title: Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain.
abstract: Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Ming F
authors.initials: MF
authors.lastname: Tam
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Tsuey-Chyi S
authors.initials: TC
authors.lastname: Tam
authors.forename: Ching-Hsuan
authors.initials: CH
authors.lastname: Tsai
authors.forename: Tong-Jian
authors.initials: TJ
authors.lastname: Shen
authors.forename: Virgil
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: Timothy N
authors.initials: TN
authors.lastname: Feinstein
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
authors.forename: Chien
authors.initials: C
authors.lastname: Ho
mesh_terms: D003911:Dextrans; D005319:Fetal Hemoglobin / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003911:Dextrans; D006451:Hemoglobin, Sickle; D011994:Recombinant Proteins; D005319:Fetal Hemoglobin; D010100:Oxygen