Role of the beta 146 histidyl residue in the alkaline Bohr effect of hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the effects of inorganic anions, such as phosphate or chloride, on the alkaline Bohr effect of normal human adult hemoglobin. By monitoring the chemical shift of the C2 proton of the beta 146 histidyl residue as a function of pH, we have determined its pK values in both ligated and unligated forms. In the presence of 0.1 M Bis-Tris buffer (with chloride ion concentration ranging from 0.005 to 0.06 M) in D2O at 27 degrees C, the pK value of the beta 146 histidine of deoxyhemoglobin is 7.98 +/- 0.03 and that of (carbon monoxy)hemoglobin is 7.85 +/- 0.03. However, in the presence of 0.2 M phosphate and 0.2 M NaCl in D2O at 27 degrees C, the corresponding pK values are 8.08 and 7.14, as previously reported by this laboratory [Kilmartin, J. V., Breen, J. J., Roberts, G. C. K., & Ho, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1246-1249]. This large difference in the pK value between the deoxy and carbon monoxy forms in the presence of 0.2 M phosphate and 0.2 M NaCl was interpreted as direct support for (1) the breaking of an intrasubunit salt bridge between beta 146 histidine and beta 94 aspartate when the hemoglobin molecule undergoes the quaternary structural transition as proposed by Perutz [Perutz, M. F. (1970) Nature (London) 228, 726-739] and (2) Perutz's suggestion that the beta 146 histidine is one of the amino acid residues responsible for the alkaline Bohr effect. The absence of a large change in the pK value of the beta 146 histidine in the presence of 0.1 M Bis-Tris buffer implies that (1) the above-mentioned intrasubunit salt bridge is not broken in going from the deoxy to the carbon monoxy form and (2) the beta 146 histidyl residue does not contribute significantly to the alkaline Bohr effect under these conditions. We have also found that in measuring the oxygen affinity of hemoglobin as a function of pH in the presence of 0.1 M Bis-Tris or 0.2 M phosphate plus 0.2 M NaCl (both in D2O), there is no significant difference in the alkaline Bohr effect in these two media. Hence, our results suggest that the detailed molecular mechanism for the Bohr effect depends on the experimental conditions.