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ARS Home » Research » Publications at this Location » Publication #101993


item Farrell, Harold
item Wickham, Edward
item Piotrowski, Edwin
item Hoagland, Peter
item Cooke, Peter

Submitted to: Journal of Protein Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Americans are consuming more yogurt and cheese each year, and these and other dairy products are becoming important sources of calcium in the diet. The major proteins of milk, called caseins, are complexed with calcium and phosphorus into small packages called micelles. When rennet, cultures, and extra calcium are added to milk, changes occur on the surfaces of these micelles that cause the milk to change from liquid to a semi-solid state. To gain better insight into the first steps in this process, a computer model of the surface of the micelle was made. By studying this model we predicted that certain portions of the casein might be strategically positioned on the surface of the micelles, and that these portions might serve as molecular attractants to speed up the process of product formation. Biochemical studies confirmed that these portions are indeed on the surface, and we can now predict how it may be possible to enhance cultured product formation through genetic manipulation of the surface or the enzymes causing the conversion. This information will aid cheese and yogurt manufactures who are attempting to streamline their processes and enhance nutritional quality of their products.

Technical Abstract: 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 percent monomer. All of these heterogenous 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 then 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 (t1/2 less than 30 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.