Oxidative damage to tissues by hydrogen peroxide has become a recurring theme as a mechanism for the induction of a variety of medical conditions including myocardial ischemia, cancer, inflammation, and aging. Proteins are a target of such damage. Most heme-containing proteins have peroxidase activity that can cause oxidative damage to a variety of biological molecules, including itself and membrane lipids. We are now in the process of investigating a series of protein-derived radicals formed by myoglobin, hemoglobin and cytochrome c oxidase that utilize our new immunological-spin trapping techniques. Our laboratory has produced polyclonal antibodies that bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were produced in rabbits immunized against the hapten, 5,5-dimethyl-2-octanoic acid-1-pyrroline N-oxide (OA-DMPO). OA-DMPO is a structural analog of DMPO with an additional carbon chain terminating in a carboxyl-group for amide linking. Antigens were synthesized by reacting OA-DMPO with chicken egg albumin via the carbodiimide method for amidation. Titer of the rabbit serum was determined using ELISA techniques. The polyclonal antibodies screened positive for protein-DMPO nitrone adducts produced on BSA via Fenton chemistry and adducts produced on metmyoglobin as a result of self-peroxidation by hydrogen peroxide.
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