The Effect of 2,3-Diphosphoglycerate on the Tetramer-Dimer Equilibrium of Liganded Hemoglobin

Abstract The effect of 2,3-diphospho-d-glycerate (P2-glycerate) on the kinetic behavior of deoxyhemoglobin generated by the rapid dissociation of gaseous ligand (either by flash photolysis of carbon monoxide hemoglobin (HbCO) or by deoxygenation of oxyhemoglobin (HbO2) in the presence of sodium dithionite) is consistent with a stabilization of the tetrameric state of liganded hemoglobin by the organic phosphate. The fraction (α) of rapidly reacting hemoglobin produced by pulsed laser photolysis of phosphate-free HbCO in 0.05 m 2,2'-bis(hydroxymethyl)-2,2',2''-nitriloethanol-0.1 m NaCl, pH 7.0, was independent of CO concentration below about 500 µm, but increased systematically with dilution of the hemoprotein. The apparent tetramer-dimer dissociation constant (Kl4,2) calculated from the dependence of α on HbCO was 3.6 ± 1.0 µm in the absence of phosphates and decreased to 1.4 ± 0.3 µm when 1 mm P2-glycerate was added. These values of Kl4,2 are similar to estimates for liganded hemoglobin obtained in sedimentation experiments conducted at pH 7.0 in 0.1 m phosphate (Edelstein, S. J., Rehmer, M. J., Olson, J. S., and Gibson, Q. H. (1970) J. Biol. Chem. 245, 4372–4381) and in 0.1 m Tris-0.09 m NaCl (Kellett, G. L. (1971) J. Mol. Biol. 59, 401–424). The magnitude of the Soret absorbance drift accompanying deoxygenation of dilute solutions of HbO2 in the presence of sodium dithionite was decreased by added 1 mm P2-glycerate, and the second order rate constant characterizing the drift phase was increased at 20° from 0.54 ± 0.09 µm-1 s-1 to 1.57 ± 0.18 µm-1 s-1 by the organic phosphate. Since the drift has been shown to result from the formation of deoxy tetramers from deoxy αβ dimers (Kellett, G. L., and Gutfreund, H. (1970) Nature 227, 921–926), this result is also consistent with the proposed phosphate inhibition of dimer formation by liganded hemoglobin.