Phosphorus Nuclear Magnetic Resonance Study of Phosphoproteins

Phosphorus nuclear magnetic resonance spectroscopy has been used to investigate the chemical nature of the phosphorus atoms in bovine αs-casein. A satisfactory 31P nuclear magnetic resonance spectrum can be obtained in a sample containing 26% by weight of the protein in a 6.5 M urea. The average 31P chemical shift of αs-casein is 221 ppm from PCl3 with a standard deviation of 1 ppm over the pH range 4 to 9. The variations with pH of the phosphorus chemical shift of model compounds adenosine 5'-triphosphate, O-phosphoserine, creatine phosphate, reduced β-diphosphopyridine nucleotide, and sodium pyrophosphate fall into two categories: (a) the chemical shifts in compounds with disubstituted pyrophosphate or phosphodiester linkages remain essentially constant in the pH range 4 to 9, whereas (b) for monoester compounds, there is a relatively large change in chemical shift (greater than 4 ppm) as the pH changes from 3 to 9, i.e. as the phosphate group goes from the singly ionized to the doubly ionized state. This difference (which is to be expected from a priori considerations) suggests that there may be phosphodiester or disubstituted pyrophosphate bonds, or both, in bovine αs-casein. The relation of this type of phosphate linkage to the structure of αs-casein and to its stability in aqueous solution is discussed.