联用酶反应过程分析法和初速度法测定谷胱甘肽-S-转硫酶活性

Integration of kinetic analyses of reaction curve with the classical initial rate method to measure glutathione-S-transferase

目的:考察联用谷胱甘肽-S-转硫酶(Glutathione-S-transferase,GST)产物抑制反应过程分析法和初速度法测定GST活性的有效性。方法:阳离子交换层析纯化猪肝碱性GST同工酶。在25℃,用1.0 mmol/L 2,4-二硝基氯苯(1-Chloro-2,4-dinitrobenzene,CDNB)和50.0 mol/L谷胱甘肽(Glutathione,GSH)跟踪产物在340 nm吸收的变化。5.0 min内底物消耗比例低于设定界限时测定初速度;否则,用以反应时间为自变量且包含产物抑制的积分速度方程分析反应过程确定最大反应速度,再换算成优化GSH浓度下的初速度。结果:优化GST产物抑制常数后,只要被分析数据中GSH消耗55%以上,所得最大反应速度对被分析数据范围不敏感。用48 mol/L GSH将最大反应速度换算成初速度,联用策略所得GST活性的线性下限略高于初速度法,而线性上限提高4倍。结论:用积分速度方程分析酶反应过程并优化最大速度换算成初速度所需底物浓度,联用策略测定酶活性线性范围更宽,有望成为测定酶活性的新方法。

Objective：To test the effectiveness of an integration strategy for measuring activities of glutathione-S-transferase（GST） that suffers strong product inhibition.Methods： The alkaline isozyme of GST was purified from porcine liver by ion chromatography.GST reaction curves were monitored by absorbance at 340 nm with 1-chloro-2,4-dinitrobenzene（CDNB） at 1.0 mmol/L and glutathione（GSH） at 50.0 μmol/L.When substrate consumption percentages were below the preset threshold,the classical initial rates were determined;otherwise,the maximal reaction rates were estimated by kinetic analyses of reaction curves using an integrated rate equation that employs the predictor variable of reaction time and considers product inhibition,and were then converted into initial rates at an optimized initial GSH concentration.Results：The maximal reaction rates of GST were resistant to variations in ranges of data as long as the product inhibition constant was optimized and the data under analyses covered the consumption of 55% GSH.By converting the maximal reaction rates into initial rates at 48 μmol/L GSH,the integration strategy had a lower limit of linear response slightly higher than that of the classical initial rates,while an upper limit of linear response that was about four times higher than that of the classical initial rates.Conclusion：By using the integrated rate equation for kinetic analysis of enzyme reaction process and an optimized substrate concentration to convert the maximal rates into initial rates,the integration strategy had wider linear ranges,and can potentially be a desirable universal method to measure enzyme activities.