Effect of β-chain surface charge on relative amounts of in vitro assembled tetramers as a function of varying amounts of input α chains. (A) Equimolar mixtures of normal (β^A), sickle (β^S), β16 Gly→Asp, β95 Lys→Glu or β120 Lys→Glu chains (75 μmol/L) were added to varying amounts of α-globin chain in 0.1 mol/L phosphate buffer, pH 7.0 at 25°C, and assembled tetramers were analyzed by FPLC. The relative ratio (y axis) of Hb S to Hb A (•) in (β^S + β^A)/α mixtures, Hb βG16D to Hb A (○) in (β16 Asp +β^A)/α mixtures, Hb βK95E to Hb A (□) in (β95 Glu +β^A)/α mixtures and Hb βK120E to Hb A (▪) in (β120 Glu +β^A)/α mixtures was calculated as a function of varying amounts of input α-globin chain. (B) Equimolar mixtures of β^s with β16 Gly→Asp, β95 Lys→Glu, β120 Lys→Glu, β16 Gly→Asp, 120 Lys→Glu or β112 Cys→Asp chains (75 μmol/L) were added to increasing amounts of α-globin chain in 0.1 mol/L phosphate buffer, pH 7.0 at 25°C. Tetramer formation was analyzed by FPLC, and the relative ratio of Hb A to Hb S (•), Hb βC112D to Hb S (▵), Hb βG16D to Hb S (o), Hb βK95E to Hb S (□), Hb βK120E to Hb S (▪), and Hb βG16D, K120E to Hb S (▴) was calculated (y axis).