Kinetic Characterisation of the R and R2 Quaternary Structures of Oxyhemoglobins Arising from Different Effects of Inositol Hexakisphosphate on Their Reactions with Ellman’s Reagent

In a previously reported equilibrium study of the reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with various carbonmonoxyhemoglobins over the pH range 5.6 to 9, we obtained contradictory results on the influence of the allosteric effector inositol hexakisphosphate (inositol-P₆) on the DTNB reaction. For this reason, we replaced the carbonmonoxyhemoglobins with oxyhemoglobins and investigated the effect of inositol-P₆ on the equilibrium and <i>kinetics</i> of their reactions with DTNB over the same pH range. We found that there are two sets of oxyhemoglobins: (i) In guinea fowl (major) and in dog oxyhemoglobin, inositol-P₆ <i>decreases</i> both the DTNB affinity and the apparent second-order rate constant of the DTNB reaction; and (ii) in the major and minor goat oxyhemoglobins, inositol-P₆ <i>increases</i> each of these two parameters. The x-ray structure of guinea pig methemoglobin shows that it has the R2 quaternary structure. Inositol-P₆ decreased the DTNB affinity of guinea pig oxyhemoglobin throughout our experimental pH range. On the basis of the guinea pig result, we associate the oxyhemoglobins in set (i) with the R2 quaternary structure and those in set (ii) with the R quaternary structure. We conclude that oxyhemoglobins that do not belong to either of these two sets - those of guinea fowl (minor), horse (major), donkey and human - contain equilibrium mixtures of the R and R2 quaternary structures.