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

Paolo Ascenzi1, Giovanna De Simone2, Fabio Polticelli2,3

  • 1Interdepartmental Laboratory of Electron Microscopy, Roma Tre University, Via Della Vasca Navale 79, 00146, Rome, Italy. ascenzi@uniroma3.it.

Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry
|April 2, 2018
PubMed

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 [Formula: see text] [(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 hemoglobinHaptoglobin1-1:hemoglobin complexHaptoglobin2-2:hemoglobin complexHuman haptoglobin 1-1Human haptoglobin 2-2KineticsReductive nitrosylation

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