Monomer shift assay to examine interactions of mutant α globins with β globin. (A) IEF (pH 6-8) of purified and in vitro-synthesized WT hemoglobins. (Left panel) Hemoglobins purified from human blood were fractionated by IEF and stained with Coomassie blue. Each lane contains 10 μg of purified protein. (Right panel) WT ³⁵S-α globin was synthesized by TNT with CN-hemin present and then unlabeled oxygenated β Hb subunit (20 nM final concentration) with or without HbA (5 μg = 3.1 μM final concentration in 25 μL) were added. The mixtures were fractionated by IEF and ³⁵S-labeled α globin complexes were detected by autoradiography. pH markers and the positions of purified hemoglobins are indicated on the left. As described in the text, purified α globin (lanes 2 and 6) migrates slightly faster than TNT-synthesized ³⁵S-α globin (lane 7) probably the result of posttranslational modification(s) that are specific to the TNT reaction. Also note that addition of excess HbA drives ³⁵S-α globin-β globin dimers into mixed HbA tetramers, which have a slightly lower pI than the αβ dimers (compare lanes 8 and 9). (B) WT and mutant ³⁵S-radiolabeled α globins were synthesized by TNT with CN-hemin present, and then unlabeled oxygenated β Hb subunit (20 nM) was added. The mixtures were fractionated by IEF (pH 6-8) and radiolabeled α globin (either free or in the context of α₁β₁ heterodimers) was visualized by autoradiography. (−) indicates no β globin added; and (+), β globin added.