Objective:To establish a method based on analysis of the changes of kinetic parameters to characterize an inhibitor of glutathione-S-transferase(glutathione-S-transferases

GST).Methods:The acidic GST isozyme was purified from the porcine liver via anion-exchange chromatography and affinity chromatography.The reaction of glutathione(glutathione

GSH) and 1-chloro-2

4-dinitrobenzene(1

-chloro-2

4-dinitrobenzene

CDNB) gave S-(2

4-dinitrobenzyl)-glutathione(GS-DNB) as a candidate inhibitor.Michaelis-Menten constant(Km) and maximal reaction rate(Vm) were estimated to determine the inhibition constant(Ki) of GS-DNB.Results:Specific activity of GST was increased by more than 146 times with overall activity yield of about 30%.GST followed random bi-substrate kinetics and had Km of 42 μmol/L for GSH

and Km of 0.86 mmol/L for CDNB.The competitive Ki of GS-DNB was(21±1) μmol/L(n=2) against CDNB

and(17±1) μmol/L(n=2) against GSH.Conclusion:GS-DNB is an effective competitive inhibitor of GST;the estimation of Ki from responses of Km and Vm to inhibitor concentrations can be a conventional method to screen GST inhibitors.