Development of a fast and efficient CE enzyme assay for the characterization and inhibition studies of α-glucosidase inhibitors

The inhibition of the -glucosidase enzyme plays an important role in the treatment of diabetes mellitus. We have established a highly sensitive, fast, and convenient CE method for the characterization of the enzyme and inhibition studies of -glucosidase inhibitors. The separation conditions were optimized; the pH value and concentration of the borate-based separation buffer were optimized in order to achieve baseline separation of p-nitrophenyl--d-glucopyranoside and p-nitrophenolate. The optimized method using 25 mM tetraborate buffer, pH 9.5, was evaluated in terms of repeatability, LOD, LOQ, and linearity. The LOD and LOQ were 0.32 and 1.32 M for p-nitrophenyl--d-glucopyranoside and 0.83 and 3.42M for p-nitrophenolate, respectively. The value of the Michaelis-Menten constant (K-m) determined for the enzyme is 0.61 mM, which is in good agreement with the reported data. The RSDs (n = 6) for the migration time was 0.67 and 1.83% for substrate and product, respectively. In the newly established CE method, the separation of the reaction analytes was completed in <4 min. The developed CE method is rapid and simple for measuring enzyme kinetics and for assaying inhibitors.