The Measurement of the Activity of Rabbit Muscle Lactic Dehydrogenase by Integrating the Classical Initial Rate Method with an Integrated Method
A new strategy to measure enzyme activity by integrating the classical initial rate method for low activities with an integrated method for high activities was investigated using rabbit muscle lactic dehydrogenase (LDH) as the model. The absorbance at 340 nm was recorded at 1 s interval for LDH reaction at 1.0 mmol/L pyruvate and 52 μmol/L NADH in 50 mmol/L sodium phosphate buffer (pH 7.2). At low enzyme activities, the classical initial rate was determined from the reaction data within the initial rate reaction stage while at high activities the maximum reaction rate (Vm) from the integrated method was converted to the calculated initial rate using Michaelis-Menten kinetics at 48 μmol/L NADH with known Michaelis-Menten constant (Km). LDH Km for NADH was (24 ± 3) μmol/L (n=5) by the integrated method, with which the data of substrate consumption > 50％ gave reliable Vm. The range of linear response were about 15-fold by either method alone. The detection limit of the calculated initial rate with data collected within 4.0 min was far below the upper limit of linear response by the classical initial rate method. There were consistent results to enzyme activities within the overlapped range measurable by the two individual methods. Moreover, the response of the calculated initial rate to the amount of LDH exhibited a slope consistent to that by the classical initial rate method alone at low activities. Under this condition, the integration of the two methods together yielded the upper limit higher than 50-fold of the lower limit for linear response. These results supported that this integration strategy was feasible for enzyme activity assay with an expanded linear range and practical efficiency at moderate substrate concentration.
Enzyme activity assay Lactic dehydrogenase The classical initial rate method The integrated method The integration strategy
Zhenli CHENG Hong CHEN Yunsheng ZHAO Xiaolan YANG Wei LU Hong LIAO Mingan YU Fei LIAO
Chongqing Key Laboratory of Biochemistry & Molecular Pharmacology, College of Pharmaceutical Sciences, Chongqing Medical University, Chongqing 400016, the Peoples Republic of China
国际会议
上海
英文
1209-1212
2008-05-16（万方平台首次上网日期，不代表论文的发表时间）