The binding of phosphorylated red cell metabolites to human hemoglobin A.

The binding affinity to hemoglobin of 2 ,J-bisphosphoglycerate, ATP, and some other phosphorylated red cell metabolites has been measured at pH 7.2 in 0.12 M KC1 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 2,3bisphosphoglycerate are 4.22 X 10e5 M at 22’ and 1.03 X 1O-4 M at 37” with deoxyhemoglobin and ~2.5 X lOma M at 22’ and 4.75 X low3 M at 37” with oxyhemoglobin. Dissociation constants for ATP are 8.54 X 10m5 M at 25” with deoxyhemoglobin and 2.55 X 10v3 M for oxyhemoglobin at 25”. Binding to ADP, 1,3-bisphosphoglycerate and glucose1, 6-PZ was measured. The dissociation constant for magnesium complexes of 2,3-bisphosphoglycerate to deoxyhemoglobin was too weak to measure (> 10m3 M at 37’) ; magnesium complexes of ATP have an apparent dissociation constant of 1.15 X 10m3na at 25”. The effects of the differential binding of cellular intermediates on red cell glycolysis and 2,3-bisphosphoglycerate metabolism are discussed.