Capillary electrophoretic separations of enzyme inhibitors with activity-based detection

A technique for separating and detecting enzyme inhibitors was developed using capillary electrophoresis with an enzyme microreactor. The on-column enzyme microreactor was constructed using one or two NdFeB magnets in two configurations to immobilize alkaline phosphatase-coated superparamagnetic beads with diameters of 2.8 µm inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with an alkaline phosphatase substrate, AttoPhos. Product generated in the enzyme microreactor was detected by laser-induced fluorescence. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation in the presence of the inhibitors. The goal of this study was to improve peak capacities for inhibitor separations relative to previous work, which combined continuous engagement electrophoretically mediated microanalysis (EMMA) and transient engagement EMMA to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Increasing the electric field strength from 100 V/cm to 500 V/cm caused a 2-3-fold decrease in peak capacity for alkaline phosphatase inhibition assays with arsenate, a reversible, competitive inhibitor. When the bead injection time was increased to increase the length of the immobilized bead plug, the peak capacity for arsenate reached a minimum value at 60.0 s for the one-magnet configuration and at 30.0 s for the two-magnet configuration. The peak capacity was enhanced to 20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. The inhibition peak width increased as the concentrations of arsenate and theophylline increased. Five reversible inhibitors of alkaline phosphatase (theophylline, orthovanadate, arsenate, L-tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures. A well-resolved, individual inhibition peak was observed for each inhibitor. Enzyme inhibition assays with a mixture of the five inhibitors were also performed with the previous EMMA method, and a peak capacity of only 3 was obtained (all 5 inhibitors could not be resolved)