MUTATIONAL ANALYSIS OF PHENYLALANINE BETA-85 IN THE VALINE BETA-6 ACCEPTOR POCKET DURING HEMOGLOBIN-S POLYMERIZATION

Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 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 beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, 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 beta 85 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 beta 85 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-F beta 85L, Hb S-F beta 85A, Hb S-F beta 85T, and Hb S-F beta 85W 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 beta 85 variants (Leu > Phe > Ala > Thr > Trp-beta 85) depends on amino acid hydrophobicity rather than stereospecificity of the side chain. These findings are in contrast to previous results for beta 88 variants. Trp-beta 85 in Hb S may affect Val-beta 6 acceptor pocket size, but may still accommodate insertion of Val-beta 6. These results also strengthen our previous conclusion that beta 88 amino acid stereospecificity is more critical than that of beta 85 for insertion of beta 6 Val.