分析谷胱甘肽.doc

分析谷胱甘肽.doc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氯2,4二硝基苯(CDNB),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GSH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90μmol/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;谷胱甘肽S转硫酶;1氯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 homogenat