A Simulation Study of Inhibition Assay With Theophylline on the Enzymatic Hydrolysis of p-Nitrophenyl Phosphate With Alkaline Phosphatase in a Continuous Mode of Electrophoretically Mediated Microanalysis.

Inhibition assays in a continuous mode of electrophoretically mediated microanalysis (EMMA) were simulated using a MATLAB matrix. The enzymatic hydrolysis of p-nitrophenyl phosphate (NPP) with alkaline phosphatase (ALP) was adopted as a model enzymatic reaction, where theophylline (THE) was used as a model inhibitor. In the simulation, the substrate NPP and the inhibitor THE were tandemly injected into a separation capillary filled with a separation buffer containing ALP. The formation rate of the product p-nitrophenol (NP) was calculated using a Michaelis-Menten constant, the inhibition constant, and the reagent concentrations. A plateau response of the product was reproduced in the simulation based on the continuous enzymatic reaction and the continuous resolution of the product in EMMA. In addition to the plateau response of the product, a depressed plateau was also simulated as a consequence of the inhibition reaction. This depressed plateau was caused by the overlapping between the injected zones of the substrate and the inhibitor. The simulated electropherograms including the inhibition-induced depression were consistent with the experimental data. The Michaelis-Menten constant, the inhibition constant, and the inhibition type were also reproduced from the simulated plateau responses. Factors affecting the plateau depression were further investigated through simulations by varying the injected zone lengths and experimental conditions. This study successfully demonstrated the utility of the simulation approach for designing inhibition assays of enzymatic reactions in a continuous mode of EMMA.