Varying apparent rate constant: determination of uptake and release of protons during tetramer-dimer dissociation in...

The effect of association-dissociation on the sulphydryl reactivity of human hemoglobin A is reported. The reactivity of CysF9(93)beta towards the sulphydryl reagent, 5,5'-dithiobis(2-nitrobenzoate), is higher at lower concentrations of hemoglobin at all pH values. This is because hemoglobin dimers have higher sulphydryl reactivity than tetramers and it is known that the proportion of dimers increases as the hemoglobin concentration decreases. This study takes advantage of this observation to determine the tetramer-dimer dissociation constant, K(4,2), of hemoglobin A and subsequently the proton uptake and the proton release during this process. The concentration dependence profiles of the apparent second-order rate constants, k(app), show that (between 2 and 20 microM heme) k(app) decreases with increasing hemoglobin concentration. Above 30 M heme k(app) remains fairly constant for all hemoglobin derivatives (oxy, carbonmonoxy and aquomethemoglobin) used. The pH dependence of the negative logarithm of tetramer-dimer dissociation constant, pK(4,2), for oxy- (and for carbonmonoxy-) hemoglobin exhibits a biphasic character with a maximum near pH 7.4 (and 6.6). For aquomethemoglobin, pK(4,20 decreases with increasing pH. The tetramer-dimer dissociation of human oxyhemoglobin A at an ionic strength of 200 mM uptakes 0.87 +/- 0.09 mole of protons between pH 6.2 to 7.4 phase and releases 0.84 0.09 mole of protons between pH 7.4 and 9.0 phase. Under a similar condition carbonmonoxyhemoglobin uptakes 0.54 +/- 0.05 mole of protons between pH 5.8 and 6.6 phase and releases 0.48 +/- 0.05 mole of protons between pH 6.6 and 9.0 phase. Aquomethemoglobin has only a single phase, it releases 0.39 +/- 0.05 mole of protons during tetramer-dimer dissociation.