Enzyme Inhibitor Screening by Capillary Electrophoresis with an on-Column Immobilized Enzyme Microreactor Created by an Ionic Binding Technique

Abstract

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.