[Purification of kappa-caseins from sheep. Analysis of the glycan and peptide components (author's transl)].
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)], kappa-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 kappa-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 kappa-casein, with separation from a contaminant representing 10-12% of the material applied. Further purification was achieved by chromatography on hydroxyapatite which eliminated the alpha-s- and beta-caseins. The purified kappa-casein had a molecular weight of about 20000, an absorption coefficient (see journal for formula) 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-acetylgalactosamine, galactose and neuraminic acid in equimolar ratio. Neuraminic acid existed mainly as the N-glycolyl derivative. The polypeptide chain of the ovine kappa-casein was composed of about 170 amino-acids residues. Compared to bovine kappa-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 kappa-casein. Chromatographic fractionation of whole kappa-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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002364:Caseins / Q000302:isolation & purification; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004586:Electrophoresis; D005260:Female; D005688:Galactosamine / Q000032:analysis; D005690:Galactose / Q000032:analysis; D046911:Macromolecular Substances; D008623:Mercaptoethanol; D008970:Molecular Weight; D011247:Pregnancy; D011485:Protein Binding; D012756:Sheep; D012794:Sialic Acids / Q000032:analysis; D014508:Urea
D000596:Amino Acids; D002364:Caseins; D046911:Macromolecular Substances; D012794:Sialic Acids; D008623:Mercaptoethanol; D005688:Galactosamine; D014508:Urea; D005690:Galactose
Purification des caséines kappa de brebis. Analyse des parties glycanne et peptidique