Isolation of κ-casein-like fractions from human whole casein by chromatography on thiol-Sepharose, hydroxyapatite and DEAE-cellulose

Abstract

SummaryDifferent fractions of human κ-casein characterized by their sensitivity to chymosin were separated, after reduction, by affinity chromatography on thiol-Sepharose in a 0·1 m-Tris-HCl buffer, pH 7·0, containing 7 M-urea, 0·3 m-NaCl and 1 mm-EDTA. The fraction retained on the column and bearing the SH-groups was then chromatographed on hydroxyapatite in a 5 mm-phosphate buffer, pH 6·8, containing 0·2 m-KC1, 4·5 m-urea and 2 mm 2-mercaptoethanol. The fraction not retained on this column was purified by chromatography on DEAE-cellulose in a 6·6 m-urea, 0·02 m-imidazole, 0·02 m 2-mercaptoethanol buffer, pH 7·0. Two chymosinsensitive fractions were identified by starch-gel electrophoresis at pH 8·6; one of low electrophoretic mobility, the other a smeared band covering the whole distance of migration, in which several bands with mobilities greater than those of β-caseins could be detected.