文档介绍：1 分析谷胱甘肽作者:赵利娜,陶佳,赵运胜,廖飞【关键词】积分法; 动力学酶法分析; 还原型谷胱甘肽; 谷胱甘肽摘要:目的以谷胱甘肽 S 转硫酶(GST) 为模型,探讨有产物抑制时用积分法分析酶反应过程测定底物量的可靠性。方法从猪肝制备 GST 。1*** 2,4 二硝基苯(CDNB) 终浓度为 ,监测 GST 催化还原型谷胱甘肽(GSH) 与 CDNB 结合产物在 340nm 吸收变化。用积分法预测反应终点的产物吸收。结果优化产物抑制常数可使此积分法所得反应终点产物吸收对剩余底物变化不敏感。此积分法测定 GS H 对常见干扰有抗性。此法测定大鼠肝匀浆中外加 GSH 回收率大于 98% ,线性响应上限接近 90μ mol/L ,下限接近 μ mol/L ,所得大鼠肝 GSH 含量与文献报道一致。结论有产物抑制时用积分法也可定量底物; 用此积分法进行动力学酶法分析具有一定普遍性,而且有明显优势。关键词: 积分法; 动力学酶法分析; 还原型谷胱甘肽; 2 谷胱甘肽 S 转硫酶; 1*** 2,4 二硝基苯 Quantification of reduced glutathione by analyzing glutathineStransferasereaction process taking into account of product inhibition) ABSTRACT: Objective To investigate the reliability of the integrated method for ic assay of substrate in the presence of product inhibition using glutathioneStransferase(GST) as model. Methods Purified GST from pig liver was used to catalyze the conjugation of reduced glutathione (GSH) to 1chloro2,4dinitrobenzene (CDNB) (final concentration at ), and reaction curve was monitored by product absorbance at 340nm. Maximal product absorbance after pletion of reaction was predicted by the integrated method. Results The optimization of product inhibition constant conferred to resistance to the variation of residual substrate concentration on the estimation of maximal product absorbance. This integrated method for ic substrate assay was also resistant mon source of errors. The recovery for extra GSH in rat liver homogenate was above 98% with linear response ranged from μ mol/L to 90μ mol/L. The 3 concentration of GSH in rat liver was consistent to previous reports. Conclusion The integrated method is valid for ic assay of substrate when there is product inhibition, and it exhibits some universality asa ic method for enzymatic analysis with obvious advantages. KEY WORDS:integrated method; ic enzymatic analysis; reduced glutathione; glutathioneStransferase; 1chloro2,4dinitrobenzene 还原型谷胱甘肽(glutathione, GSH) 有重要生理功能[1] 。用 Ellman 试剂(2, 2双(4 硝基 2 羧基) 二硫化物, DTNB) 反应或高效液相层析(HPLC