ROLE OF HYDROPHOBIC AMINO-ACIDS AT BETA-85 AND BETA-88 IN STABILIZINGF-HELIX CONFORMATION OF HEMOGLOBIN-S

Three Hb S variants containing Glu substitutions at Phe-beta 85 and/or Leu-beta 88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conf ormation of Hb S. Helix stability of tetrameric Hb beta F8E, beta L88E was measured by CD and compared with those of Hb S beta F85E, Hb S be ta L88E, Hb A, and Hb S, The CD spectra of these Hb S variants were si milar to those of Hb S and Hb A at 10 degrees C, However, changes in e llipticity at 222 nm for Hb S beta F85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S be ta L88E and Hb S beta F85E, beta L88E were similar and about 30-fold g reater than Hb S, Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unst able than Hb S, and stability of Hb S beta F85E, beta L88E was similar to that of Hb S beta L88E rather than Hb S beta F85E. These results s uggest that Glu insertion at both beta 85 and beta 88 makes heme inser tion into the heme pocket more difficult; however, once inserted, stab ility of Hb S beta F85E, beta L88E is similar to Hb S beta L88E rather than Hb S beta F85E, Furthermore, these results suggest that both Phe -beta 85 and Leu-beta 88 are critical for F helix stabilization and th at Glu insertion at beta 88 leads to more destabilization than inserti on at beta 85.