peakⅡ and 23 5 kD,22 5 kD,18 kD,16 kD in the peakⅢ by PBE 94 Sepharose chromatofocusing.