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 qualitativ e and quantitative determination of enzyme kinetics has been investiga ted. The enzyme used in this study was cu-chymotrypsin (ACHT) which wa s immobilized by absorption into a commercially available immobilized artificial membrane (IAM) interphase. The resulting IAM-ACHT phases we re enzymatically active and catalyzed the hydrolysis of L-tryptophan m ethyl ester to L-tryptophan. The interaction between the IAM-ACHT phas e and known reversible inhibitors of ACHT has been studied with hydroc innamic acid (HCA) and beta-phenylethylamine (BPEA), and the results d emonstrate that displacement chromatography can determine the type and degree of enzyme/inhibitor interactions. In addition, an inhibition c onstant (K-I) of 1.8 mM for the competitive inhibition by HCA was calc ulated which is consistent with the previously reported value of 4.5 m M determined using non-immobilized ACHT. For BPEA the calculated K-I w as 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/inhibito r interactions. The same immobilized enzyme was repeatedly used over a 10-day period to study enzyme kinetics.