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.
Miyagi
Masaru
M
Case Center for Proteomics, Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4988, USA. masaru.miyagi@case.edu
Nakazawa
Takashi
T
D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D002417:Cattle; D041961:Deuterium Exchange Measurement; D006639:Histidine / Q000032:analysis* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012997:Solvents / Q000737:chemistry
D011506:Proteins; D012997:Solvents; D000804:Angiotensin II; D006639:Histidine; D012259:Ribonuclease, Pancreatic