Reversed-phase high-performance liquid chromatographic separation of bovine kappa-casein macropeptide and characterization of isolated fractions

From complex mixtures of non-glycosylated and differently glycosylated caseinomacropeptides (CMP; kappa-casein fragment 106-169; M(r) approximate to 7000) various fractions were isolated and further purified by reversed-phase HPLC. The fractions were characterized by mass determination and composition analysis and also used in gel-permeation chromatography and NMR studies to investigate their molecular size behaviour as a function of pH, ionic strength, peptide concentration and degree of glycosylation. No evidence was found for association of any CMP fraction as a function of the experimental conditions applied, which is in contrast with suggestions made in the literature. The increased molecular size (apparent molecular mass approx. 30-45 kDa) is rather explained by a large voluminosity of the molecular species due to internal electrostatic and steric repulsion, Furthermore, the susceptibility of some non-glycosylated and glycosylated CMP fractions to enzymic attack by the Glu-specific endopeptidase from Staphylococcus aureus V8 was studied, Initial rates of proteolysis by this enzyme were independent of the degree of glycosylation. Only in the case of highly glycosylated CMP was further hydrolysis to smaller fragments inhibited. Hydrolytic products were identified by electrospray ionization and fast-atom bombardment mass spectrometry.