Correlation of exchangeable (NH) and nonexchangeable (C2H) histidine resonances in the proton NMR spectrum of ribonuclease A in aqueous solution.

The ionization characteristics of the hydrogen-bonded His 12 N₁ proton observed to titrate between 11 to 13 ppm in the nmr spectrum of ribonuclease A in H₂O solution are compared with the ionization characteristics of the four histidine C₂ protons in the enzyme. Comparison of the pK_a's of the enzyme in H₂O and D₂O in the absence and presence of cytidine monophosphate (?5′, ?3′, and ?2′) inhibitors, line widths in the presence of Cu II at pH 3.6 and 5.6, and chemical shifts in the presence of AgNO₃ permit a correlation of the exchangeable His 12 N₁ proton with the active site histidine C₂ proton exhibiting the lower ionization pK_a. The histidines with pK_a of 5.1 and 5.6 in ribonuclease A in the absence of salt are assigned in this study to His 12 and His 119, respectively.