| 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 |