Reactivity of peptide imidazole, amino, and phenolic groups towards p-nitrophenyl acetate.

Previous studies have shown that the reaction of p-nitrophenyl acetate with imidazole and amino groups may be studied under pseudo-first order conditions (2, 3). The reaction with an imidazole derivative is catalytic, the imidazole being regenerated by the spontaneous decomposition of the acylimidazole. The reaction with an amine yields a stable acylamino derivative but is most readily studied with an excess of the amine so that the kinetics is pseudo-first order with respect to the ester. The measurement of the kinetics of reaction with p-nitrophenyl acetate provides a convenient second parameter to be manipulated along with the hydrogen ion association constant in assessing the reactivity of imidazole or amino groups in a peptide (2-6). It has been applied successfully to the interpretation of the hydrogen ion titration curve of sperm whale metmyoglobin (7) and of certain metal-binding studies with that protein (1). The task of distinguishing the contributions of groups, such as imidazole and amino, which may show some degree of overlapping behavior in their interaction with hydrogen ions is made easier by the fact that the second, kinetic parameter will be different in general for the different groups (2, 8). The present study extends the previous observations on amino group reactivity to the e-amino type with e-aminocaproate and on imidazole reactivity with imidazole-Cacetate. The reaction with the phenolate group is explored with N-acetyltyrosine. The lack of reactivity under comparable conditions of the guanidinium group in y-guanidinobutyrate is established. The two peptides, glycyl-L-histidine and glycyl-n-tyrosine, are studied as examples of peptides carrying two different groups which are reactive toward p-nitrophenyl acetate. The very slight reactivity of the amino group of the valyl residue previously shown with n-valylglycine (5) is confirmed with n-valyl-n-alanine. Values for glycylsarcosylglycine and a copolymer of n-aspartic acid and n-histidine (9) are reported under the same conditions. Last, the dependence of the rate constants for imidazole and amino groups on ionic strength is shown to be small.