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Updated: Feb 3, 2026

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
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Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Published on: March 18, 2012

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Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle

Sirsendu Jana1, Michael Brad Strader1, Fantao Meng1

  • 1Laboratory of Biochemistry and Vascular Biology and.

JCI Insight
|November 3, 2018
PubMed

Insights

Oxidized sickle hemoglobin (HbS) and altered proteins drive red blood cell microparticle (MP) formation in sickle cell disease (SCD). Hydroxyurea treatment reduced HbS oxidation and MP formation, suggesting new antioxidant therapies for SCD.

Area of Science:

  • Hematology
  • Biochemistry
  • Cell Biology

Background:

  • Intracellular hemoglobin (Hb) oxidation's role in red blood cell (RBC)-derived microparticle (MP) formation in sickle cell disease (SCD) is not well understood.
  • Sickle Hb (HbS) oxidation and associated cytosolic antioxidative protein changes are linked to membrane alterations and MP formation in Townes-SS mice.

Purpose of the Study:

  • To investigate the contribution of intracellular Hb oxidation to RBC-derived MP formation in SCD.
  • To explore the effects of HbS oxidation on RBC membrane proteins and MP generation.
  • To evaluate the therapeutic potential of hydroxyurea in mitigating HbS oxidation and MP formation.

Main Methods:

  • Photometric and proteomic analyses to detect Hb oxidation intermediates and posttranslational modifications.
  • Assessment of Hb complex formation with RBC membrane proteins.
  • In vitro incubation of Townes-SS MPs with human endothelial cells to evaluate cellular damage.
  • Analysis of MPs from hydroxyurea-treated Townes-SS mice and in vitro hydroxyurea effects on ferryl Hb.

Main Results:

  • High levels of Hb oxidation intermediates (ferric/ferryl) and β-globin modifications (oxidized βCys93, ubiquitinated βLys96/145) were confirmed in Townes-SS mice.
  • Ferryl Hb formed complexes with band 3 and other RBC membrane proteins.
  • Townes-SS MPs induced greater endothelial cell damage (loss of integrity, apoptosis, heme oxygenase-1 induction, bioenergetic imbalance) compared to control MPs.
  • Hydroxyurea treatment reduced Hb posttranslational modifications in vivo and decreased ferryl Hb levels in vitro, with S-nitrosylation shielding βCys93.

Conclusions:

  • Sickle Hb oxidation and subsequent protein modifications contribute significantly to RBC-derived MP formation in SCD.
  • These MPs exert detrimental effects on endothelial cells, contributing to SCD pathophysiology.
  • Hydroxyurea demonstrates potential as an antioxidant therapy by reducing Hb oxidation and shielding critical Hb residues, suggesting novel therapeutic strategies for SCD.
Keywords:
Cardiovascular diseaseHematologyProteomicsUbiquitin-proteosome systemVascular Biology

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