Integration of kinetic analysis of reaction curve with a proper classical approach for enzymatic analysis

Abstract

For enzymatic analysis to quantify a substrate or enzyme, kinetic analysis of reaction curve can be integrated with a proper classical approach. For their integration, they should have consistent slopes and intercepts of linear response and an overlapped region of analyte quantities measurable under optimized conditions. To quantify a substrate after optimizations of tool enzyme activity and reaction duration, the equilibrium method works when the reaction is completed within the reaction duration; otherwise, kinetic analysis of reaction curve applies providing at least seven data with sufficient consumption of substrate. To quantify an enzyme after optimizations of initial substrate concentration and reaction duration, the classical initial rate method works when an estimated initial rate locates within the linear range; otherwise, kinetic analysis of reaction curve applies after the conversion of the quantification index with optimized parameters. This integration strategy has ideal linear ranges and practical efficiency for quantifying an enzyme at moderate substrate levels and for quantifying a substrate at moderate cost on tool enzyme; it has promise to simultaneous assays of multiple enzymes in one reaction vessel each time and ,thus, potential applications to concurrently quantify multiple serum enzymes, screen inhibitors against multiple enzyme targets, and detect multiple serum components by enzymeimmunoassay.

Figure 1

Conversion of V _m into initial rate (V _i) to meet the second prerequisite for an integration strategy to quantify an enzyme.

Figure 2

Optimization of activities of a tool enzyme to increase upper limit 1 to upper limit 2 by the equilibrium method for meeting the second prerequisite for an integration strategy to quantify a substrate.

Figure 3

Optimizations of both S ₀ to increase the upper limit of the classical initial rate method and reaction duration to reduce the lower limit by kinetic analysis of reaction curve for meeting the second prerequisite for an integration strategy to quantify an enzyme (V _i).

Figure 4

Association of standard deviations (SD) and relative SD (RSD) with averaged ALT activities by the integration strategy. Data were obtained by methods as reported before [24].