Fig. 1. The integration strategy for enzyme initial rate assay (Modified from Liu et al. (2009)).
After the integration strategy for enzyme initial rate assay is validated, a switch point should be determined for changing from the classical initial rate method to kinetic analysis of reaction curve. The estimation of Vm by kinetic analysis of reaction curve usually prefers substrate consumption percentages reasonably high. Therefore, the substrate consumption percentage that gives an enzyme activity from 90% to 100% of the upper limit of linear response by the classical initial rate method can be used as the switch point.
It should be noted that the lower limit of linear response is difficult to be defined for enzyme initial rate assay by an integration strategy. For most methods, their lower limits of linear response are usually defined as three times the standard errors of estimate (Miller, J. C. & Miller, J. N., 1993). Usually, enzyme initial rate assay utilizes just one method for data processing and the difference between the lower limit and the upper limit of linear response is seldom over 30-fold. By the integration strategy, the measurable ranges of enzyme quantities cover two magnitudes and the detection limit is reduced to that by the classical initial rate method. By manual operation, different dilution ratios of a stock solution of the enzyme have to be used and any dilution error will increase the standard error of estimate for regression analysis. The measurement of higher enzyme activities will inevitably have larger standard deviation. Thus, regression analysis of the response of all measurable enzyme initial rates by the integration strategy to quantities of the enzyme will give higher standard error of estimate and thus an unfavourable lower limit of linear response. By this new integration strategy, we arbitrarily use twice the lower limit of linear response by the classical initial rate method as the lower limit if the overall standard error of estimate is more than twice that by the classical initial rate method alone; or else, the lower limit of linear response is still three times the overall standard error of estimate.
Taken together, for measuring initial rates of enzyme acting on single substrate by the integration strategy based on NLSF and data transformation, there are the following basic steps different from those by the classical initial rate method. The first is to work out the