LIAO Juan1, ZHAO Li-na2, LONG Gao-bo2, et al. Integration of kinetic analyses of reaction curve with the classical initial rate method to measure glutathione-S-transferase. [J]. Journal of Chongqing Medical University 36(12):1436-1439(2011)
LIAO Juan1, ZHAO Li-na2, LONG Gao-bo2, et al. Integration of kinetic analyses of reaction curve with the classical initial rate method to measure glutathione-S-transferase. [J]. Journal of Chongqing Medical University 36(12):1436-1439(2011)DOI:
Integration of kinetic analyses of reaction curve with the classical initial rate method to measure glutathione-S-transferase
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.
关键词
谷胱甘肽-S-转硫酶积分速度方程反应曲线联用策略线性范围
Keywords
glutathione-S-transferaseintegrated rate equationreaction curvesintegration strategylinear range