Assessment of the enzymatic activity and inhibition using HPFA with a microreactor, trypsin, absorbed on immobilized artificial membrane

Abstract

The purpose of this study was to develop a new method to assess the activity and inhibition of the immobilized enzyme trypsin based on the frontal analysis of enzymatic reaction products. This novel technique was performed by on-line monitoring of the absorption at 253 nm of N-benzoyl-L-arginine (BA) from the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE). A microreactor was constructed by immobilizing trypsin on an immobilized artificial membrane (IAM)-packed column. Trypsin was non-covalently and dynamically immobilized on-line in the hydrophobic interface of an IAM liquid chromatographic stationary phase. The trypsin-IAM stationary phase was bioactive. Due to the enzymatic reaction, the substrate of BAEE was completely hydrolyzed when the BAEE concentration was below 0.5 mmol/L and partly hydrolyzed when the BAEE concentration ranged from 0.75 to 1.0 mmol/L. By the addition of soybean trypsin inhibitor (STI), phenylmethane sulfonyl fluoride (PMSF), and benzamidine into the substrate solution, the results obtained from the frontal analysis showed that the activity of trypsin on IAM was strongly inhibited not only by STI but by both benzamidine and PMSF.