Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

Abstract

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis-Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.

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]. …”

“…The mechanism used to prevent cancer progression is to reduce the enzyme activity on the pathway that promotes cancer cell growth. In order to find a potent inhibitor, on-line analysis is generally adapted to screen the selected compounds, such as natural extracts [23,56,63,76] or traditional Chinese medicines [18,57,68,73]. In the process of screening the inhibition, the selected inhibitor is evaluated as the relative difference in the product peak area in the presence and in the absence of the inhibitor.…”

“…Noncovalent coatings that employ electrostatic [22,76,77] interactions through the use of a cationic coating or hydrophobic [18] interactions through the use of a neutral coating, are simple to employ. However, they are less stable than coatings based on covalent immobilization to the silica wall, such as silanol [57] and glutaraldehyde chemistry [78].…”

Applications of polydopamine modifications in capillary electrophoretic analysis

“…Biomaterials have been used as stationary phase not only for chiral separation but also as promising materials for screening enzyme inhibitors. Cheng et al [66] used trypsin physically adsorbed on the capillary modified with PDA layer to study enzyme kinetics of trypsin and online screening of trypsin inhibitors. The parameters based on Michaelis-Menten constant and half-maximal inhibitory concentration were investigated.…”