The Reaction of Hydrogen Peroxide with Hemoglobin Induces Extensive Alpha-Globin Crosslinking and Impairs the Interaction of Hemoglobin with Endogenous Scavenger Pathways



Florence Vallelian1, Tatiana Pimenova2, Claudia P. Pereira1, Bindu Abraham3, Malgorzata G. Mikolajczyk3, Gabriele Schoedon1, Renato Zenobi2, Abdu I. Alayash3, Paul W. Buehler3, and Dominik J. Schaer1


  1. Internal Medicine Research Unit, University of Zurich, Zurich, Switzerland
  2. Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
  3. Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration (FDA), Bethesda, MD, USA


Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained α−chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H2O2-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between α−globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.

CovalX Technology Used

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Hb (HbA0 purity >99%), αα-crosslinked Hb (αXLHb), and polymerized αXLHb) (PolyαXLHb) were obtained and stored at -80 °C. HbA0 was prepared in 50 mM potassium phosphate buffer (pH 7.4) at a concentration of 250 μM and then treated with H2O2 concentration ratios of 1:0, 1:1, and 1:10 in 1-mL volumes. The solutions were reacted at room temperature for 1 hour and then the H2O2 was removed using five buffer exchanges in equal volumes of potassium phosphate buffer before being filtered through 30 kDa cutoff centrifuge tubes and then returned to storage on ice or at -80 °C.

Protein complexes were stabilized using 1 μL of disuccinimidyl suberate (DSS) in a 10 μL sample (1 -5 μM) in 0.01 M triethylammonium bicarbonate buffer (pH 9.2). The samples were incubated at room temperature for 2 hours and then 1 μL was removed and mixed with 1 μL of sinapic acid matrix (10 mg/ml in 50% acetonitrile/0.1% TFA). 1 μL of the final mixture was spotted on a MALDI plate before being analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system. The data were background subtracted and smoothed using the CovalX Complex Tracker software.

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