Determination of enzyme substrates with an extended range of linearity

This paper examines the possibility of using a one-component kinetic model for determination of l-amino acids having concentrations near or greater than their Michaelis constants. A commercial nonlinear regression program is used to fit data for absorbance and rate vs. time to the model and to the Michaelis-Menten equation. Coupled l-amino acid oxidase reactions are used to estimate such kinetic parameters as the maximum velocity, Vm, the Michaelis constant, Km, and the forward rate constant, k1. A conventional mixing method and an air-segmented continuous flow (ASCF)/stopped-flow method are applied to monitor the time course of the reaction. The correlation of results obtained between the two methods is studied. The accuracy and precision of the analytical results obtained by the proposed method are reported and compared to those obtained from the Michaelis-Menten model and from double reciprocal plots.