title: Oxidative instability of hemoglobin E (β26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/β-thalassemia.
abstract: When adding peroxide (H2O2), β subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired α subunits within red cells and/or co-inheritance of another β subunit mutant, HbE (β26 Glu→Lys) have been implicated in the pathogenesis and severity of β thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of βCys93 to reflect oxidative instability of β subunits. In the presence of free α subunits and H2O2, both HbA and HbE showed βCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the α-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/β thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own β subunit (i.e., βCys93) than HbA. Secondly, in the presence of excess free α-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Michael Brad
authors.initials: MB
authors.lastname: Strader
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Tigist
authors.initials: T
authors.lastname: Kassa
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Fantao
authors.initials: F
authors.lastname: Meng
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Francine B
authors.initials: FB
authors.lastname: Wood
authors.affiliation: Department of Medicine, Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Rhoda Elison
authors.initials: RE
authors.lastname: Hirsch
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Friedman
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA. Electronic address: abdu.alayash@fda.hhs.gov.
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
mesh_terms: D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism / Q000473:pathology; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006446:Hemoglobin E / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D017086:beta-Thalassemia / Q000378:metabolism* / Q000473:pathology
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D018832:Molecular Chaperones; D006418:Heme; D006446:Hemoglobin E; D006861:Hydrogen Peroxide
keywords: Alpha-hemoglobin stabilizing protein; Hemoglobin E; Oxidation; Thalassemia