Singlet Oxygen Mediated Inactivation of Glutathione Peroxidase and Thioredoxin Reductase

5,5-dimethyl-1-pyrroline N-oxide (DMPO), in which the initially formed nitroxide radical adduct subsequently decays to a far longer-lived nitrone adduct that is detected using an anti-DMPO nitrone adduct antibody. the sequences of DNA that are damaged are likely to be as important as the total level of damage. Therefore, the development of techniques allowing for the separation of different DNA sequences and the detection of DMPO adducts on the DNA would be the first step in assessing the extent of DNA damage mediated by radicals throughout the genome. for development of this method, we used an in vitro copper-Fenton system to generate DNA radicals in the presence of DMPO. This DNA was electrophoresed on agarose or polyacrylamide gels under denaturing conditions and transferred to nitrocellulose or PVDF membranes, respectively. the DNADMPO adducts were detected using standard immuno-blotting techniques with an anti-DMPO nitrone antibody. This technique was used for the first time on polynucleotides containing only cytosine and guanine bases and DNA-DMPO adducts were detected indicating that other radicals could be trapped aside from an adenine radical, previously the only DNA radical identified using the copper-Fenton system. These radical intermediates may be important in the mutation of guanine and cytosine bases to thymine, a common mutation catalyzed by copper-Fenton oxidizing systems.

doi : 10.1016/j.freeradbiomed.2011.10.077

54 Singlet Oxygen Mediated Inactivation of Glutathione Peroxidase and Thioredoxin Reductase Aldwin Suryo Rahmanto1, and Michael J Davies1,2 1 2 The Heart Research Institute, Sydney, Australia, Faculty of Medicine, University of Sydney, Australia 1 Singlet oxygen ( O2) is a reactive oxygen species known to be generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g. myeloperoxidase and 1 eosinophil peroxidase). O2 oxidizes the amino acids Trp, Tyr, His, Cys and Met present on proteins, with this resulting in the inactivation of various metabolic and antioxidant enzymes. Impairment of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathological conditions including photoaging and atherosclerosis. in this study, the effects 1 of O2 on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (a 1 source of O2) resulted in significant, time-dependent reductions in enzyme activity (10 – 40%, p < 0.05). a greater extent of inhibition (ca. 2-fold) was detected when the reactions were 1 carried out in D2O, consistent with the intermediacy of O2. No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts 1 7 -1 -1 rapidly with O2 (k ~ 10 M s ), significantly reduced the extent of photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH4, DTT, GSH, or 1 NADPH) added after the cessation of O2 formation, were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of non-reducible protein aggregates and / or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes 1 GPx and TrxR can be irreversibly inactivated by O2.

doi : 10.1016/j.freeradbiomed.2011.10.078

S32

55 Specific Oxidation of Torpedo Californica Acetylcholinesterase by Singlet Oxygen: Identification of N-Formyl-Kynurenine Tryptophan Derivatives by Mass Spectrometry Mathilde Triquigneaux1, Marilyn Ehrenshaft1, Lev Weiner2, Esther Roth2, Israel Silman2, Ronald Mason1, and Leesa Deterding1 1 National Institute of Environmental Health Sciences, USA, 2 Weizmann Institute of Science, Israel The principal biological role of acetylcholinesterase (AchE) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the acetylcholine neurotransmitter. the active site of AchE contains a subunit allowing the catalytic function and peripheral anionic sites that bind the acetylcholine in the first step of the catalytic pathway. the presence of aromatic residues in the active site of AchE was previously described as an array of negative charges to guide the positive charge of the acetylcholine to the active site cavity. 1 It has been previously demonstrated that generation of O2 by illumination of methylene blue causes irreversible inactivation of AchE Torpedo Californica (TcAchE). Further evidence suggests that oxidative damage to tryptophan residues is perhaps responsible for the inactivation. in the present work, structural modifications of the TcAchE tryptophan residues induced by methylene blue (MB)-sensitized oxidation were investigated by anti-N-formyl-kynurenine and mass spectrometry. From these analyses, we determined that N-formyl-kynurenine derivatives were specifically localized on Trp 84 and Trp 279 – the two tryptophan residues in the active-site gorge of TcAchE. Moreover, enzymatic peptides that contain these two tryptophan residues could not be detected when the competitive inhibitors edrophonium and propidium were included as ligands of the active-site gorge of TcAchE. These data suggest that the inhibitors efficiently protect the enzyme from photo-inactivation 1 caused by O2. Our results demonstrate the specificity of the TcAchE MB-sensitized oxidation and suggest the main role of Trp 84 and Trp 279 in the catalytic activity of the enzyme.

doi : 10.1016/j.freeradbiomed.2011.10.079

56 Oxidatively Induced DNA Base Damage and DNA Repair Enzyme Expression Levels in Alzheimer's Disease Gamze Tuna1, Feriha Ozkaya1, Miral Dizdaroglu2, and Güldal Kirkali1 1 Department of Medical Biochemistry, Dokuz Eylul University, 2 Izmir, Turkey, Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD, USA There is evidence that free radical-mediated oxidative damage to DNA may play a role in basic neurodegenerative mechanisms in Alzheimer's disease (AD). Oxygen-derived free radicals, most notably the hydroxyl radical, generate a plethora of modifications in DNA. in living organisms, DNA repair mechanisms exist to repair this type of DNA damage. Defects in DNA repair may be a contributing factor to disease processes. in mammals, OGG1 and NEIL1 constitute the two major DNA glycosylases. the aim of this study was to investigate the level of the oxidatively induced DNA base damage and the mRNA expression of OGG1 and NEIL1 in peripheral blood of AD patients and healthy individuals. DNA samples of AD patients (n = 33) and healthy volunteers (n = 37) were isolated from blood samples. the levels of DNA base lesions were measured by gas chromatography-isotope dilution mass spectrometry. the mRNA expression levels of OGG1 and NEIL1 were determined in the blood samples of AD patients (n = 15) and control individuals (n = 20). Total RNA was extracted and

SFRBM 2011