Heme chain exchange was employed to investigate the dimer dissociation reaction of human apohemoglobin in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees C. Incubation of apohemoglobin (alpha 0 beta 0) with either alpha h (or beta h) allowed the monitoring of the formation of a semihemoglobin alpha h beta 0 (or alpha 0 beta h) species with time. Analysis revealed that the rate of formation of both semihemoglobins was essentially identical and coincided with the disappearance of heme chain. Time courses were exponential and followed first order kinetics yielding a dimer dissociation rate constant of 0.54 (+/- 0.07) h-1. A study over the pH range from 6.5 to 8.0 revealed that this dissociation rate exhibited a maximum at pH 7.0 (implicating a histidyl residue). The effect of temperature (6-37 degrees C) on this dimer dissociation rate yielded a linear Arrhenius Plot and an energy of activation of 7.2 kcal/mol. These results are consistent with alpha G-beta G helical pairing being a major contributor to apohemoglobin dimer integrity.
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