As demonstrated in the definition of $M_1$, $M_2$ and $M_3$, kinetic parameters preset as constants for kinetic analysis of GST reaction curve should have strong covariance. Except $K_{iq}$ as an unknown kinetic parameter for optimization, other kinetic parameters are those reported (Kunze, 1997; Pabst, et al, 1974). To optimize $K_{iq}$, two criteria are used. The first is the consistency of predicted $A_m$ at a series of GSH concentrations using data of 6.0-min reaction with that by the equilibrium method after 40 min reaction (GST activity is optimized to complete the reaction within 40 min). The second is the resistance of $V_m$ to reasonable changes in data ranges for analyses. After stepwise optimization, $K_{iq}$ is fixed at 4.0 µmol/L; $A_m$ predicted for GSH from 5.0 µmol/L to 50 µmol/L is consistent with that by the equilibrium method (Zhao, L.N., et al. 2006); the estimation of $V_m$ is resistant to changes of data ranges (Fig. 5). Therefore, $K_{iq}$ is optimized and fixed as a constant at 4.0 µmol/L.
Fig. 6. Response of GSH concentration determined to preset GSH concentrations (the equilibrium method uses data with 6.0 min reaction).
Fig. 7. Response of initial rates to quantities of purified porcine alkaline GST.
Kinetic analysis of GST reaction curve can predict $A_m$ for GSH over 4.0 µmol/L, but there are no sufficient data for analyses at GSH below 3.0 µmol/L; after optimization of GST activity for complete conversion of GSH at 5.0 µmol/L within 6.0 min, reaction curve within 5.0 min for GSH at 5.0 µmol/L can be used for kinetic analysis of reaction curve to predict $A_m$. With the optimized GST activity for reaction within 5.0 min, the linear range for GSH assay is from 1.5 µmol/L to over 90.0 µmol/L by the integration strategy while it is from 4.0