Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

Free Radic Biol Med. 2012 Sep 15;53(6):1317-26. doi: 10.1016/j.freeradbiomed.2012.07.023. Epub 2012 Jul 27.

Abstract

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb-Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb-Hp exhibited active-site heterogeneity and noncooperative, high-affinity O(2) binding (n(1/2)=0.88, P(1/2)=0.33 mm Hg in inorganic phosphate buffer at pH 7 and 25 °C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O(2) dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb-Hp had significantly reduced absorption at the λ(max) of 430 nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E(1/2)) of Hb-Hp was found to be +54 mV, showing that it is much more easily oxidized than uncomplexed Hb (E(1/2)=+125 mV). The Nernst plots for Hb-Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb-Hp was unchanged by pH over the range of 6.4-8.3. Exposure of Hb-Hp to excess hydrogen peroxide (H(2)O(2)) produced ferryl heme, which was found to be more kinetically inert in the Hb-Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb-Hp complex.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Carbon Monoxide / chemistry
  • Cyclic N-Oxides / chemistry
  • Free Radical Scavengers / chemistry
  • Haptoglobins / chemistry*
  • Hemoglobins / chemistry*
  • Humans
  • Hydrogen Peroxide / chemistry*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Oxidants / chemistry*
  • Oxidation-Reduction
  • Oxygen / chemistry
  • Protein Binding
  • Protein Multimerization
  • Protein Stability
  • Protein Subunits / chemistry

Substances

  • Cyclic N-Oxides
  • Free Radical Scavengers
  • HP protein, human
  • Haptoglobins
  • Hemoglobins
  • Oxidants
  • Protein Subunits
  • 5,5-dimethyl-1-pyrroline-1-oxide
  • Carbon Monoxide
  • Hydrogen Peroxide
  • Oxygen