High-resolution 1H-NMR spectroscopy has been used to investigate the molecular basis of the Bohr effect in human normal adult hemoglobin in the presence of anions which serve as heterotropic effectors, i.e., Cl-, Pi, and 2,3-diphosphoglycerate. The individual H+ equilibria of 22-26 histidyl residues of hemoglobin in both deoxy and carbonmonoxy forms have been measured under buffer conditions chosen to demonstrate the effects of anion binding. The results indicate that β2His residues are binding sites for Cl- and Pi, in both dcoxy and carbonmonoxy forms, and that the affinity of this site for these anions is greater in the deoxy form. Recently assigned, the resonance of β146His does not show evidence of involvement in anion binding. The results also indicate that the binding of 2,3-diphosphoglycerate at the central cavity between the two β-chains in deoxyhemoglobin involves the β2His residues, and that the 2,3-diphosphoglycerate-binding site in carbonmonoxyhemoglobin may remain similar to that in deoxyhemoglobin. The interactions of Cl-, Pi and 2,3-diphosphoglycerate also result in changes in the pK values for other surface histidyl residues which vary in both magnitude and direction. The array of pK changes is specific for the interaction of each effector. The participation of β2His in the Bohr effect demonstrates that this residue can release or capture protons, depending on its protonation properties and its linkage to anion binding, and therefore provides an excellent illustration of the variable roles of a given amino acid. Although β146His does not bind anions, its contributions to the Bohr effect are substantially affected by the presence of anions. These results demonstrate that long-range electrostatic and/or conformational effects of anions binding play significant roles in the molecular basis of the Bohr effect of hemoglobin. © 1990.
