An integration strategy to estimate the initial rates of enzyme reactions with much expanded linear ranges using uricases as models.

ABSTRACT

A new strategy was proposed to estimate the initial rates of reactions catalyzed by Michaelis-Menten enzymes via integrating the classical initial rate method for low activities with an improved integrated method for high activities. Between these two individual methods, this integration strategy required (a) the consistent linear response slopes, acquired with an optimized preset substrate concentration (PSC) to derive the initial rates from the maximal reaction rates estimated by the improved integrated method; (b) an overlapped region of the initial rates measurable with consistent results, realized with an optimized reaction duration to record reaction curves for analyses by the improved integrated method; (c) a switch cutoff, preset as the instantaneous substrate concentration slightly above that after a given lag time when the enzyme activity was just below the upper limit for the linear response of the classical initial rates. By simulation with uricases at a given initial substrate concentration (S(0)), the optimized PSC was 93% S(0), the optimized reaction duration at S(0) from 0.35-fold to 11.0-fold Michaelis-Menten constant (K(m)) was within 6.0 min and the switch cutoff was available at the given S(0) after 30-s lag time, all of which were combined to produce 300-fold linear ranges. By experimentation with one uricase of K(m) at 6.6 microM and the other uricase of K(m) at 220 microM under optimized conditions, this integration strategy with S(0) at 75 microM produced 100-fold linear ranges. Thus, this integration strategy exhibited much expanded linear ranges and practical efficiency over wide ratios between S(0) and K(m).