BOHR EFFECT IN HEMOGLOBIN DEOXY/CYANOMET INTERMEDIATES

The Bohr protons released by oxygen exposure of the unliganded subunits of intermediates (alpha(+CN)-beta>(alpha(+CN)-beta> and (alpha beta(+CN-))(Crp+CN3 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, (alpha beta)(alpha beta), (alpha(+CN-)beta)(alpha(+CN-)beta), (alpha beta(+CN-))(alpha beta(+CN-)), and (alpha(+CN-)beta(+CN-))(alpha(+CN-)beta(+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 (alpha(+CN-)beta)(alpha beta) and (alpha beta(+CN-))(alpha beta) 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 (alpha(+CN-)beta(+CN-))(alpha beta), 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 (alpha(+CN-)beta)(alpha(+CN-)beta) and one liganded subunit of intermediate (alpha beta(+CN-))(alpha beta(+CN-)). The Bohr effect of intermediate (alpha(+CN)-beta)(alpha beta(+CN-)) was not determined, but the Bohr protons released by oxygen exposure of the equilibrium mixture of this intermediate and the parental species (alpha(+CN-)beta)(alpha(+CN-)beta) and (alpha beta(+CN-))(alpha beta(+CN-)) suggest independent contributions to the Bohr effect of intermediate (alpha(+CN-)beta)(alpha beta(+CN-)) from the Bohr effects of one liganded subunit of each parental species. These findings focus on the functional and structural asymmetry of the diliganded intermediates (alpha(+CN-)beta(+CN-))(alpha beta) and (alpha(+CN-)beta) (alpha beta(+CN-)), which is predicted by the energetics of the same species [Smith, F. R., and 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 (alpha(+CN-)beta(+CN-))(alpha(+CN-)beta) was different from the curve calculated by adding the Bohr effects of intermediate (alpha(+CN-)beta)(alpha(+CN-)beta) and one liganded beta subunit of intermediate (alpha beta(+CN-))(alpha beta(+CN-)). Similarly the curve of the Bohr effect of intermediate (alpha(+CN-)beta(CN-))(alpha beta(+CN-)) was different from the curve calculated by adding the Bohr effects of intermediate (alpha beta(+CN-)(alpha beta(+CN-)) and one liganded alpha subunit of intermediate (alpha(+CN-)beta)(alpha(+CN-)beta). This suggests that the tertiary structures of the liganded subunits in intermediates (alpha(+CN-)beta)(alpha(+CN-)beta) and (alpha beta(+CN-))(alpha beta(+CN-)) and the triply-liganded intermediates are different, despite the energetics, which indicates that all these species are in the quaternary R structure.