Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twe...Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the other's and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation In [Am/(Am -Ai)] + Ai/ ( ξ× Km ) = ( Vm/Km )×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the concentration of one substrate twenty-fold above the other's, this integrated method was reliable to measure the activity of enzyme on two substrates , and substrate concentration of the lower one close to its apparent Km was able to be used.展开更多
基金National Natural Science Foundation of China (No.30200266)
文摘Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the other's and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation In [Am/(Am -Ai)] + Ai/ ( ξ× Km ) = ( Vm/Km )×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the concentration of one substrate twenty-fold above the other's, this integrated method was reliable to measure the activity of enzyme on two substrates , and substrate concentration of the lower one close to its apparent Km was able to be used.
