µmol/L to over 90.0 µmol/L by kinetic analysis of reaction curve alone (Fig. 6, unpublished). By the equilibrium method alone for reaction within 5.0 min, the assay of 80.0 µmol/L GSH requires GST activity that is 50 folds higher due to the inhibition of GST by the accumulated product. Therefore, the integration strategy for GSH assay is obviously advantageous.
The integration strategy for measuring GST initial rates is tested. For convenience, $S_0$ of the final GSH is fixed at 50 µmol/L and the duration to monitor reaction curve is optimized. After the analyses of reaction curves recorded within 10 min, it is found that reaction for 6.0 min is sufficient to provide the required overlapped region of GST activities measurable by both methods. By using $K_{iq}$ fixed at 4.0 µmol/L as a constant, the reaction duration of 6.0 min and PSC at 48 µmol/L to convert $V_m$ to initial rates, the integration strategy gives a linear range from 2.0 U/L to 60 U/L; kinetic analysis of reaction curve alone gives the linear range from 5.0 U/L to 60 U/L while the classical initial rate method alone gives a linear range from 1.0 U/L to 5.0 U/L (Fig. 7, unpublished). Clearly, with enzyme suffering strong product inhibition, the integration strategy for enzyme initial rate assay is advantageous.
2.5.3 Alcohol dehydrogenase reaction
ADH is widely used for serum ethanol assay. ADH kinetics is sophisticated due to the reversibility of reaction and the inhibition by both acetaldehyde and NADH as products. To simplify ADH kinetics, some special approaches are employed to make ADH reaction apparently irreversible on single substrate (alcohol). Thus, reaction pH is optimized to 9.2 to scavenge hydrogen ion; semicabarzide at final 75 mmol/L is used to remove acetaldehyde as completely as possible; final nicotinamide adenine dinucleotide (NAD+) is 3.0 mmol/L; final ADH is about 50 U/L (Liao, et al., 2007a). By assigning the maximal absorbance at 340 nm for reduced nicotinamide adenine dinucleotide (NADH) by the equilibrium method to $A_{me}$ and that by kinetic analysis of reaction curve to $A_{mk}$, kinetic analysis of ADH reaction curve should predict $A_{mk}$ consistent with $A_{me}$, but requires some special efforts.
Fig. 8. Response of $F$ values to preset $C_{ald}$ for kinetic analysis of reaction curve for 0.31 mmol/L ethanol (reproduced with permission from Liao, et al, 2007a).
The use of semicabarzide reduces concentrations of acetaldehyde ($C_{ald}$) to unknown levels, and thus complicates the treatment of acetaldehyde inhibition on ADH. The integration rate equation with the predictor variable of reaction time can be worked out for ADH (Liao, et