Methodological principles in the enzymatic determination of substrates illustrated by the measurement of uric acid.

The enzymatic method of Haeckel for the determination of uric acid in serum has been used to compare three different kinetic approaches with the corresponding equilibrium (end-point) procedure. The kinetic methods were: measurement of the difference in absorbance between two pre-selected fixed instants during the course of the reaction under first-order conditions; measurement of the initial rate of reaction at substrate concentrations sufficiently low for first-order kinetics to apply, and determination of initial rate with substrate concentrations approximating to, or greater than, the Michaelis constant (pseudo-zero order kinetics). In the third method results were obtained by solving the Michaelis-Menten equation. The results emphasise the superiority of the two-point kinetic approach, which is apparent even when the timed absorbance values are obtained from an analogue signal displayed on a recorder chart. Conditions are described for the determination or uric acid in serum according to this principle.