分析谷胱甘肽-S-转硫酶的产物抑制反应过程测定还原型谷胱甘肽

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)终浓度为1.0 mmol/L,监测GST催化还原型谷胱甘肽(GSH)与CDNB结合产物在340 nm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,下限接近2.0μmol/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。

Quantification of reduced glutathione by analyzing glutathine-S-transferase reaction process taking into account of product inhibition

Objective To investigate the reliability of the integrated method for kinetic assay of substrate in the presence of product inhibition using glutathione-S-transferase(GST) as model.Methods Purified GST from pig liver was used to catalyze the conjugation of reduced glutathione(GSH) to 1-chloro-2,4-dinitrobenzene(CDNB)(final concentration at 1.0mmol/L),and reaction curve was monitored by product absorbance at 340nm.Maximal product absorbance after the completion 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 kinetic substrate assay was also resistant to common source of errors.The recovery for extra GSH in rat liver homogenate was above 98% with linear response ranged from 2.0μmol/L to 90 μmol/L.The concentration of GSH in rat liver was consistent to previous reports.Conclusion The integrated method is valid for kinetic assay of substrate when there is product inhibition,and it exhibits some universality as a kinetic method for enzymatic analysis with obvious advantages.