Characterization of the pH titration shifts of ribonuclease A by one- and two-dimensional nuclear magnetic resonance spectroscopy.

One- and two-dimensional 1H NMR experiments were used to determine the chemical shifts of resonances arising from 29 residues of RNase A in H2O at 17 pH values ranging from 1.2 to 7.9 Nearly all resonances displaying pH-induced changes in chemical shift greater than 0.1 ppm were monitored. Individual plots of the chemical shift as a function of pH were fit by nonlinear least squares methods to Henderson-Hasselbalch models yielding pK alpha values which were then analyzed using a set of criteria to determine their reliability. The criteria included statistics from the curve fitting analysis as well as the distance of the reporter proton to ionizing groups. Only the most reliable pK alpha values were assigned to specific ionizing groups within RNase A based upon the proximity of the reporter proton to the ionizing group as determined from the X-ray crystal structure. Only 2 of the 15 groups expected to undergo ionization within the pH range investigated could not be assigned pK alpha values within the highest two levels of reliability. Of the 11 carboxylate groups, 5 have pK alpha values less than 3.0. Many of the low pK alpha values can be interpreted as resulting from favorable hydrogen bonds between the carboxylate group and other moieties within the protein. The pK alpha values for the four histidine residues are similar to earlier literature reports. Two resonances underwent particularly large pH-induced shifts of approximately 2.3 ppm and corresponded to nitrogen-bound protons involved in hydrogen bonds with carboxylate groups.