A problem in hemoglobin studies is separating the allosteric and affinity factors that account for differences in the oxygen partial pressure at 50% saturation among various hemoglobins. Like oxygen binding to the hemes, the reaction of Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate)-DTNB for short-with the 93β sulfhydryl groups of hemoglobin is a reversible process. However, the reaction of DTNB with Cys93β of hemoglobin is devoid of allosteric factors. We present the results of equilibrium studies of the reaction of DTNB with thirty-eight heme-liganded hemoglobins between pH 5.6 and 9. Over a considerable pH range, inositol hexakisphosphate (inositol-P6) decreases the DTNB affinities of hemoglobins that have the R2 quaternary structure. By contrast, inositol-P6 increases the DTNB affinities of hemoglobins that have the R quaternary structure. Theoretical analyses of the pH dependence profiles show that inositol-P6 lowers the DTNB affinities of R2-state hemoglobins by shifting the equilibrium between two tertiary structures, r and t, in favor of the r structure. By contrast, inositol-P6 increases the DTNB affinities of R-state hemoglobins by shifting the r⇌t tertiary equilibrium in favor of the t structure. We suggest that the differences in the oxygen affinities of various hemoglobins may be accounted for, partially, by differences in their tertiary equilibria.
Okonjo et al. (Mon,) studied this question.