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

We have determined the second-order reverse rate constant, kR, 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, kF, and the equilibrium constant, Kequ, at 25 °C: kR=kFKequ. We demonstrate that (i) inositol hexakisphosphate (inositol-P6) decreases kF and kR by increasing Krt, the r ⇌ t 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 kF data a t isomer population of 34.6% (± 14) prior to reaction with DTNB; from the kR data we calculate a t isomer population of 44.8% (± 4) following reaction with DTNB. In the presence of inositol-P6 the latter value is increased to 79.5% (± 2). These results demonstrate that an allosteric transition occurs on reaction with DTNB and on inositol-P6 binding.