Enzymatic Hydrolysis of a Chemisorbed Peptide Film Using Beads Activated with Covalently Bound Chymotrypsin

Abstract

Silica and polyacrylamide microspheres were modified with chemisorbed chymotrypsin and used to enzymatically hydrolyze a peptide thin film which was covalently bound to a flat silica surface. Chymotrypsin was covalently cross-linked to 500 nm silica spherical beads and 30-50 µm polyacrylamide spherical beads and shown to be enzymatically active against thin films of a fluorescent peptide, succinyl-ala-ala-phe-7-amido-4-methylcoumarin (SAAP-AMC), and an unlabeled peptide, t-BOC-phe. SAAP-AMC and t-BOCphe were covalently coupled to an aminosilane film on silica and aluminum substrates through an amide linkage. Control experiments showed that free chymotrypsin in solution was able to hydrolyze the amide bond between the phenylalanine and the AMC groups of the chemisorbed peptide, resulting in the release of the AMC group into solution. When they were placed into contact with the SAAP-AMC surface, the chymotrypsin-modified beads also hydrolyzed the phe-AMC bond and released AMC into solution, demonstrating that covalently immobilized enzymes can be used to hydrolyze immobilized organic thin films. The hydrolytic activity of the chymotrypsin beads was also confirmed for a second peptide film, t-BOC-phe, by external reflectance IR spectroscopy.

Phosphatase responsive peptide surfaces

“…Most of these existing examples focus on the use of enzymes to produce surface patterns. Enzymes were immobilized on AFM tips, 23-28 stamps 29,30 or silica beads 31 to create patterns on surfaces modified with DNA, 28,29 peptides, 23,31 phospholipids 25 or polymers. 26,27,30 Examples of enzymes used include serine protease, 23 proteinase K, 26 trypsin, 30 chymotrypsin, 31 horseradish peroxidase, 27 phospholipase A 2 , 25 DNAse I 28 and exonuclease I.…”

Bioactivation of Metal Oxide Surfaces. 1. Surface Characterization and Cell Response

“…1,2,10 Chemisorption of biomolecules does not suffer from the aforementioned disadvantages and provides a durable linkage between the molecule and the surface. 11 For example, the detection of enterotoxin B by a surface-bound antibody was dependent on covalent grafting of the antibody to silica surfaces. Antibodies that were physisorbed were not well-structured and acted like insulators interfering with capacitancebased detection methods.…”

“…Deposition of proteins, peptides, or enzymes on solid supports such as silica may be obtained by simply incubating the surface with the desired organic molecules. However, such a crude strategy results in surfaces which suffer from denaturation, leaching and inconsistency in the activity of the adsorbed layer, and these problems lead to difficulties in both understanding structure−function relationships and accurate surface characterization of the adsorbed layer. ,, Chemisorption of biomolecules does not suffer from the aforementioned disadvantages and provides a durable linkage between the molecule and the surface . For example, the detection of enterotoxin B by a surface-bound antibody was dependent on covalent grafting of the antibody to silica surfaces.…”

Controlling protein retention on enzyme‐responsive surfaces

“…The reduction for chymotrypsin suggests that the enzyme may have cleaved the bond on the carboxylic acid side of Phe, an observation previously reported by Turner et al using SIMS and XPS in a similar system, although this work used chymotrypsin covalently bound to silica beads. 19 For Fmoc-Phe-Gly, chymotrypsin . XPS C 1s core level spectra for both Fmoc-dipeptide surfaces following incubation with both enzymes and the water controls using Method 3.…”

“…Earlier work by Turner et al demonstrated that enzymatic modification of surface-tethered molecules is possible, in principle, by the cleavage of surface-bound phosphate esters by a lipase; however, it was observed that non-specific adsorption of protein molecules to the surface limited the effectiveness of this system 18. This problem was solved by using enzymes (chymotrypsin) covalently bound to silica beads,19 a method that both eliminated enzyme adsorption to the surface and allowed for enzyme recovery. Yeo and Mrksich have shown that polyethylene glycol (PEG) surfaces modified with 4-hydroxyphenyl valerate could be used to enzymatically switch a redox-inactive surface to a redox-active surface by de-acylation 20.…”