On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

Abstract

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9 cm of the capillary inlet (total length of capillary 60.2 cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0 min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551 μmol L(-1) and 1.52 μmol L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…[128][129][130][131][132][133][134][135]. Another suitable approach was the adsorption of either the enzyme [136][137][138][139][140][141] or enzyme previously immobilized to gold nanoparticles [142][143][144] to a capillary which had been coated by polybrene or poly(ethyleneimine). Alginate encapsulation of the enzymes prior to adsorption to the capillary has also been applied [145].…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…Most of the on-line incubations described in the paper either use the same pH [13,18,23,24,34,43,56–62], or a pH close to enzyme reaction [26,32,33,41,55]. In some instances a wide difference of pH existed between the enzyme incubation buffer and the separation buffer [31,63].…”

“…The on-line mixing of enzyme, substrate, and inhibitor has been accomplished through electrophoretically mediated microanalysis [58,59,62,63,66,73], transverse diffusion of laminar flow profiles [21,31,41,74,75], and pressure mediated microanalysis [45]. On-line analysis with the use of immobilized enzyme for inhibitor studies have also been developed and high throughput analysis was achieved [18,22,23,56,68,76–78]. …”

“…Calculating the K I value requires knowledge of the Michaelis-Menten constant so that the appropriate equation is used in the determination. During the time frame of this review, capillary electrophoresis has been used to calculate K I for inhibitors that were competitive [13,20,22,23,25,31,79], mixed [56,77], and noncompetitive [72] inhibition. When the goal is to identify the mechanism of inhibition, Eq.…”

“…When the goal is to identify the mechanism of inhibition, Eq. (4) can be linearized so that the data can easily be visualized using a Lineweaver-Burk plot, as demonstrated by several reports published during the time period of this review [13,22,25,56,72,77]. …”