1H-NMR study on the tautomerism of the imidazole ring of histidine residues : I. Microscopic p K values and molar ratios of tautomers in histidine-containing peptides

Abstract

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N 1-methylimidazole, l-histidine, N 1- methyl- l- histidine , N 3- methyl- l- histidine , and other related compounds. With these results, the macroscopic p K values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the p K values of imidazole and N 1-methylimidazole, the perturbation for the p K of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic p K values of the individual tautomers of the imidazole ring were estimated with the p K values of N 1- methyl- l- histidine , N 3- methyl- l- histidine , and perturbation due to methyl substitution. The estimated p K values were 6.73 for the N 1-H tautomer and 6.12 for the N 3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic p K value (6.02) calculated using the estimated microscopic p K values agreed with that (6.03) observed for the imidazole ring of l-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic p K values of tautomers were also obtained for N α- acetyl- l- histidine and N α- acetyl- l- histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic p K values of tautomers. The intrinsic (or unperturbed) p K value of imidazole ring and perturbations due to the CO 2 − and NH 3 + were obtained for each of the N 1-H and N 3-H tautomers.