3] Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to immobilon

Abstract

This chapter discusses the determination of phosphoamino acid composition by acid hydrolysis of protein blotted to immobilon. The identification of phosphorylated amino acids in a protein labeled biosynthetically with [ 32 P]P i is accomplished by chemical or enzymatic hydrolysis of amide bonds and resolution of the liberated phosphoamino acids by chromatographic techniques. Complete enzymatic hydrolysis represents the best approach for obtaining accurate determinations of the relative abundance and identity of phosphoamino acids within a protein, because it avoids the problem of hydrolysis of the phosphoester bond, which occurs during acid-catalyzed hydrolysis. The rates of acid-catalyzed hydrolysis of both peptide and phosphoester bonds are affected by the nature of adjacent bonds and, consequently, the relative proportions of free phosphoamino acids after a partial hydrolysis are not necessarily an accurate reflection of their native abundance in the original protein.