Characterization of the Particles of Purified κ-Casein: Trypsin as a Probe of Surface-Accessible Residues
| Autor: | Harold J. Dower, Edward D. Wickham, Merton L. Groves, Peter H. Cooke, Peter D. Hoagland, E. G. Piotrowski, Harold M. Farrell |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Protein Conformation Surface Properties Dimer Molecular Sequence Data Biochemistry Hydrolysis chemistry.chemical_compound Biopolymers Dynamic light scattering Casein medicine Animals Humans Trypsin Amino Acid Sequence Chymosin Histone octamer Chromatography High Pressure Liquid Chromatography Sequence Homology Amino Acid Caseins Kinetics Microscopy Electron Monomer chemistry Molecular Probes Cattle Electrophoresis Polyacrylamide Gel medicine.drug |
| Zdroj: | Journal of Protein Chemistry. 18:637-652 |
| ISSN: | 1573-4943 0277-8033 |
| Popis: | kappa-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the kappa-casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t1/230 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for kappa-casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin. |
| Databáze: | OpenAIRE |
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