Protein Derived Radicals

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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 and some copper-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 superoxide dismutase that utilize our new immunological/spin-trapping techniques. Polyclonal antibodies bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The ESR signal of the myoglobin-DMPO radical decays within minutes, however a persistent adduct can be detected using mass spectroscopy. This adduct can be visualized using Western blot techniques at a sensitivity 10-fold greater than ESR. Western blot analysis demonstrates that myoglobin specifically forms a protein-derived radical in rat heart supernatant. For the first time, we were able to demonstrate the formation of free radicals inside a cell. When red blood cells were exposed to 50-micromolar hydrogen peroxide, hemoglobin-derived radicals were detected inside the red blood cells. The sites of free radical formation on hemoglobin have been determined by the mass spectroscopy group who discovered a novel histidine free radical.