Two enzymatic methods for determination of the phosphate content of phosphoproteins

Two new enzymatic methods are described for the determination of the amount of inorganic phosphate covalently bound to macromolecules. In the first method, the inorganic matter obtained after ashing is incubated with [1-14C] pyruvate in the presence of pyruvate oxidase and the enzymatically formed 14CO2 is collected. The other method uses glycogenolytic enzymes to generate NADH which is then measured fluorometrically. The use of these two phosphate-dependent enzymes allows the measurement of phosphate at up to 1000 times greater sensitivity than is obtainable with the phosphomolybdic acid method, thus permitting phosphate analyses to be performed on microgram quantities of proteins.