Determination of protein-bound glutamine and asparagine.

The correct assignment of amide groups to the glutamyl and aspartyl residues determined from analyses of acid hydrolysates continues to be an important problem in the delineation of protein and peptide structure. This problem is illustrated by recent discussions by Potts et al. (1) and Smyth, Stein, and Moore (2) on the elucidation of ribonuclease structure and by Chibnall et al. (3) on the estimation of glutaminyl and asparaginyl residues by the lithium borohydride method. In the course of studies on protein metabolism in brain and other tissues, one of us (J. R. W.) has adapted and modified several existing procedures, so that glutamine and asparagine can each be liberated from proteins and peptides without deamidation and determined quantitatively by specific assay methods. In this procedure, protein or peptide samples are hydrolyzed enzymatically by Viokase (4), an activated, desiccated, and defatted whole pancreas preparation, and the glutamic acid, glutamine, aspartic acid, and asparagine contents of the hydrolysates are determined by specific enzymatic assays (5-8). Enzymatic hydrolysis of peptides is complete, but hydrolysis of proteins seldom exceeds SO%, as judged from data obtained on acid hydrolysates. The validity of the procedure, and particularly the estimation of protein amide content from partial hydrolysis data, have been evaluated on a number of pure proteins and peptides of known composition. A number of other proteins of widely differing glutamic acid, aspartic acid, and amide group content have also been analyzed as an additional evaluation. Enzymatic hydrolysis of proteins for constituent amide groups dates from at least 1932, when enzymatic digests of edestin and gliadin were used to demonstrate the existence of protein-bound asparagine (9) and glutamine (lo), respectively. A variety of proteolytic enzyme preparations is now becoming available, notably those reported by Barry (ll), by Nomoto, Narahashi, and Murakami (12), and by Hill and Schmidt (13), each of which appears to be capable of liberating glutamine and asparagine from proteins without deamidation. However, with the exception of the studies by Barry (II), little attention has been focused directly on elucidation of protein or peptide amide content by these methods. The procedure presented here would appear to offer a valuable adjunct to other methods currently available and to permit definite assignment of amide groups to glutamic acid and aspartic acid residues in proteins and in peptide sequences. It must be emphasized that in the present procedure and in others (11, 12) in which enzymatic hydrolysis is incomplete, the assumption is made that digestion is complete for a portion of each protein sample subjected to hydrolysis so that correct compositions can be calculated therefrom. This assumption has been validated for the proteins studied here, but