The Assignment of an Exchangeable Low-Field NH Proton Resonance of Ribonuclease A to the Active-Site Histidine-119

High-resolution proton magnetic resonance spectra between 11 and 14 ppm of bovine ribonuclease A in H₂O show that a single resonance titrates with changes in pH. This resonance arises from a histidine ring nitrogen proton and exhibits an apparent pK_a of 5.8. This pK_a value can be correlated with that of only one of the four histidine ring C-2 protons, namely, that which has been assigned to the active site histidine-119. Additional evidence for the correlation is obtained from the selective shifts of the apparent pK_a, values caused by temperature variation and by the binding of the inhibitors cytidine 2′-monophosphate, cytidine 3′-monophosphate, cytidine 5′-monophosphate, and inorganic phosphate. These results suggest that a specific histidine ring nitrogen proton of residue 119 is relatively inaccessible to solvent. Solvent inaccessibility of histidine residues as defined by proton magnetic resonance studies of proteins in H₂O is discussed.