title: Determination of extinction coefficients of human hemoglobin in various redox states.
abstract: The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss. Highly purified and well characterized redox forms of human Hb were used in this study and the extinction coefficients of each Hb species (ferrous/oxy, ferric/met and ferryl) were determined. A new set of equations were established to improve accuracy in determining the transient ferryl Hb species. Additionally, heme concentrations in solutions and in human plasma were determined using a novel reversed phase HPLC method in conjugation with our photometric measurements. The use of more accurate redox-specific extinction coefficients and method calculations will be an invaluable tool for both in vitro and in vivo experiments aimed at determining the role of Hb-mediated vascular pathology in hemolytic anemias and when Hb is used as oxygen therapeutics.
authors.affiliation: Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, United States.
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, United States. Electronic address: abdu.alayash@fda.hhs.gov.
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
mesh_terms: D006418:Heme / Q000032:analysis* / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D006418:Heme; D010100:Oxygen
keywords: Extinction coefficients; HPLC measurements; Hemoglobin; Spectrometric