A proton nuclear magnetic resonance investigation of the anion Bohr effect of human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the molecular mechanism of the Bohr effect of human normal adult hemoglobin in the presence of two allosteric effectors, i.e., chloride and inorganic phosphate ions. The individual hydrogen ion equilibria of 22-26 histidyl residues of hemoglobin have been measured in anion-free 0.1 M HEPES buffer and in the presence of 0.18 M chloride or 0.1 M inorganic phosphate ions in both deoxy and carbonmonoxy forms. The results indicate that the β2-histidyl residues are strong binding sites for chloride and inorganic phosphate ions in hemoglobin. The affinity of the β2-histidyl residues for these anions is larger in the deoxy than in the carbonmonoxy form. Nevertheless, the contribution of these histidyl residues to the anion Bohr effect is small due to their low pK value in deoxyhemoglobin in anion-free solvents. The interactions of chloride and inorganic phosphate ions with the hemoglobin molecule also result in lower pK values and/or changes in the shapes of the hydrogen ion binding curves for several other surface histidyl residues. These results suggest that long-range electrostatic interactions between individual ionizable sites in hemoglobin could play an important role in the molecular mechanism of the anion Bohr effect