The group continued to implement and validate the technique called immuno-spin trapping, which combines the specific free radical reactivity of the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin trap with nitrone-antibody sensitivity. Anti-DMPO has proven itself to be a highly specific antibody with the no-DMPO control easily identifying any non specificity issues. This type of control is uncommon among antibody work, but is of great utility. Unlike electron spin resonance (ESR) detection of radical intermediates, anti-DMPO immuno-spin trapping is not dependent on transient free radical intermediates and is about 10,000 times more sensitive than ESR and 100 times more sensitive than MS. In the last four years we have continued using the immuno-spin trapping approach to investigate the formation of protein-centered free radicals in vitro, in cells, and in vivo. The immunoassay has many advantages compared to ESR spin-trapping: 1) immuno-spin trapping requires much less material (micrograms of protein rather than milligrams); 2) the stability of the final oxidation product, the DMPO-nitrone adduct (a stable non-radical species), is much greater than that of the paramagnetic DMPO-radical adducts (t1/2 = sec-min) required for ESR, resulting in greatly enhanced sensitivity, and 3) immuno-spin trapping can be performed using standard ELISA, Western blotting techniques, and immuno-histological techniques, making immuno-spin trapping far more accessible and versatile than ESR spin trapping. This advance greatly expanded the utility of the spin-trapping technique, freeing it from the quantum mechanical complexity of ESR spectroscopy. The major limitation of the anti-DMPO methodology is the inability to identify the low abundance site(s) of protein radical formation even with MS in any sample other than purified proteins. As an alternative approach to this problem, we have decided to try to develop amino acid-specific anti-DMPO antibodies. Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Recently we have measured DNA damage induced by a Cu(II)-H2O2 oxidizing system using not only immuno-spin trapping, but ESR, MS, and MS/MS. The LC-MS/MS analysis of total DNA digests characterized an adenosine-DMPO adduct in both calf thymus and cellular DNA. The MS detection of this nucleoside nitrone adduct from cells is notable because we have always failed in attempting to identify protein nitrone adducts from cells. The ease of purification of DNA in contrast to a specific protein and the fact that DNA has only four components are major advantages in the MS investigation of DNA products. We are narrowing our MS search for nucleoside nitrone adducts by using competitive ELISA to identify the HPLC fractions containing the DMPO nitrone adducts. Once this method is perfected we will search for nucleoside nitrone adducts in a cell model of arsenic carcinogenicity, which is thought to have multiple but poorly defined modes of action. DMPO does not generally form adducts with tryptophan radicals. To address this problem, we have developed immuno-detection of oxidatively modified tryptophan residues in proteins, comparable to anti-DMPO immuno-spin trapping of radicals, with the aim of providing a more accessible means for localizing radical and singlet oxygen post-translational protein modifications. The initial study describing the development and validation antiserum to the tryptophan oxidation product N-formylkynurenine (NFK) showed that the antiserum was specific for detection of proteins containing tryptophan residues with cleaved indole rings and that it is sensitive enough to detect NFK in proteins with as few as one or two tryptophan residues and in mixtures of proteins. Subsequent work has used the anti-NFK antiserum to study subcellular localization of NFK-containing proteins in cultured skin cells, photosensitization in human lens epithelial cells, and oxidation of proteins in mice. Other work has led to the publication of two papers detailing the effects on acetylcholinesterase of oxidation of site-specific residues of tryptophan to NFK.
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