Bohr effect in hemoglobin deoxy/cyanomet intermediates

The Bohr protons released by oxygen exposure of the unliganded subunits of intermediates (α+CN-β)(α+CN-β) and (αβ+CN-)(αβ+CN-) were obtained by titrations of concentrated solutions of these species. The Bohr protons released by oxygen exposure of the other intermediates were obtained from titrations of equilibrium mixtures of two parental species, (αβ)(αβ), (α+CN-β)(α+CN-β), (αβ+CN-)(αβ+CN-), and (α+CN-β+CN-)(α+CN-β +CN-), in which the concentration of the hybrid intermediate was determined by cryogenic electrophoretic techniques. The Bohr effect of the intermediates was calculated by subtracting the Bohr protons released by oxygen exposure of the intermediates from the total Bohr protons of deoxyhemoglobin at the same pH. The Bohr effects of intermediates (α+CN-β)(αβ) and (αβ+CN-)(αβ) were similar and vanished at pH 8 where the total Bohr effect of deoxyhemoglobin is still significant. This suggests that the Bohr effect in these intermediates is tertiary in the quaternary T structure. The curve of the Bohr effect of intermediate (α+CN-β+CN-)(αβ), which was close to the curve obtained by adding the Bohr effects of the two monoliganded intermediates at acidic and physiological pH values, was significantly different from the curve obtained by adding the Bohr effects of one liganded subunit of intermediate (α+CN-β) (α+CN-β) and one liganded subunit of intermediate (αβ+CN-) (αβ+CN-). The Bohr effect of intermediate (α+CN-β)(αβ+CN-) was not determined, but the Bohr protons released by oxygen exposure of the equilibrium mixture of this intermediate and the parental species (α+CN-β)(α+CN-β) and (αβ+CN-)(αβ+CN-) suggest independent contributions to the Bohr effect of intermediate (α+CN-β)(αβ+CN-) from the Bohr effects of one liganded subunit of each parental species. These findings focus on the functional and structural asymmetry of thediliganded intermediates (α+CN-β+CN-)(αβ) and (α+CN-β)(αβ+CN-), which is predicted by the energetics of the same species [Smith, F. R., & Ackers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351; Perrella, M., et al. (1990) Biophys. Chem. 35, 97-103; Daugherty, M. A., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. The triply-liganded intermediates retained a significant Bohr effect up to physiological pH. The curve of the Bohr effect of intermediate (α+CN-β+CN-)(α+CN-β) was different from the curve calculated by adding the Bohr effects of intermediate (α+CN-β)(α+CN-β) and one liganded β subunit of intermediate (αβ+CN-)(αβ+CN-). Similarly the curve of the Bohr effect of intermediate (α+CN-βCN-)(αβ+CN-) was different from the curve calculated by adding the Bohr effects of intermediate (αβ+CN-)(αβ+CN-) and one liganded α subunit of intermediate (α+CN-β)(α+CN-β). This suggests that the tertiary structures of the liganded subunits in intermediates (α+CN-β)(α+CN-β) and (αβ+CN-)(αβ+CN-) and the triply-liganded intermediates are different, despite the energetics, which indicates that all these species are in the quaternary R structure.