2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S

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We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (c(sat)) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic c(sat) assay system, a steep dependence of c(sat) on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that c(sat) increased steeply in the same pH range (6.8 to 7.6). The effect of 2,3-DPG on c(sat) under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, c(sat) values for Hb and Hb-DPG were 19.56 ± 0.14 and 17.95 ± 0.45 g/dL, respectively, indicating that the solubility of Hb-DPG is lower than that of Hb by 8.2% ± 2.3%. Thus, binding of 2.3-DPG in the β-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be ~0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH, the corresponding c(sat) for Hb-DPG is 20.22 g/dL, which is the best estimate of the intrinsic solubility of T-state Hb S under conditions that approximate closely those of Ph, temperature, ionic strength, and 2,3-DPG saturation in the fully desaturated sickle erythrocyte.

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