Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure

Abstract

The effects of the mutation ␤9(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 ␤9 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 ␣-and ␤-chains, whereas the oligomeric fraction contained only normal ␣-chain and disulfide-linked ␤ 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 Å 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.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

“…In human Hb mutants Mississippi (␤9Ser¡Cys) and Hb Ta-li (␤83Gly¡Cys), polymerization is induced by the incorporation of a single Cys residue (6).…”

Two New β0-Thalassemic Mutations: A Deletion (−CC) at Codon 142 or Overlapping Codons 142-143, and an Insertion (+T) at Codon 45 or Overlapping Codons 44-45/45-46 of the β-Globin Gene

“…However, no abnormal β-globin chain was observed in reversed phase HPLC, and as in the majority of the described cases, the corresponding Hb variant was not detectable (10)(11)(12)(13)(14)(15). In addition, no elongated β-globin chain (theoretical monoisotopic mass 17325.01 Da) or more complex species were detected by electrospray ionization mass spectrometry (16,17). The increase of the Hb F level is difficult to understand.…”

Asymptomatic child heterozygous for hemoglobin S and hemoglobin Pôrto Alegre

“…Hemoglobin Pôrto Alegre (PA) is a rare hemoglobin resulting from a mutation in β9(A6)Ser→Cys, which results in high molecular weight oligomers through covalent [disulfide] bonds [3]. The normal electrophoretic mobility of a fresh un-oxidized hemolysate of homozygous patients indicates that the mutation causing Hb PA [does] not alter the net charge of the molecule, and that in vivo the molecule exists as a tetramer [4].…”

“…Hemoglobinopathies are the most common inherited disorders in humans; the most frequent are hemoglobins S and C [2]. Hemoglobin Pôrto Alegre results from a mutation in β9(A6)Ser→Cys which induces hemoglobin polymerization by forming interchain disulfide bonds via the extra cysteine [3]. Previous studies report that homozygous and heterozygous Hb PA forms polymers in vitro, but exists as a tetramer in vivo [4].…”