Nuclear‐Magnetic‐Resonance Study of the Histidine Residues of S‐Peptide and S‐Protein and Kinetics of 1H‐2H Exchange of Ribonuclease A

Abstract

1H NMR spectroscopy at 100 MHz was used to determine the 1st-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35.degree. C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35.degree. C and pH meter reading 11 caused irreversible denaturation of RNase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by J. Ohe, H. Matsuo, F. Sakiyama and K. Narita. The rate data obtained by various authors are summarized and the reasons for the poor agreement between the data are discussed. The 1st-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5. The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK'' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK'' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26.degree. C. His-48 in S-protein titrates normally and has a lower pK than in RNase-A probably because of the absence of Asp-14, which in RNase-A forms a H-bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.