以CDNB为探针测定大鼠肝微粒体中GST活性并优化反应体系
The optimization of the determination of the GST enzyme activity in rat liver microsomes with CDNB as probe
作者:张有金;郭延垒;张艳辉;李文娟;于超;
Author:
收稿日期: 年卷(期)页码:2013,28(03):-259-261
期刊名称:华西药学杂志
Journal Name:WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字:高效液相色谱法;1-氯-2,4-二硝基苯;大鼠肝微粒体;谷胱甘肽硫转移酶;反应条件
Key words:
基金项目:国家科技重大专项(编号:2010ZX09401-306-1-1);;
重庆科技创新能力建设项目(CSTC,编号:2010AA5058)
中文摘要
目的以1-氯-2,4-二硝基苯(CDNB)为探针,优化大鼠肝微粒体中谷胱甘肽硫转移酶(GST)催化反应的条件,为准确测定GST活性提供依据。方法采用Welch Materials Ultimate TM XB C18反相柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(7∶3),流速0.8 mL.min-1,检测波长238 nm。首先于0.05 mg.mL-1蛋白浓度下,孵育10 min,检测CDNB能否被GST催化发生反应,然后分别在CDNB及CDNB+GSH两种反应条件下,比较不同浓度蛋白、孵育时间、底物浓度的反应差异,选出合理反应条件。结果 CDNB在选定色谱条件下实现了快速分离,且无内源性干扰。CDNB在GST催化下发生了反应;在不同浓度蛋白、孵育时间、底物浓度下,两种反应条件下CDNB反应存在明显差异(P
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