Allosteric effects in cobaltohaemoglobin as studied by precise oxygen equilibrium measurements

Accurate oxygen equilibrium curves of cobalt-substituted haemoglobin (cobalto-haemoglobin) were determined by the automatic recording method of Imai et al. (1970) in a wide range of pH values and in the presence and absence of allosteric effectors such as chloride ion, 2,3-diphosphoglycerate, inositol hexaphosphate, and orthophosphate. Cobaltohaemoglobin preserves a full Bohr effect in accordance with earlier observations. As in ferrohaemoglobin, the Adair constants (k₁, i=1 to 4), which measure the oxygen affinity for the four individual oxygenation steps, depend non-uniformly on pH, indicating that the number of protons released upon oxygenation differs for each step. The effectors altered the overall oxygen affinity, the co-operativity of oxygen binding, and the Adair constants for cobaltohaemoglobin. As in ferrohaemoglobin, chloride ion, 2,3-diphosphoglycerate, and orthophosphate lower the overall oxygen affinity and increase the co-operativity by reducing k₁, k₂ and k₃ without causing significant change of k₄, while inositol hexaphosphate lowers the overall oxygen affinity by reducing all k values. In the absence of inositol hexaphosphate, the k₄ values are similar to the oxygen association constants for free chains, implying that cobaltohaemoglobin becomes free of structural constraints prior to the last step of oxygenation. Analysis according to the allosteric model of Monod et al. (1965) indicates that the metal substitution of cobalt for iron causes a drastic shift of the allosteric equilibrium towards the R structure and an increase of the oxygen affinity of the T structure. The present results are consistent with the distributed energy model of Hopfield (1973) and, by implication, support the trigger mechanism of Perutz (1970) for co-operative oxygen binding.