Measurement of Chemical Phosphate in Proteins

Analytical procedures for measuring the phosphate content in proteins have suffered in general from a lack of sensitivity. This problem has required preparation of large amounts of a purified protein for phosphate analysis, a task that cannot easily be accomplished for many phosphoproteins. Such preparation is particularly difficult in cases in which the protein must be purified from tissue biopsy samples obtained for investigations of protein phosphorylation in vivo. This chapter discusses the measurement of chemical phosphate in proteins. This procedure incorporates two methods. First, the purified protein sample is ashed to convert protein-bound phosphate to inorganic phosphate. Second, the inorganic phosphate is measured after complexation of phosphomolybdate with the triphenylmethane dye, malachite green. The sensitivity of this procedure is times greater than the standard Fiske-SubbaRow procedure for measuring inorganic phosphate and measures as low as 0.2 nmol phosphate.