R and R2 quaternary structures of carbonmonoxyhemoglobins: Differential effect of inositol hexakisphosphate on their affinity for Ellman's reagent

The reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with hemoglobin sulthydryl groups is linked to three negatively contributing Bohr effect groups: His2 beta is present in all avian hemoglobins but absent in some mammalian hemoglobins; His77 beta and His143 beta are absent in avian but present in nearly all mammalian hemoglobins. To probe the consequences of these differences, we determined the influence of inositol hexakisphosphate (inositol-P-6) on the DTNB affinities of avian and mammalian carbonmonoxyhemoglobins. Inositol-P-6-decreases by two orders of magnitude the DTNB affinity of guinea pig hemoglobin, which has His2 beta and the R2 quaternary structure. It decreases, or has no effect on, the DTNB affinities of hemoglobins that have His2 beta and whose structures lie along the R2 reversible arrow R quaternary equilibrium. Finally, inositol-P(6)increases by one to two orders of magnitude the DTNB affinities of hemoglobins that lack His2 beta. Thus His2 beta, DTNB and inositol-P6, in combination, distinguish the R2 from the R quaternary structure.