title: Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure.
abstract: The effects of the mutation beta9(A6)Ser --> Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the beta9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher-molecular-weight oligomeric fraction (30%). Reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ESI-MS) under denaturing conditions showed that the tetrameric fraction contained only normal alpha- and beta-chains, whereas the oligomeric fraction contained only normal alpha-chain and disulfide-linked beta(PA) dimer. Under native conditions, ESI-MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 A diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA-CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer-dimer allosteric interface. None of the other variants investigated formed such a large oligomer.
authors.affiliation: INSERM, Unité 473, 94276 Le Kremlin-Bicêtre Cedex, France. baudin@kb.inserm.fr
authors.forename: Véronique
authors.initials: V
authors.lastname: Baudin-Creuza
authors.forename: Christophe
authors.initials: C
authors.lastname: Fablet
authors.forename: Franck
authors.initials: F
authors.lastname: Zal
authors.forename: Brian N
authors.initials: BN
authors.lastname: Green
authors.forename: Danielle
authors.initials: D
authors.lastname: Promé
authors.forename: Michael C
authors.initials: MC
authors.lastname: Marden
authors.forename: Josée
authors.initials: J
authors.lastname: Pagnier
authors.forename: Henri
authors.initials: H
authors.lastname: Wajcman
mesh_terms: D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D006455:Hemoglobins, Abnormal / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
chemical_list: D004220:Disulfides; D006455:Hemoglobins, Abnormal; D012694:Serine; C034991:hemoglobin Porto Alegre; D003545:Cysteine