Chemical phosphorylation of bovine casein: relationships between the reacting mixture and the binding sites of the phosphoryl moiety

Abstract

The aim of this work was to determine the influence of pH on the chemical phosphorylation of bovine casein by POCl 3. Experiments were performed at pH 5, pH 7 and pH 9. A pH-stat apparatus was used to maintain the pH at a fixed value. The phosphorus content of modified casein was measured by the Fiske and Subbarow method. The amount of unreacted ϵ-amino groups of lysyl residues was determined using 2,4,6-trinitrobenzene sulfonic acid. Polyacrylamide gel electrophoresis in a dissociating medium showed that many of the modified species were linked together by stable bonds. This observation is consistent with the very high viscosity of the product in dissociating medium. An amino acid analysis was performed to check the possible production of a lysino-alanine linkage. The stability of the modified casein was investigated by partial hydrolysis at pH 2, pH 5 and pH 10.5, followed by polyacrylamide gel electrophoresis. The results showed that the number and nature of phosphate bonds depend on the pH at which the reaction was carried out.