测定谷胱甘肽-S-转硫酶动力学参数的变化表征其抑制剂

Characterization of an inhibitor on glutathione-S-tranferase by analyzing the changes of kinetic parameters

目的:测定谷胱甘肽-S-转移酶(Glutathione-S-transferases,GST)动力学参数变化,建立表征其抑制剂的方法。方法:从猪肝经阴离子交换层析和亲和层析制备GST酸性同工酶,用还原型谷胱甘肽(Glutathione,GSH)和1-氯-2,4-二硝基苯(1,-chloro-2,4-dinitrobenzene,CDNB)合成S-(2,4-二硝基苯基)-谷胱甘肽(GS-DNB)为候选抑制剂,以GSH与CDNB为底物测定GST在GS-DNB作用下的米氏常数(Km)和最大反应速度(Vm),从而确定GS-DNB对GST的抑制常数(Ki)。结果:此GST被纯化146倍以上,活性总收率近30%。该GST对GSH和CDNB的Km分别为42μmol/L和0.86 mmol/L,属于随机双底物动力学模型。GS-DNB对CDNB竞争性Ki为(21±1)μmol/L(n=2);对GSH竞争性Ki为(17±1)μmol/L(n=2)。结论:产物GS-DNB是GST的高亲和力竞争性抑制剂;测定GST动力学参数随抑制剂浓度的变化可筛选其抑制剂。

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.