Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support
| Autor: | Daniel Horák, Jaroslava Turková, Zuzana Bílková, Jaroslav Churáček, J Mazurová |
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| Rok vydání: | 1999 |
| Předmět: |
Chromatography
Chymotrypsin biology Immobilized enzyme Organic Chemistry Active site General Medicine Methacrylate Biochemistry Antibodies Analytical Chemistry Catalysis Hydrolysis chemistry.chemical_compound Kinetics Affinity chromatography chemistry biology.protein Microscopy Electron Scanning Adipic acid dihydrazide |
| Zdroj: | Journal of chromatography. A. 852(1) |
| ISSN: | 0021-9673 |
| Popis: | 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 microm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 micromol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 microg 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. |
| Databáze: | OpenAIRE |
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