DETERMINATION OF ENZYME SUBSTRATES WITH AN EXTENDED RANGE OF LINEARITY

This paper examines the possibility of using a one-component kinetic m odel for determination of L-amino acids having concentrations near or greater than their Michaelis constants. A commercial nonlinear regress ion program is used to fit data for absorbance and rate vs. time to th e model and to the Michaelis-Menten equation. Coupled L-amino acid oxi dase reactions are used to estimate such kinetic parameters as the max imum velocity, V-m the Michaelis constant, K-m, and the forward rate c onstant, k(1). A conventional mixing method and an air-segmented conti nuous flow (ASCF)/stopped-now method are applied to monitor the time c ourse 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 t hose obtained from the Michaelis-Menten model and from double reciproc al plots.