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

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journal contribution
posted on 01.09.2008, 00:00 by Masaru Miyagi, Takashi Nakazawa
We developed a mass spectrometric method to determine the pKa values of individual histidine residues in proteins. The method is based on the fact that the imidazole C2-proton undergoes pH-dependent hydrogen−deuterium exchange reaction, of which the rate constant (kφ) reflects the pKa for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D2O 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 kφ of the H−D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The pKa value is then determined from a plot of kφ versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the pKa values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The pKa 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 1H 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.