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Protein Engineering, Vol. 13, No. 2, 113-120, February 2000
© 2000 Oxford University Press

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin {alpha} and ß chains

Abdul Hassan Mohammed Mawjood, Gentaro Miyazaki1, Rina Kaneko2, Yoshinao Wada2 and Kiyohiro Imai3

Department of Physiology and Biosignaling, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, 1 Department of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531 and 2 Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan

The cysteine residue at F9(93) of the human hemoglobin (Hb A) ß chain, conserved in mammalian and avian hemoglobins, is located near the functionally important {alpha}1–ß2 interface and C-terminal region of the ß chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the {alpha} chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4,4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala->Cys at 88{alpha} (rHb A88{alpha}C), Cys->Ala at 93ß (rHb C93ßA) and Cys->Thr at 93ß (rHb C93ßT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2–3.5 for rHb A88{alpha}C compared with 2.2 for Hb A, whereas those for rHb C93ßA and rHb C93ßT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88{alpha}C was smaller than that in Hb A. Our experimental data have shown that the residues at 88{alpha} and 93ß have definite roles but they have no functional homology. Structure–function relationships in our mutant Hbs are discussed.


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