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 alpha-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 N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-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 alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.
Chui
W K
WK
Department of Oncology, McGill University, Montreal, Quebec, Canada.
Wainer
I W
IW
D001586:Benzoylarginine Nitroanilide; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D002863:Chromogenic Compounds; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008567:Membranes, Artificial; D013696:Temperature; D014357:Trypsin / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology*
D002863:Chromogenic Compounds; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D014361:Trypsin Inhibitors; D001586:Benzoylarginine Nitroanilide; D002918:Chymotrypsin; D014357:Trypsin