substrate assay always has unsatisfactory precision. Therefore, this new kinetic method itself is still much beyond satisfaction for substrate assay.
To concomitantly have wider linear ranges, desirable analysis efficiency and favourable precision for enzyme substrate assay, the integration of kinetic analysis of reaction curve with the equilibrium method can be used. The indexes of substrate quantities by the two methods have exactly the same physical meanings, and thus the integration strategy can be easily realized for enzyme substrate assay. By this integration strategy, there should still be an overlapped range of concentrations of the substrate measurable consistently by both methods, besides a switch threshold within such an overlapped region to change from the equilibrium method to kinetic analysis of reaction curve. Additionally, this overlapped region of substrate concentration measurable by both methods with consistent results should localize in a range of substrate concentration high enough for reasonable precision of substrate assay based on kinetic analysis of enzyme reaction curve. These requirements can be met as described below. (a) The upper limit of linear response by the equilibrium method should be optimized to be high enough, so that the difference between the initial signal before enzyme action and the last recorded signal for about 80% of this upper limit is 50 times higher than the random noise of an instrument to record enzyme reaction curves; such a difference can be used as the switch threshold. (b) The activity of a tool enzyme and the duration to monitor reaction curve as experimental conditions should be optimized; kinetic parameters except Vm for kinetic analysis of reaction curve are optimized as well. The resistance of the predicted last signal to reasonable variations in data ranges for analysis can be a criterion to judge the optimized set of preset parameters. For favourable analysis efficiency in clinical laboratories, reaction duration can be about 5.0 min. This reaction duration results in a minimum activity of the tool enzyme for the integration strategy so that the upper limit of linear response by the equilibrium method can be high enough to switch to kinetic analysis of reaction curve. This integration strategy after optimizations can simultaneously have wider linear ranges, higher analysis efficiency and lower cost, better precision and stronger resistance to factors affecting enzyme activities.
Similarly, with the integration strategy for enzyme substrate assay, we also use twice the lower limit of the equilibrium method as the lower limit by the integration strategy if the standard error of estimate is much larger; or else, three times the standard error of estimate by the integration strategy is taken as the lower limit of linear response.
In general, the following steps are required to realize this integration strategy for enzyme substrate assay: (a) to work out the integrated rate equation with the predictor variable of reaction time; (b) to optimize individually the (kinetic) parameters preset as constants for kinetic analysis of reaction curve; (c) to optimize the activity of the tool enzyme so that data for the upper limit of linear response by the equilibrium method within about 5.0-min reaction are suitable for kinetic analysis of reaction curve. As demonstrated later, this integration strategy is applicable to enzymes suffering from strong product inhibition.
2.5 Applications of new methods to some typical enzymes
We investigated kinetic analysis of reaction curve with arylesterase (Liao, et al., 2001, 2003a, 2007b), alcohol dehydrogenase (ADH) (Liao, et al., 2007a), gama-glutamyltransfease (Li, et al., 2011), uricase (Liao, 2005; Liao, et al., 2005a, 2005b, 2006; Liu, et al., 2009; Zhao, Y.S., et