Kinetic Study of α-Chymotrypsin by Electrophoretically Mediated Microanalysis Combined with Partial Filling Technique

Abstract

A novel method combining electrophoretically mediated microanalysis methodology and partial filling technique was developed and validated for kinetic study of α-chymotrypsin with N-benzoyl-l-tyrosine ethyl ester (l-BTEE) as substrate. The in-line enzymatic reaction was performed in 20 mmol L⁻¹ Tris with 5 mmol L⁻¹ calcium chloride (pH 7.8), while 20 mmol L⁻¹ Tris (pH 7.8) was used as a background electrolyte. A plug–plug injection sequence of incubation buffer, enzyme, substrate, and incubation buffer was applied due to the lower electrophoretic mobility of α-chymotrypsin than that of l-BTEE. The reaction was initiated by the application of 15 kV for 0.2 min. The voltage was turned off to increase the product amount (zero-potential amplification) and turned on again at a constant voltage of 18 kV to separate all the components. Incubation and subsequent analyte separation were carried out in a 37/30 cm (50 μm i.d.) fused-silica capillary at 25 °C. In the developed in-capillary α-chymotrypsin assay, the Michaelis–Menten constant (K _m) and an inhibition and activation study were performed. The kinetic parameters are in accordance with that determined by a spectrophotometric method.