Abstract
Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at β85 were made and expressed in yeast in an effort to evaluate the role of Phe‐β85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these β385 substitutions do not significantly affect heme‐globin interactions and tetramer helix content. Hb S containing Trp‐β85 had decreased oxygen affinity, whereas those with Leu‐, Ala‐, and Thr‐β85 had increased oxygen affinity. All four supersaturated β85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe‐β88, Ala‐β88, Glu‐β88, or Glu‐β85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477–17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563–31566). Leu substitution at β85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these β85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb S^Fβ85L^, Hb S^Fβ85A^, Hb S^Fβ85T^, and Hb s^Fβ85W^ are 0.65‐, 2.2‐, 2.5‐ and 3‐fold higher, respectively, than Hb S. These results suggest that the relative order for polymerization of β85 variants (Leu > Phe > Ala > Thr > Trp‐β85) depends on amino acid hydrophobicity rather than stereospecificity of the side chain. These findings are in contrast to previous results for β88 variants. Trp‐β85 in Hb S may affect Val‐β6 acceptor pocket size, but may still accommodate insertion of Val‐β6. These results also strengthen our previous conclusion that β88 amino acid stereospecificity is more critical than that of β85 for insertion of β6 Val.