Phthalides are more potent modulators than 2,3-BPG in lowering oxygen affinity of HbF. (A) Molecular structure of z-butylidenephthalide (z-But). (B) Molecular structure of z-ligustilide (z-Lig). (C) OEC curves of purified HbF (orange), purified HbA (gray), HbF treated with 0.6 mM 2,3-BPG (green), HbA treated with 0.6 mM 2,3-BPG (purple), HbF treated with 0.6 mM z-butylidenephthalide (pink), and HbF treated with 4.0 mM z-butylidenephthalide (blue). (C) Inset shows an enlarged view of the area specified by gray dashed lines. (D) P₅₀ values for HbF treated with 2,3-BPG (gray columns), z-butylidenephthalide (orange columns) and z-ligustilide (purple columns) at varying levels of treatment ranging between 0.6 and 4.0 mM. (E) A comparison of P₅₀ for pure HbA (i), HbA treated with 4 mM 2,3-BPG (ii), pure HbF (iii), HbF treated solely with 4 mM 2,3-BPG (iv), and HbF cotreated with 4 mM 2,3-BPG and 2.5 to 4 mM phthalides (v-vii), which explicitly show that additional phthalide treatments can help raise the P₅₀ for HbF to a level similar to that of P₅₀ for HbA treated solely with the same level of 2,3-BPG. (F) P₅₀ evolution for HbF treated with 2,3-BPG only (gray curve) and cotreated with z-butylidenephthalide at 0.6 mM (green curve), 1.2 mM (orange curve), 2.5 mM (lime green curve), and 4.0 mM (purple curve), along with varying levels of 2,3-BPG. (G) P₅₀ evolution for HbF treated with 2,3-BPG only (gray curve), and cotreated with z-ligustilide of 0.6 mM (green curve), 1.2 mM (orange curve), and 4.0 mM (purple curve) along with varying levels of 2,3-BPG.