Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway.
Hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) 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.
Jia
Yiping
Y
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, and the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
Buehler
Paul W
PW
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, and the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
Boykins
Robert A
RA
Laboratory of Biophysics, Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and.
Venable
Richard M
RM
Membrane Biophysics Section, Laboratory of Computational Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Alayash
Abdu I
AI
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, and the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892. Electronic address: abdu.alayash@fda.hhs.gov.
D001802:Blood Substitutes / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000276:immunology; D006418:Heme / Q000737:chemistry* / Q000276:immunology; D006441:Hemoglobin A / Q000737:chemistry* / Q000276:immunology; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000276:immunology; D008715:Methionine / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
D001802:Blood Substitutes; D006418:Heme; D006441:Hemoglobin A; D008715:Methionine; D006861:Hydrogen Peroxide; D003545:Cysteine