ENZYME-BASED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY SUPPORTS AS PROBES OF ENZYME-ACTIVITY AND INHIBITION - THE IMMOBILIZATION OF TRYPSIN AND ALPHA-CHYMOTRYPSIN ON AN IMMOBILIZED ARTIFICIAL MEMBRANE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY SUPPORT

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes α-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA), and the IAM-α-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized α-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions. © 1992.