K. Adachi et al., MUTATIONAL ANALYSIS OF PHENYLALANINE BETA-85 IN THE VALINE BETA-6 ACCEPTOR POCKET DURING HEMOGLOBIN-S POLYMERIZATION, Protein science, 4(7), 1995, pp. 1272-1278
Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at be
ta 85 were made and expressed in yeast in an effort to evaluate the ro
le of Phe-beta 85 in the acceptor pocket during polymerization of deox
y Hb S. The four Hb S variants have the same electrophoretic mobility
as Hb S, and these beta 85 substitutions do not significantly affect h
eme-globin interactions and tetramer helix content. Hb S containing Tr
p-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-
, and Thr-beta 85 had increased oxygen affinity. All four supersaturat
ed 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 ti
me (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 polymeriza
tion, whereas Ala, Thr, or Trp substitution inhibited polymerization.
The length of the delay time and total polymer formed for these beta 8
5 Hb S variants depended on hemoglobin concentration in the same fashi
on as for deoxy Hb S: the higher the concentration, the shorter the de
lay 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 orde
r for polymerization of beta 85 variants (Leu > Phe > Ala > Thr > Trp-
beta 85) depends on amino acid hydrophobicity rather than stereospecif
icity of the side chain. These findings are in contrast to previous re
sults 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.