Phosphorus Nuclear Magnetic Resonance Study of Phosphoproteins

I. CHEMICAL NATURE OF PHOSPHORUS ATOMS IN BOVINE _s-CASEIN

From the ¹ From the Department of Biophysics, University of Pittsburgh, and the Department of Chemistry, Carnegie Institute of Technology, Pittsburgh, Pennsylvania 15213

Phosphorus nuclear magnetic resonance spectroscopy has been used to investigate the chemical nature of the phosphorus atoms in bovine _s-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 _s-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 ß-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 _s-casein. The relation of this type of phosphate linkage to the structure of _s-casein and to its stability in aqueous solution is discussed.

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