Effect of deoxygenation on ATP release from wild-type and transgenic mouse erythrocytes. Washed RBCs were oxygenated or deoxygenated and kept for 30 minutes at room temperature, and gently pelleted. The supernatant was collected and evaluated for ATP and Hb content. The pelleted RBCs were then lysed with ddH2O and the Hb concentration was measured to calculate erythrocyte numbers. ATP arising from hemolysis was subtracted from the total ATP in supernatant to yield net ATP released by deoxygenation. Absolute numbers of ATP released from intact erythrocytes containing the murine deoxyHb-binding site (wild-type), the human deoxyHb-binding site (w/ human Hb site), and no deoxyHb site (w/o Hb site) in oxygenated vs deoxygenated condition are 44 ± 4.0 pmol/108 cells vs 67 ± 5.1 pmol/108 cells, 39 ± 3.1 pmol/108 cells vs 54 ± 1.4 pmol/108 cells, and 53 ± 1.5 pmol/108 cells vs 60 ± 3.0 pmol/108 cells.
