Phosphorylation of casein and lysozyme by phosphorus oxychloride

Casein and lysozyme were phosphorylated by phosphorus oxychloride at pH 6-8 and 3-20 degree C. Up to 7.4 and 6.2 mol of phosphate/mol of protein were covalently attached to casein and lysozyme, respectively. super(31)P NMR spectral data and pH stability studies of the phosphate residues indicated that in phosphorylated casein, the phosphate was exclusively bound to hydroxyl oxygen as monophosphate and diphosphate. The phosphate linkages were stable at pH 2.0-8.5. In contrast, the majority of the phosphate in phosphorylated lysozyme appeared to be bound to nitrogen. In addition to mono- and diphosphate, triphosphate bonds were present. Gel electrophoresis in the presence of sodium dodecyl sulfate and urea indicated that protein cross-linking occurred during phosphorylation.