Tertiary conformational transition in sheep hemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate

Abstract

We have determined the second-order reverse rate constant, k(R), for the reaction of 5,5 -dithiobis(2-nitrobenzoate) - DTNB - with sheep hemoglobins as a function of pH from values of the second-order forward rate constant, k(F), and the equilibrium constant, K(equ), at 25 degrees C: k(R)=k(F)K(equ). We demonstrate that (i) inositol hexakisphosphate (inositol-P(6)) decreases k(F) and k(R) by increasing K(rt), the rright harpoon over left harpoont tertiary conformation transition constant; (ii) the conformation favored for both the forward and reverse reactions is the r conformation. For stripped hemoglobin we obtain from the k(F) data a t isomer population of 34.6% (+/-14) prior to reaction with DTNB; from the k(R) data we calculate a t isomer population of 44.8% (+/-4) following reaction with DTNB. In the presence of inositol-P(6) the latter value is increased to 79.5% (+/-2). These results demonstrate that an allosteric transition occurs on reaction with DTNB and on inositol-P(6) binding.