Thermodynamic aspects of the co-operativity in four-step oxygenation equilibria of haemoglobin.

Accurate oxygen equilibrium curves of human haemoglobin (concentration, 600 μm as haem) were determined by an automatic recording method (Imai et al., 1970) under a variety of conditions combining six different temperatures with seven sets of solute conditions, producing wide-ranging structural constraints on haemoglobin. The heat and entropy change of oxygenation for four individual steps (ΔHtand ΔSi, i = 1 to 4) were evaluated by a least-squares method directly from each set of six equilibrium curves without knowing the values of the four equilibrium constants kt. As shown in previous studies with dilute haemoglobin solutions (Imai & Tyuma, 1973; Imai & Yonetani, 1975b) ΔHi depended strongly on i; small amounts of heat were liberated at oxygenation steps involving the release of H+ and anions such as Cl−, 2,3-diphosphoglycerate, and inositol hexaphosphate, while large amounts of heat were liberated on the oxygenation of the R state or highly constrained T state, from which no or few non-haem ligands are released. The observed amounts of heat, when corrected for the heat of H+ and anion release associated with oxygenation, became uniform to a good approximation, indicating that the intrinsic heat of haem oxygenation is essentially equal for the four oxygenation steps, and a large part of the non-uniformity of ΔHi may be ascribed to the oxygen-linked release of the non-haem ligands. ΔSi exhibited similar behaviour. The relation, k1 ≲ k2 ≲ k3 ⪡ k4 which usually holds under physiological conditions, is a consequence of the presence of an enthalpy-entropy compensation process at the first three steps and its absence at the fourth step. The compensation temperature was around 300 K. The origin of the co-operativity cannot be specified as either an enthalpic or entropic effect. In the presence of 0.1 m-Cl− and 2 mm-2,3-diphosphoglycerate, the T to R transition at any oxygenation step is an endothermic process and haemoglobin gains entropy on the transition. The deoxy T structure is stabilised by the enthalpy term, while the oxy R structure is stabilised by the entropy term, so that the T to R transition occurs at a stop where the entropy contribution exceeds the enthalpy contribution. The present study shows that the oxygen-linked binding of non-haem ligands is very important in co-operative oxygen binding by haemoglobin, as predicted by Perutz, (1970).