Inhibition Assay of Theophylline by Capillary Electrophoresis/Dynamic Frontal Analysis on the Hydrolysis of <i>p</i>-Nitrophenyl Phosphate with Alkaline Phosphatase
Masanori Mine, Hitoshi Mizuguchi, Toshio Takayanagi
Abstract
A novel inhibition assay by capillary electrophoresis/dynamic frontal analysis (CE/DFA) is proposed. When substrate p-nitrophenyl phosphate and inhibitor theophylline were tandemly introduced into a capillary containing alkaline phosphatase enzyme, two plateau signals were detected in the electropherogram. A higher plateau is based on the CE/DFA without inhibition, and a suppressed plateau is formed while the substrate zone passes through the inhibitor zone. Inhibition constant was successfully determined through the two plateau heights. Inhibition of enzymatic reaction in CE/DFA is demonstrated by the tandem injections of a substrate and an inhibitor solutions. The inhibition occurs when the zones of a substrate (S) and an inhibitor (I) overlap by the electrophoretic migration. Two plateau heights correspond either with or without the inhibitor.