ENZYMATIC-HYDROLYSIS OF A CHEMISORBED PEPTIDE FILM USING BEADS ACTIVATED WITH COVALENTLY BOUND CHYMOTRYPSIN

Silica and polyacrylamide microspheres were modified with chemisorbed chymotrypsin and used to enzymatically hydrolyze a peptide thin film w hich was covalently bound to a flat silica surface. Chymotrypsin was c ovalently cross-linked to 500 nm silica spherical beads and 30-50 mu m polyacrylamide spherical beads and shown to be enzymatically active a gainst thin films of a fluorescent peptide, succinyl-ala-ala-phe-7-ami do-4-methylcoumarin (SAAP-AMC), and an unlabeled peptide, t-BOC-phe. S AAP-AMC and t-BOC-phe were covalently coupled to an aminosilane film o n silica and aluminum substrates through an amide linkage. Control exp eriments showed that free chymotrypsin in solution was able to hydroly ze the amide bond between the phenylalanine and the AMC groups of the chemisorbed peptide, resulting in the release of the AMC group into so lution. When they were placed into contact with the SAAP-AMC surface, the chymotrypsin-modified beads also hydrolyzed the phe-AMC bond and r eleased AMC into solution, demonstrating that covalently immobilized e nzymes can be used to hydrolyze immobilized organic thin films. The hy drolytic activity of the chymotrypsin beads was also confirmed for a s econd peptide film, t-BOC-phe, by external reflectance IR spectroscopy .