Quantification of reduced glutathione by analyzing glutathine-S-transferase reaction process taking into account of product inhibition / 西安交通大学学报(医学版)

ABSTRACT

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?mol/L to 90 ?mol/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.