A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

N. B J Vollaard, Brandon J. Reeder, Jerry P. Shearman, Patrick Menu, Michael T. Wilson, Chris E. Cooper

    Research output: Contribution to journalArticlepeer-review

    72 Citations (Scopus)


    Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins. © 2005 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)1216-1228
    Number of pages13
    JournalFree Radical Biology and Medicine
    Issue number9
    Publication statusPublished - 1 Nov 2005


    • Assay
    • Blood substitute
    • Chlorin
    • Exercise
    • Free radical
    • Hemoglobin
    • Myoglobin
    • Oxidative modification
    • Oxidative stress
    • Peroxide


    Dive into the research topics of 'A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo'. Together they form a unique fingerprint.

    Cite this