Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support

• Published in: Journal of Chromatography A 1999-08, Vol.852 (1), p.141-149
• Main Authors: BILKOVA, Z, MAZUROVA, J, CHURACEK, J, HORAK, D, TURKOVA, J
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Antibodies; Antibodies - immunology; Biological and medical sciences; Chymotrypsin - immunology; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Kinetics; Microscopy, Electron, Scanning; Proteins
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00234-4

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid support was developed and used for oriented immobilization of chymotrypsin by means of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol–toluene mixture stabilized with cellulose acetate butyrate. The resulting particles were 1.2 μm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 μmol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 μg of chymotrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practically 100% of its native proteolytic activity. Kinetic parameters of catalysis by chymotrypsin immobilized via this way were improved due to the good steric accessibility of the enzyme active site for high-molecular-mass substrates, when digestion of proteins in batch experiments was used.