APPLICATION OF AN ENZYME-BASED STATIONARY-PHASE TO THE DETERMINATION OF ENZYME-KINETIC CONSTANTS AND TYPES OF INHIBITION - NEW HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC APPROACH UTILIZING AN IMMOBILIZED ARTIFICIAL MEMBRANE CHROMATOGRAPHIC SUPPORT

The application of an immobilized enzyme HPLC column to the qualitative and quantitative determination of enzyme kinetics has been investigated. The enzyme used in this study was cu-chymotrypsin (ACHT) which was immobilized by absorption into a commercially available immobilized artificial membrane (IAM) interphase. The resulting IAM-ACHT phases were enzymatically active and catalyzed the hydrolysis of L-tryptophan methyl ester to L-tryptophan. The interaction between the IAM-ACHT phase and known reversible inhibitors of ACHT has been studied with hydrocinnamic acid (HCA) and beta-phenylethylamine (BPEA), and the results demonstrate that displacement chromatography can determine the type and degree of enzyme/inhibitor interactions. In addition, an inhibition constant (K-I) of 1.8 mM for the competitive inhibition by HCA was calculated which is consistent with the previously reported value of 4.5 mM determined using non-immobilized ACHT. For BPEA the calculated K-I was 8.5 mM and the inhibition was mostly noncompetitive. This indicates that the IAM-ACHT can be used to quantitatively determine the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions. The same immobilized enzyme was repeatedly used over a 10-day period to study enzyme kinetics.