Electron Paramagnetic Resonance Detection of Free Tyrosyl Radical Generated by Myeloperoxidase, Lactoperoxidase, and Horseradish Peroxidase

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259

180

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o,o-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.

supporting

confidence: 59%

Mass Spectrometric Quantification of 3-Nitrotyrosine, ortho-Tyrosine, and o,o′-Dityrosine in Brain Tissue of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Mice, a Model of Oxidative Stress in Parkinson's Disease

Pennathur¹,

Jackson‐Lewis²,

Przedborski³

et al. 1999

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180

“…Previously, MNP has been used to detect and characterize tyrosyl radical formed during MPO/H 2 O 2 /NO 2 Ϫ -catalyzed oxidation of tyrosine in solution (45). As shown in Fig.…”

Section: Spin Trapping Of Tyrosyl Radical In Membrane-mentioning

confidence: 99%

Transmembrane Nitration of Hydrophobic Tyrosyl Peptides

Zhang

Bhargava

Keszler

et al. 2003

“…As a specific marker of oxidation, dityrosine has been detected in human atherosclerotic plaques (41,42) in the brain of elderly humans (43) or patients affected with Alzheimer's disease (44), in age-related nuclear cataract (45) and other pathologies thought to be associated with oxidative stress. Formation of dityrosine has been attributed mainly to the activation of the myeloperoxidase/H 2 O 2 system of neutrophils and macrophages (46), but other peroxidases (47) and peroxynitrite (36, 48) have been recognized as additional sources of dityrosine. The present results agree with previous studies suggesting that dityrosine formation together with increased NO synthase expression may be a useful marker for peroxynitrite formation in tissues (49,50).…”

Section: Peroxynitrite-mediated Tyrosine Modificationmentioning

confidence: 99%

Dityrosine Formation Outcompetes Tyrosine Nitration at Low Steady-state Concentrations of Peroxynitrite

Pfeiffer

Schmidt

Mayer

2000

155

106

Tyrosine nitration is a well established protein modification occurring in vivo in a number of inflammatory diseases associated with oxidative stress and increased activity of NO synthases (1, 2). Nitration of specific tyrosine residues has been reported to affect protein structure and function (3), suggesting that 3-nitrotyrosine formation may not only be a disease marker but could be causally involved in the pathogenesis of certain disease states.Peroxynitrite, formed in a nearly diffusion-controlled reaction from NO and O 2 . , is considered a potent pathophysiologically relevant cytotoxin. Besides oxidation reactions resulting in dysfunction of various biomolecules, nitration of free and protein-bound tyrosine to yield 3-nitrotyrosine is a well established reaction of peroxynitrite that may contribute to NO cytotoxicity (1). The nitration reaction has been extensively studied in vitro by bolus addition of synthetic peroxynitrite to tyrosine-containing samples including purified proteins, cells, and tissues (3-6) . In situ, 3-nitrotyrosine was most frequently visualized with monoclonal or polyclonal antibodies (2), but the identity of the product has been confirmed by several laboratories using sophisticated gas chromatography/mass spectroscopy and HPLC 1 methods (7,8). Thus, there is general agreement that (i) authentic peroxynitrite is a potent nitrating agent that converts free and proteinbound tyrosine to the corresponding 3-nitro derivative, and that (ii) 3-nitrotyrosine does occur in vivo. The conclusion that peroxynitrite is the main cause for in vivo nitration may thus seem obvious, but is not supported by experimental data. In fact, several recent studies have identified alternative pathways of tyrosine nitration (9), and we found that nitration by simultaneously generated NO and O 2 . is much less efficient than the reaction triggered by authentic peroxynitrite (10). The interpretation of the latter results has been disputed, and a number of points have been raised questioning their validity. One point was related to the possibility that urate formed in the XO reaction might have scavenged peroxynitrite and thus prevented tyrosine nitration in long term (12 h) experiments. Concerning data interpretation, we had suggested that NO and O 2 . may combine to trans-peroxynitrite, the rapid protonation of which prevents formation of the nitrating CO 2 . adduct. However, thermodynamic calculations have unambiguously identified the cis-rotamer as the more stable conformation of peroxynitrite in both gas phase (11) and aqueous solution, 2 rendering our initial hypothesis untenable. We hope to settle both issues with the present study in which we extend our earlier findings to other, urate-free NO/O 2 .-generating systems and demonstrate that the low nitrating efficiency of NO/O 2 . is explained by an unexpected dependence of nitration yields on peroxynitrite steady-state concentrations. It is shown that the reaction between tyrosine and peroxynitrite yields almost exclusively dityrosine at peroxynitrit...