Mingan Yu

The measurement of serum cholinesterase activities by an integration strategy with expanded linear ranges and negligible substrate-activation.

OBJECTIVES To measure serum cholinesterase (SCHE) with an integration strategy. DESIGN AND METHODS At 54.0 micromol/L butyrylthiolcholine, SCHE initial rates were calculated with 50.0 micromol/L butyrylthiolcholine and maximal rates via an improved integrated method if substrate consumptions within 5.0 min were over 60%, or were determined by the classical initial rate method. RESULTS The linear range was from 16 to 1560 nkat/L, and SCHE in clinic sera showed negligible substrate-activation. CONCLUSION This strategy was effective.

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 mumol/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 mumol/L NADH with known Michaelis-Menten constant (Km). LDH Km for NADH was (24 plusmn 3) mu mol/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.