Use of photoacoustic Fourier-transform infrared spectroscopy to study phosphates in proteins

Photoacoustic Fourier-transform infrared spectroscopy was used to study phosphoamino acids and phosphoproteins. Using this method, we have found that the spectral properties of phosphate esters depend on the nature of the linkage, pH, and binding of metal ion. At high pH values, dianionic symmetric stretching of phosphotyrosine and phosphoserine occurs at 984 and 974 reciprocal centimeters, respectively. Analysis of the IR bands of bound phosphate in phosvitin at different pH values gives the pKa value for the esterified phosphates. Addition of aluminum ions to phosvitin at different pH values causes a shift in the phosphate band consistent with a direct binding of aluminum ions to the esterified phosphate. The phosphate signal for 40 micrograms of Pepsin (1 P/mole) is detectable by this method.