Selective Removal of Phenylalanine Impurities from Commercial κ-Casein Glycomacropeptide by Anion Exchange Chromatography

Abstract

Bovine κ-casein glycomacropeptide (GMP) found in sweet whey is a 64 amino acid residue phosphorylated glycopeptide. Because it lacks aromatic amino acids including phenylalanine, GMP is thought to be an important dietary source of amino acids for patients suffering from phenylketonuria. There is, however, very little information available concerning preparation of phenylalanine-free GMP for human consumption. This study was, therefore, undertaken to remove phenylalanine containing impurities from commercially available crude GMP by anion exchange chromatography on diethylaminoethyl (DEAE)-Sephacel. The results demonstrated that phenylalanine containing proteins or peptides do not bind to the column, while most GMP accounting for 93% of total recovered sialic acid can bind to the column. The purified GMP, which accounted for average 43% of dry weight of crude GMP, contained undetectable level of phenylalanine. Analyses and cellulose acetate electrophoresis showed that the purified GMP is a product with high sialic acid content (average 15.5% dry weight). Gel filtration chromatography on Sephacryl S-100 and size exclusion HPLC on Superdex 75 confirmed our previous findings that GMP monomers form aggregates and elute as a single peak with its elution volume close to the elution volume of dimeric β-lactoglobulin (36.6 kDa). It was concluded that the crude preparation of GMP can be highly refined by selectively removing phenylalanine impurities using DEAE-Sephacel chromatography.