The Release of Protons and Anions during Ligand Binding to Human Deoxyhemoglobin

A direct comparison has been made of the time courses of ligand binding to, anion release from, and proton release from deoxyhemoglobin A. All three processes exhibit different kinetic features. To a first approximation, protons are expelled as each ligand molecule is bound, but anions are released only after several ligand molecules have been bound. Under conditions when the α and β chains react at the same rates with ligands (carbon monoxide binding in the absence of phosphates or n-butyl isocyanide binding at low ionic strength), the time course of proton release is proportional to the time course of ligand binding. However, when the β chains react more rapidly with ligands than the α chains (carbon monoxide binding in the presence of inositol hexaphosphate or n-butyl isocyanide binding in the presence of salts), a lag is observed in the time course of proton release. About 20% of the total amount of protons displaced are released during ligand binding to the β chains and the remaining 80% are released during ligand binding to the α chains. These results suggest that many microscopic conformational equilibria are required for an adequate description of the functional behavior of hemoglobin.