Effects of Different β73 Amino Acids on Formation of 14-Stranded Fibers of Hb S versus Double-Stranded Crystals of Hb C-Harlem^†

Hb S (α₂β₂^6Glu→Val) forms polymers, while Hb C-Harlem (α₂β₂^{6Glu→Val,73Asp→Asn}) forms crystals upon oversaturation. Since the only difference between the two is the β73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. β73 Asp in Hb S forms a hydrogen bond with β4 Thr, while β73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix β6 Val acceptor sites. Two new β73 Hb S variants (β73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the β73−β4 hydrogen bond (β73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix β6 Val acceptor sites (β73 Leu). β73 His Hb S formed fibers but at a lower concentration than Hb S, while β73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of β73 His Hb S was ¹/₇ of that of Hb S, while the solubility of β73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for β73 His Hb S was 10⁵-fold shorter than that for Hb S, while that for β73 Leu Hb S was 10⁵-fold longer in 1.0 M phosphate buffer. NMR results indicate β73 amino acid changes induce alteration in the β-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the β73−β4 hydrogen bond and/or induced changes in the heme pocket by the β73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the β73−β4 hydrogen bond and enhancing the hydrophobicity of the EF helix β6 Val acceptor sites by the β73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.