The Binding of Phosphorylated Red Cell Metabolites to Human Hemoglobin A

The binding affinity to hemoglobin of 2,3-bisphosphoglycerate, ATP, and some other phosphorylated red cell metabolites has been measured at pH 7.2 in 0.12 m KCl to simulate physiological conditions. The dissociation constant with 2,3-bisphosphoglycerate is independent of hemoglobin concentration in the range 0.06 to 4 mm. There is one binding site per deoxyhemoglobin tetramer. Phosphorylated compounds bind weakly to one site per tetramer of oxyhemoglobin. Dissociation constants determined for 2,3-bisphosphoglycerate are 4.22 x 10-5 m at 22° and 1.03 x 10-4 m at 37° with deoxyhemoglobin and ∼2.5 x 10-3 m at 22° and 4.75 x 10-3 m at 37° with oxyhemoglobin. Dissociation constants for ATP are 8.54 x 10-5 m at 25° with deoxyhemoglobin and 2.55 x 10-3 m for oxyhemoglobin at 25°. Binding to ADP, 1,3-bisphosphoglycerate and glucose-1,6-P2 was measured. The dissociation constant for magnesium complexes of 2,3-bisphosphoglycerate to deoxyhemoglobin was too weak to measure (g 10-3 m at 37°); magnesium complexes of ATP have an apparent dissociation constant of 1.15 x 10-3 m at 25°. The effects of the differential binding of cellular intermediates on red cell glycolysis and 2,3-bisphosphoglycerate metabolism are discussed.