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J. Biol. Chem., Vol. 282, Issue 7, 4894-4907, February 16, 2007

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Structural Basis of Peroxide-mediated Changes in Human Hemoglobin

A NOVEL OXIDATIVE PATHWAY^*

Yiping Jia¹, Paul W. Buehler¹, Robert A. Boykins, Richard M. Venable^¶, and Abdu I. Alayash²

From the Laboratory of Biochemistry and Vascular Biology, Division of Hematology, and the Laboratory of Biophysics, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and ^¶Membrane Biophysics Section, Laboratory of Computational Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Hydrogen peroxide (H₂O₂) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H₂O₂ to highly purified human hemoglobin (HbA₀) induced structural changes that primarily resided within subunits followed by the internalization of the heme moiety within subunits. These modifications were observed when an equal molar concentration of H₂O₂ was added to HbA₀ yet became more abundant with greater concentrations of H₂O₂. Mass spectrometric and amino acid analysis revealed for the first time that Cys-93 and Cys-112 were oxidized extensively and irreversibly to cysteic acid when HbA₀ was treated with H₂O₂. Oxidation of further amino acids in HbA₀ exclusive to the -globin chain included modification of Trp-15 to oxyindolyl and kynureninyl products as well as Met-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the subunits as a result of the H₂O₂ attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two -peptide fragments (128 - 139) and a heme moiety with the loss of iron, cross-linked between Ser-138 and the porphyrin ring. The novel oxidative pathway of HbA₀ modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Received for publication, October 24, 2006 , and in revised form, December 15, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bldg. 29, Rm. 112, Bethesda, MD 20892. Tel.: 301-827-3813; E-mail: abdu.alayash{at}fda.hhs.gov.

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