Transition of haemoglobin between two tertiary conformations: Inositol hexakisphosphate increases the transition constant and the affinity of sheep haemoglobin for 5,5′-dithiobis(2-nitrobenzoate)

The equilibrium constant (Kequ) for the reaction of 5,5′-dithiobis(2-nitrobenzoate) - DTNB - with the CysF9[93]β sulphydryl group of the haemoglobins of the sheep decreases by about two orders of magnitude between pH ≈ 5.6 and 9.2: from a mean of 7.2 ± 1 to a mean of 0.044 ± 0.01. Calculations from the pH dependence of Kequ show that in the r ⇌ t tertiary conformational transition of haemoglobin the t isomer population is 50.7 and 61.8% for the major and minor haemoglobins, respectively. In the presence of inositol hexakisphosphate (inositol-P6), Kequ increases for both haemoglobins by about an order of magnitude through most of the pH range. The t isomer population also increases to 82.1 and 79.6% for the major and minor haemoglobins, respectively. These results indicate that inositol-P6 increases the affinity of the sulphydryl for DTNB by increasing the population of the t isomer. It is highly probable that a minimum four-state model that includes the r ⇌ t transition is required for a full understanding of haemoglobin function.