title: Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical.
abstract: To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.
authors.affiliation: Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27713, USA. ramirez1@niehs.nih.gov
authors.forename: Dario C
authors.initials: DC
authors.lastname: Ramirez
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D015153:Blotting, Western; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000378:metabolism; D005609:Free Radicals; D005978:Glutathione / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007120:Immunochemistry / Q000379:methods*; D008956:Models, Chemical; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D005609:Free Radicals; D006454:Hemoglobins; D014443:Tyrosine; D006861:Hydrogen Peroxide; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid