The molecular function of hemoglobin as reflected in ligand binding data: analysis of data on erythrocytes.

Hemoglobin oxygen binding data on erythrocytes at diffrent pH, PCO2 and bisphosphoglycerate concentrations have been analyzed in terms of an extended version of the Herzfeld-Stanley model of 1972. The binding of oxygen to subunits when the tetramer is in the quaternary oxy conformation was found to be insensitive to moderate changes in pH and pCO2 (0.71 +/- 0.05 mm Hg-1). Utilizing this circumstance it has been possible to obtain, for the first time, unique estimates of energy parameters related to hemoglobin cooperativity and effector action. At 37 degrees C, pH 7.2 and pCO2 22 mm Hg the following parameter values were obtained: The allosteric constant: (1.5 + 0.4)-10(4); the oxygen binding constant of the deoxy state: (5.4 +/- 0.3).10(-3) mm Hg-1; the 2,3-bisphosphoglycerate binding constants: (3.3 +/- 1.3).10(3)1. mol-1(deoxy), (1.3 +/- 0.5).10(2)1. mol-1 (oxy). Quarternary transition most likely takes place after binding of the second O2 molecule. Following the concepts of Perutz the results suggest that (1) protons and carbon dioxide act as constraint effectors and/or as quaternary effectors; (2) the difference in total conformational energy between the two quaternary ligand-free states is almost exclusively confined to molecular constraints and very little to the difference in quaternary conformational energy. The consistency of the results indicate that the model may be regarded as a useful tool for the description of the functional interrelations in the hemoglobin oxygenation process as reflected in oxygen binding data.