Okonjo, K. O. and Adeogun, I. A. and Babalola, J. O,
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: kR¼ kF Kequ. 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.
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