Effect of inositol hexakisphosphate on sulphydryl reactivities of low (cat) and high (human) oxygen affinity haemoglobins
Keywords:Cat and human haemoglobins, oxygen affinities, sulphydryl reactivities, organic phosphate, 5,5?-dithiobis (2-nitrobenzoate)
The effect of organic phosphate binding on the reactivities of low-affinity cat haemoglobin in comparison to high-affinity
human haemoglobin was determined. Kinetics of the reaction of the CysF9? sulphydryl groups in both haemoglobins
with 5,5?-dithiobis(2-nitrobenzoate) – DTNB – which has been linked to oxygen binding, was used to probe this effect.
Plots of the pseudo-first order rate constant, kobs, as a function of the DTNB concentration were linear and had positive
intercepts. This is indicative of the fact that the organic phosphate used, inositol hexakisphosphate (inositol-P6), does not
abolish reversibility in both haemoglobins. Cat haemoglobins gave simple pH-dependence profiles for the apparent
second order forward rate constants, kF. The presence of inositol-P6 increased kF, hence reactivity, by two-fold throughout
the experimental pH range for cat oxyhaemoglobin. In contrast, inositol-P6 decreased kF for human oxyhaemoglobin.
Around the physiological pH, kF decreased from a maximum value of 31.9 ? 0.6 mol-1dm3s-1 to 26.7 ? 0.3 mol-1dm3s-1 for
human haemoglobin, while it increased from a minimum of 17.2 ? 0.4 mol-1dm3s-1 to 20.6 ? 0.6 mol-1dm3s-1 for cat minor