Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Oversaturated deoxy-α2β2T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α2β2E6V,D73H. Solubility of deoxy-α2β2T4V was ∼10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α2β2E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α2β2E6V,D73N), while α2β2T4V and α2β2D73H as well as HbS, α2β2E6V,D73H and α2β2E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.