Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and β hemoglobin chains

  • Paolo AscenziEmail author
  • Alessandra di Masi
  • Giovanna De Simone
  • Magda Gioia
  • Massimo Coletta
Original Paper


Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αβ dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(–N3) and Hp2-2:Hb(III)(–N3) follow a two-step process. However, azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric β and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

Graphical abstract


Human haptoglobin 1-1:hemoglobin complex Human haptoglobin 2-2:hemoglobin complex Azide binding Fluoride binding Kinetics Thermodynamics 


CCP domain

Complement control protein domain


Human hemoglobin


Ferric Hb


Human haptoglobin


Phenotype 1-1 of Hp


Phenotype 2-2 of Hp


Ferric Hp1-1:Hb complex


Ferric Hp 2-2:Hb complex

SP-like domain

Serine protease-like domain



The Grant of Dipartimenti di Eccellenza, MIUR (Legge 232/2016, Articolo 1, Comma 314-337) is gratefully acknowledged.


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Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  • Paolo Ascenzi
    • 1
    Email author
  • Alessandra di Masi
    • 2
  • Giovanna De Simone
    • 2
  • Magda Gioia
    • 3
    • 4
  • Massimo Coletta
    • 3
    • 4
  1. 1.Interdepartmental Laboratory for Electron MicroscopyRoma Tre UniversityRomaItaly
  2. 2.Department of SciencesRoma Tre UniversityRomaItaly
  3. 3.Department of Clinical Sciences and Translational MedicineUniversity of Roma “Tor Vergata”Roma,Italy
  4. 4.Interuniversity Consortium for the Research on the Chemistry of Metals in Biological SystemsBariItaly

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