Effects of 2,3-diphosphoglycerate and other organic phosphate compounds on oxygen affinity and intracellular pH of human erythrocytes.

Abstract

The effects of changes of the 2,3-DPG content and of the total concentration of organic phosphates on the oxygen affinity and the intracellular pH of human erythrocytes were studied. The oxygen affinity as characterized by the P50 (oxygen tension at 50% O2 saturation) increases from 15 to 45 mm Hg when the 2,3-DPG concentration is elevated from 0.1 to 24 μmoles/g by incubation of erythrocytes in the presence of inosine, pyruvate and phosphate. In cells containing normal concentrations of 2,3-DPG, but accumulating high amounts of other organic phosphates during incubation with inosine and phosphate, the P50 was found to rise up to 36 mm Hg. This effect as well as a considerable part of the 2,3-DPG effect on the oxygen affinity of intact erythrocytes is due to a shift of the Donnan equilibrium induced by the accumulation of non-penetrating phosphate anions and consecutive changes of the intracellular pH, which in turn alter the oxygen affinity via the Bohr effect of hemoglobin. The intracellular pH is related to the intracellular concentration of organic phosphates (extracellular pH 7.40) by the equation: $${\text{pH}}_i {\text{ }} = {\text{ 7}}.306{\text{ }} - {\text{ }}0.0083{\text{ }} \cdot {\text{ }}P_{org} {\text{ }}(\mu moles organic P/g).$$ This dependency agrees closely with the theoretical relationship between the intracellular pH and the concentration of organic phosphates calculated from the osmolarities and the net charges of non-penetrating cell constituents. After correction of the oxygen affinities to a constant intracellular pH the P50 does not further increase in cells containing 2,3-DPG concentrations above 8 μmoles/g and remains unaltered in erythrocytes accumulating other organic phosphates.