Three Hb S variants containing Glu substitutions at Phe-beta85 and/or Leu-beta88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S. Helix stability of tetrameric Hb S betaF85E,betaL88E was measured by CD and compared with those of Hb S betaF85E, Hb S betaL88E, Hb A, and Hb S. The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10 degrees C. However, changes in ellipticity at 222 nm for Hb S betaF85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S betaL88E and Hb S betaF85E,betaL88E were similar and about 30-fold greater than Hb S. Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S betaF85E,betaL88E was similar to that of Hb S betaL88E rather than Hb S betaF85E. These results suggest that Glu insertion at both beta85 and beta88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S betaF85E, betaL88E is similar to Hb S betaL88E rather than Hb S betaF85E. Furthermore, these results suggest that both Phe-beta85 and Leu-beta88 are critical for F helix stabilization and that Glu insertion at beta88 leads to more destabilization than insertion at beta85.