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-P) on the DTNB reaction. For this reason, we replaced the carbonmonoxyhemoglobins with oxyhemoglobins and investigated the effect of inositol-P on the equilibrium and of their reactions with DTNB over the same pH range.

1-Benzyl-3-[(4-meth-oxy-phen-yl)imino]-indolin-2-one.

The title compound, CHNO, is a Schiff base obtained by condensing -arnisidine (4-meth-oxy-aniline) with -benzyl-isatin (1-benzyl-1-indole-2,3-dione), which crystallizes in the triclinic space group. The benzyl and phenyl rings subtend dihedral angles of 76.08 (7) and 60.

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 sulfhydryl groups is linked to three negatively contributing Bohr effect groups: His2β is present in all avian hemoglobins but absent in some mammalian hemoglobins; His77β and His143β 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) on the DTNB affinities of avian and mammalian carbonmonoxyhemoglobins. Inositol-Pdecreases by two orders of magnitude the DTNB affinity of guinea pig hemoglobin, which has His2β and the R2 quaternary structure.