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. With immuno-spin trapping method we have detected totally unsuspected protein radicals. For instance when LPO was reacted with glutathione (GSH) in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct with ELISA and Western blot. These results suggest that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO. The sensitivity of immuno-spin trapping enables the investigation of low abundance proteins from expression systems. For instance, neuroglobin (Ngb) is a recently discovered protein that belongs to the globin family of proteins. A particularly high Ngb concentration has been reported in the mammalian retina, one of the highest-oxygen-consuming tissues of the body. We were able to determine whether Ngb a hexacoordinated globin also forms a free radical intermediate when reacting with H2O2. Research Accomplishments: Our laboratory developed and validated a new technique, immuno-spin trapping (IST), which combines the specific free-radical reactivity of nitrone spin traps with nitrone-antibody sensitivity and specificity. Immuno-spin trapping has now been used not only by this laboratory but also in several other laboratories. Among many advances, the two most noteworthy immuno-spin trapping developments have been 1) the use of immuno-spin trapping for detection of DNA-derived free radicals, providing a more sensitive, specific and reproducible method for DNA-radical measurements than previously available and 2) the imaging of free radicals using immunofluorescence and histochemistry in cells and tissues. We have now developed and validated antiserum to the tryptophan oxidation product (N-formylkynurenine) NFK. These investigations showed that the antiserum is specific for detection of proteins containing tryptophan residues with cleaved indole rings and that it can detect these products in proteins with as few as one or two tryptophan residues, in mixtures of proteins and in cells.
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