EPR spin-trapping of protein radicals to investigate biological oxidative mechanisms

Augusto, Ohára; Vaz, Sandra M.

doi:10.1007/s00726-006-0429-4

Cited by 23 publications

(15 citation statements)

References 55 publications

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“…EPR spectroscopy is a widely used methodology to detect and identify free radicals, with the possibility to unravel oxidative mechanisms, and also to discriminate radical from non-radical mechanisms [82,83]. In addition, in combination with analytical methods allows identify the protein residues that are the target of oxidants.…”

Section: Methods To Study the Reactivity Of Peroxynitritementioning

confidence: 99%

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Carballal

Bartesaghi

Radí

2014

Biochimica et Biophysica Acta (BBA) - General Subjects

137

122

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Background Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant of short half-life and low steady-state concentration in biological systems; it promotes nitroxidative damage. Scope of Review We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable state-state levels in living systems. Major Conclusions The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1 % of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s−1 and its steady-state concentration ~ 1 nM. General Significance The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite.

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Section: Methods To Study the Reactivity Of Peroxynitritementioning

confidence: 99%

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Carballal

Bartesaghi

Radí

2014

Biochimica et Biophysica Acta (BBA) - General Subjects

137

122

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“…Direct EPR spectrometry can be used to detect and identify relatively stable protein-centered free radicals. However, the short lifetimes of these reactive species have generally required either that the samples containing the radical be rapidly frozen after formation [1] or that the radical be spin trapped to give an adduct more stable than the primary radical [4][5]. Nitrone spin traps, of which 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) is the most commonly employed, often form long-lived adducts that can be seen by EPR for minutes.…”

Section: Introductionmentioning

confidence: 99%

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

Lardinois

Detweiler

Tomer

et al. 2008

Free Radical Biology and Medicine

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An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein-DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography-ELISAmass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals upon treatment with H 2 O 2 . The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments.

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“…To test the hypothesis that UV‐induced CL is a radical reaction, exposure to the laser was carried out in the presence of spin trap molecules. Spin traps are often used to allow the visualization of transient free radical populations by reacting with short‐lived radicals to produce persistent spin adduct radicals that can be studied by electron paramagnetic resonance (EPR) or MS since the spin trap molecules covalently label the radical site in the molecule . Therefore, the peptides were exposed to the UV laser as described above, for either 10 s or 1 min, but in the presence of either DMPO or MNP.…”

Section: Resultsmentioning

confidence: 99%

Ultraviolet laser‐induced cross‐linking in peptides

Altucci

Bourgoin-Voillard

Gravagnuolo

et al. 2013

Rapid Comm Mass Spectrometry

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RATIONALE The aim of this study was to demonstrate, and to characterize by high resolution mass spectrometry, that it is possible to preferentially induce covalent cross-links in peptides by using high energy femtosecond UV laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS Three peptides, xenopsin, angiotensin I, interleukin, individually or in combination, were exposed to high energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS High resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS High energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

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EPR spin-trapping of protein radicals to investigate biological oxidative mechanisms

Cited by 23 publications

References 55 publications

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

Ultraviolet laser‐induced cross‐linking in peptides

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