Thermodynamic analysis of precisely measured oxygen equilibria of tench (Tinca tinca) hemoglobin and their dependence on ATP and protons.

Precise oxygen equilibria including extreme, high and low saturation values were determined for hemoglobin (Hb) from the freshwater teleost Tinca tinca at three temperatures, each at two pH levels and in the presence and absence of the erythrocytic cofactor ATP, at twofold molar excess over Hb. Analysis of the data in terms of Adair's successive oxygenation theory shows that in the absence of ATP, each of the four oxygenation steps are exothermic, but that net heat release decreases as pH falls from 8.2 to 7.4. ATP greatly depresses the temperature sensitivity of oxygenation particularly at physiological erythrocytic pH, where endothermic cofactor dissociation finds expression in a reverse temperature sensitivity for binding of the 3rd oxygen molecule to the tetrameric Hb. Enthalpy (delta Hi) and entropy (delta Si) changes of oxygenation vary with oxygenation step, i, as well as with pH and ATP addition, but the variations of delta Hi are similar to those of delta Si reflecting enthalpy-entropy compensation. The data show that the cooperative effects in tench Hb can be dominated either by entropic or enthalpic contributions, depending on the experimental condition and the oxygenation step.