Original Contribution IMMUNOCHEMICAL DETECTION OF HEMOGLOBIN-DERIVED RADICALS FORMED BY REACTION WITH HYDROGEN PEROXIDE: INVOLVEMENT OF A PROTEIN-TYROSYL RADICAL DARIO C. RAMIREZ,YEONG-RENN CHEN, and RONALD P. MASON Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA (Received 23 August 2002; Accepted 23 December 2002) Abstract—To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/ H 2 O 2 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 H 2 O 2 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/H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 . Published by Elsevier Science Inc. Keywords—Hemoglobin, Hydrogen peroxide, Immuno-spin trapping, Red blood cells, Free radicals INTRODUCTION Considerable attention has been focused on the forma- tion, stability, and reactions of hemoprotein-centered free radicals in biological systems [1,2]. Particular bio- chemical studies have been focused on hemoprotein- centered radicals [3–5] such as the prostaglandin H syn- thase-tyrosyl radical [6], the mitochondrial cytochrome c oxidase-thiyl radical [7], the cytochrome c peroxidase- tryptophanyl radical [8], the cytochrome c-tyrosyl radical [9], the horse metmyoglobin-tyrosyl radical [10], and the hemoglobin-thiyl [11–13] and methemoglobin(metHb)- tyrosyl [14,15] radicals. Hemoprotein-mediated redox reactions have been suggested to contribute to tissue damage and/or organ dysfunction that occurs in some pathological states, char- acterized by the release of myoglobin or hemoglobin into the extracellular environment as with cerebral hemor- rhage, ischemia/reperfusion, rhabdomyolysis, intravas- cular hemolytic anemia, atherosclerosis, and the infusion of hemoglobin-based blood substitutes [2,16,17]. The reaction of hydrogen peroxide (H 2 O 2 ) with hemoproteins has been known for decades [5], and the development of specific and sensitive methods to characterize and deter- mine their intermediates is an important concern in free radical biochemistry. Indeed, the general interest in the Address correspondence to: Dr. Dario C. Ramirez, NIEHS/NIH, 111 T. W. Alexander Drive, Room F048 - MD F0-02, Research Triangle Park, NC 27713, USA; Tel: (919) 541-3866; Fax: (919) 541-1043; E-Mail: ramirez1@niehs.nih.gov. Free Radical Biology & Medicine, Vol. 34, No. 7, pp. 830 – 839, 2003 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter doi:10.1016/S0891-5849(02)01437-5 830