BOHR EFFECT IN HEMOGLOBIN DEOXY CYANOMET INTERMEDIATES/

The Bohr protons released by oxygen exposure of the unliganded subunit s of intermediates (alpha(+CN)-beta>(alpha(+CN)-beta> and (alpha beta( +CN-))(Crp+CN3 were obtained by titrations of concentrated solutions o f these species. The Bohr protons released by oxygen exposure of the o ther intermediates were obtained from titrations of equilibrium mixtur es 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 te chniques. The Bohr effect of the intermediates was calculated by subtr acting the Bohr protons released by oxygen exposure of the intermediat es 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 t otal Bohr effect of deoxyhemoglobin is still significant. This suggest s that the Bohr effect in these intermediates is tertiary in the quate rnary T structure. The curve of the Bohr effect of intermediate (alpha (+CN-)beta(+CN-))(alpha beta), which was close to the curve obtained b y adding the Bohr effects of the two monoliganded intermediates at aci dic and physiological pH values, was significantly different from the curve obtained by adding the Bohr effects of one liganded subunit of i ntermediate (alpha(+CN-)beta)(alpha(+CN-)beta) and one liganded subuni t of intermediate (alpha beta(+CN-))(alpha beta(+CN-)). The Bohr effec t of intermediate (alpha(+CN)-beta)(alpha beta(+CN-)) was not determin ed, but the Bohr protons released by oxygen exposure of the equilibriu m mixture of this intermediate and the parental species (alpha(+CN-)be ta)(alpha(+CN-)beta) and (alpha beta(+CN-))(alpha beta(+CN-)) suggest independent contributions to the Bohr effect of intermediate (alpha(+C N-)beta)(alpha beta(+CN-)) from the Bohr effects of one liganded subun it of each parental species. These findings focus on the functional an d structural asymmetry of the diliganded intermediates (alpha(+CN-)bet a(+CN-))(alpha beta) and (alpha(+CN-)beta) (alpha beta(+CN-)), which i s predicted by the energetics of the same species [Smith, F. R., and A ckers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351; Perre lla, 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-l iganded intermediates retained a significant Bohr effect up to physiol ogical pH. The curve of the Bohr effect of intermediate (alpha(+CN-)be ta(+CN-))(alpha(+CN-)beta) was different from the curve calculated by adding the Bohr effects of intermediate (alpha(+CN-)beta)(alpha(+CN-)b eta) and one liganded beta subunit of intermediate (alpha beta(+CN-))( alpha beta(+CN-)). Similarly the curve of the Bohr effect of intermedi ate (alpha(+CN-)beta(CN-))(alpha beta(+CN-)) was different from the cu rve 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 s tructures of the liganded subunits in intermediates (alpha(+CN-)beta)( alpha(+CN-)beta) and (alpha beta(+CN-))(alpha beta(+CN-)) and the trip ly-liganded intermediates are different, despite the energetics, which indicates that all these species are in the quaternary R structure.