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Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure
Author(s) -
BaudinCreuza Véronique,
Fablet Christophe,
Zal Franck,
Green Brian N.,
Promé Danielle,
Marden Michael C.,
Pagnier Josée,
Wajcman Henri
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.35702
Subject(s) - tetramer , chemistry , dimer , size exclusion chromatography , oligomer , cysteine , crystallography , trimer , chromatography , polymer chemistry , organic chemistry , enzyme
The effects of the mutation β9(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 β9 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 α‐ and β‐chains, whereas the oligomeric fraction contained only normal α‐chain and disulfide‐linked β 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.