Reductive nitrosylation of ferric human hemoglobin bound to human haptoglobin 1-1 and 2-2

  • Paolo Ascenzi
  • Giovanna De Simone
  • Fabio Polticelli
  • Magda Gioia
  • Massimo Coletta
Original Paper
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Abstract

Haptoglobin (Hp) sequesters hemoglobin (Hb) preventing the Hb-based damage occurring upon its physiological release into plasma. Here, reductive nitrosylation of ferric human hemoglobin [Hb(III)] bound to human haptoglobin (Hp) 1-1 and 2-2 [Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively] has been investigated between pH 7.5 and 9.5, at T=20.0 °C. Over the whole pH range explored, only one process is detected reflecting NO binding to Hp1-1:Hb(III) and Hp2-2:Hb(III). Values of the pseudo-first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation (k) do not depend linearly on the ligand concentration but tend to level off. The conversion of Hp1-1:Hb(III)-NO to Hp1-1:Hb(II)-NO and of Hp2-2:Hb(III)-NO to Hp2-2:Hb(II)-NO is limited by the OH−- and H2O-based catalysis. In fact, bimolecular NO binding to Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(II), and Hp2-2:Hb(II) proceeds very rapidly. The analysis of data allowed to determine the values of the dissociation equilibrium constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation [K = (1.2 ± 0.1) × 10−4 M], which is pH-independent, and of the first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) conversion to Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively (k′). From the dependence of k′ on [OH−], values of hOH– [(4.9 ± 0.6) × 103 M−1 s−1 and (6.79 ± 0.7) × 103 M−1 s−1, respectively] and of \( h_{{{\text{H}}_{ 2} {\text{O}}}} \) [(2.6 ± 0.3) × 10−3 s−1] were determined. Values of kinetic and thermodynamic parameters for Hp1-1:Hb(III) and Hp2-2:Hb(III) reductive nitrosylation match well with those of the Hb R-state, which is typical of the αβ dimers of Hb bound to Hp.

Keywords

Ferric human hemoglobin Human haptoglobin 1-1 Human haptoglobin 2-2 Haptoglobin1-1:hemoglobin complex Haptoglobin2-2:hemoglobin complex Reductive nitrosylation Kinetics 

Abbreviations

CCP

Complement control protein

Hb

Hemoglobin

Hb(II)

Ferrous Hb

Hb(III)

Ferric Hb

Hb(II)-NO

Nitrosylated Hb

Hp

Haptoglobin

Hp1-1

Phenotype 1-1 of Hp

Hp2-2

Phenotype 2-2 of Hp

Hp1-1:Hb

Hp1-1:Hb complex

Hp1:Hb(II)

Hp1-1:Hb(II) complex

Hp1-1:Hb(III)

Hp1-1:Hb(III) complex

Hp1-1:Hb(II)-NO

Hp1-1:Hb(II)-NO complex

Hp2-2:Hb

Hp2-2:Hb complex

Hp2-2:Hb(II)

Hp2-2Hb(II) complex

Hp2-2:Hb(III)

Hp2-2:Hb(III) complex

Hp1-1:Hb(II)-NO

Hp1-1:Hb(II)-NO complex

Mb

Myoglobin

Mb(III)

Ferric Mb

Ngb

Neuroglobin

SP-like domain

Serine protease-like domain

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Notes

Acknowledgements

The grant of Excellence Departments, MIUR (Legge 232/2016, Articolo 1, Comma 314-337), is gratefully acknowledged.

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Copyright information

© SBIC 2018

Authors and Affiliations

  • Paolo Ascenzi
    • 1
  • Giovanna De Simone
    • 2
  • Fabio Polticelli
    • 2
    • 3
  • Magda Gioia
    • 4
    • 5
  • Massimo Coletta
    • 4
    • 5
  1. 1.Interdepartmental Laboratory of Electron MicroscopyRoma Tre UniversityRomeItaly
  2. 2.Department of SciencesRoma Tre UniversityRomeItaly
  3. 3.Roma Tre SectionNational Institute of Nuclear PhysicsRomeItaly
  4. 4.Department of Clinical Sciences and Translational MedicineUniversity of Roma “Tor Vergata”RomeItaly
  5. 5.Interuniversity Consortium for the Research on the Chemistry of Metals in Biological SystemsBariItaly

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