Modification of HbS with iMet and acetyl-iMet increases oxygen affinity and delays polymerization. Purified samples of modified HbS (Table 2; supplemental Figure 4) were tested for oxygen binding and polymerization under complete hypoxia. (A) Oxygen affinity determined from OECs in the presence of 5 mM 2,3-BPG at 37°C. 2,3-BPG stabilizes deoxyhemoglobin and is added to mimic levels in RBCs. OEC of unmodified HbS (red line, 21.1 torr) shows that p50 for pure HbS is lower than p50 measured in intact RBCs in whole blood (∼28 torr; Figure 5D). Modified HbS samples have higher affinities for oxygen, as shown by OEC for modified HbS containing unmodified β^S-globin with a mixture of acetyl-iMet–α-globin and iMet–α-globin (blue line, 19.0 torr), OEC for iMet–β^S-globin with a mixture of acetyl-iMet–α-globin, iMet–α-globin, and unmodified α-globin (purple line, 18.2 torr), and OEC for acetyl-iMet–β^S-globin with a mixture of acetyl-iMet–α-globin, iMet–α-globin, and unmodified α-globin (green line, 13.1 torr). (B) Acetyl-iMet–β^S-globin modification of HbS delays polymerization under hypoxia, as shown by turbidity increases at 700 nm of samples assayed at 1.2 mg/mL in 1% sodium metabisulfite and 1.895 M potassium phosphate, pH 7.5, in sealed cuvettes at 37°C. The delay time for acetyl-iMet–β^S-globin with a mixture of acetyl-iMet–α-globin, iMet–α-globin, and unmodified α-globin (green lines) is increased by 80 seconds (17%), and the midpoint time is increased by 100 seconds (20%) relative to HbS (red lines). Polymerization of unmodified β^S-globin with a mixture of acetyl-iMet–α-globin and iMet–α-globin (blue lines) or iMet–β^S-globin with a mixture of acetyl-iMet–α-globin, iMet–α-globin, and unmodified α-globin (purple lines) are similar to unmodified HbS. Similar results were obtained in 2 independent studies. (C) For 0.9 mg/mL HbS samples, the delay times of acetyl-iMet–β^S-globin with the mixture of α-globins (green lines) is increased by 54 seconds (10%) and the midpoint time increased by 83 seconds (14%) relative to HbS (red lines). (D) Dilution of purified acetyl-iMet–β^S-globin into pure HbS to a final concentration of 0.9 mg/mL significantly delays polymerization of the bulk hemoglobin mixture starting at 30% acetyl-iMet–β^S-globin and 70% HbS. The delay time to initiate turbidity increase and the time to reach the midpoint of maximal turbidity were determined using a modified high phosphate buffer assay of HbS polymerization.²⁷  ***P < .005, **P < .01, *P < .05, 2-tailed Student t test.