Determination of pKa Values of Individual Histidine Residues in Proteins Using Mass Spectrometry

We developed a mass spectrometric method to determine the p K a values of individual histidine residues in proteins. The method is based on the fact that the imidazole C 2-proton undergoes pH-dependent hydrogen-deuterium exchange reaction, of which the rate constant ( k phi) reflects the p K a for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D 2O solvent at various pH values, (2) protein digestion by proteolytic enzyme(s), during which all the rapidly exchanging deuterons such as those in amide and hydroxyl groups are back-exchanged for protons, and (3) measurement of the mass spectrum of each histidine-containing peptide by LC/ESI-MS. The k phi of the H-D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The p K a value is then determined from a plot of k phi versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the p K a values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The p K a values of three of four histidine residues (His12, -105, and -119) in RNase A were successfully determined by this method and were in good agreement with those determined by (1)H NMR and hydrogen-tritium exchange methods. The method uses subnanomole quantities of protein, allowing measurement at a much lower concentration than that of 1 mM required for the conventional NMR approach that is currently almost exclusively the method of choice.