Purification des caséines χ de brebis

Purification of χ-Caseins from Sheep. Analysis of the Glycan and Peptide Components Starting from whole individual ovine casein prepared according to the method of Shahani, K. M. & Sommer, H. H. [J. Dairy Sci. 34, 1003–1009 (1951)], χ-casein was isolated and purified by successive steps of chromatography on columns of dextran gel and hydroxyapatite. On filtration through Sephadex G-150 in a buffer containing urea, the bulk of the χ-casein behaved as aggregates appearing in the void volume. Dissociation of these aggregates by reductive cleavage of disulfide bonds with 2-mercaptoethanol, followed by a second filtration step on Sephadex G-150 in the presence of both urea and 2-mercaptoethanol, resulted in retardation of the χ-casein, with separation from a contaminant representing 10–12% of the material applied. Further purification was achieved by chromatography on hydroxyapatite which eliminated the χs- and β-caseins. The purified χ-casein had a molecular weight of about 20000, an absorption coefficient A1%1cm at 280 nm of 10.85 and a sialic acid and phosphorous content of 0.3% (w/w) each. The sugar fraction liberated on acid hydrolysis of the caseinomacropeptide showed the presence of N-acetyl-galactosamine, galactose and neuraminic acid in equimolar ratio. Neuraminic acid existed mainly as the N-glycolyl derivative. The polypeptide chain of the ovine χ-casein was composed of about 170 amino-acids residues. Compared to bovine χ-caseins, the most notable difference was the presence of one additional cysteinyl and four additional aspartyl residues. Starch-gel and polyacrylamide-gel electrophoresis clearly revealed the heterogeneity of ovine χ-casein. Chromatographic fractionation of whole χ-casein on DEAE-cellulose also led to the separation of several fractions, the main characteristics of which are presented. Analysis of these fractions indicated that only those components which were firmly bound to DEAE-cellulose were glycosylated.