Kinetic analysis of an enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase by pressure-assisted capillary electrophoresis/dynamic frontal analysis

Abstract

An enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p-nitrophenol by the CE/DFA applying in-capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis–Menten analysis, and a Michaelis–Menten constant was determined as K_M = 0.83 mmol L⁻¹. An enzyme inhibition was also examined with bis(p-nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC₅₀ = 0.79 μmol L⁻¹. The values of K_M and IC₅₀ obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product.