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)

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

“…We recently reported that the organic phosphate inositol hexakisphosphate -inositol-P 6 -increases the affinity of the CyF9[93]β sulphydryl group of sheep hemoglobins for DTNB by about an order of magnitude in the pH range 5.6 to 9 [14]. Here we report the determination of the apparent forward second-order rate constant, k F , over the same pH range.…”

“…The separation of the major and minor hemoglobins was achieved as follows in a cold room at 5°C [14]. A 3 cm (diameter) by 30 cm column of Whatman CMC-52 carboxymethyl cellulose, a microgranular, preswollen cation exchanger, was used.…”

“…We employed the method previously described [14] to study the kinetics of the reaction of DTNB with hemoglobin derivatives under pseudo-first order conditions. This was achieved by reacting each of the hemoglobin samples with at least a 60-fold excess of DTNB per sulphydryl group.…”

“…This abolition arises because iodoacetamide binds irreversibly to the CysF9[93]β sulphydryl and shifts the r ⇌ t equilibrium completely to the right, in favor of the t conformation [9]. By contrast, the reaction of this sulphydryl group with 5,5´-dithiobis(2-nitrobenzoate) -DTNB -is a reversible process [10][11][12][13][14] and therefore does not abolish the transition.…”

“…Here we report the determination of the apparent forward second-order rate constant, k F , over the same pH range. Using the K equ values that we recently published [14], together with the k F values reported here, we have calculated values of k R as a function of pH for the oxy, carbonmonoxy and aquomet derivatives of the major and minor hemoglobins of the sheep. We find that inositol-P 6 reduces the k R values of each of the hemoglobin derivatives by about an order of magnitude throughout the pH range 5.6 to 9.…”