Spin Trapping

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Ronald P Mason - One of the best experts on this subject based on the ideXlab platform.

  • immuno Spin Trapping of macromolecules free radicals in vitro and in vivo one stop shopping for free radical detection
    Free Radical Biology and Medicine, 2019
    Co-Authors: Ronald P Mason, Douglas Ganini
    Abstract:

    Abstract The only general technique that allows the unambiguous detection of free radicals is electron Spin resonance (ESR). However, ESR Spin Trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-Spin Trapping technique, which combine the specificity of the Spin Trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.

  • imaging free radicals in organelles cells tissue and in vivo with immuno Spin Trapping
    Redox biology, 2016
    Co-Authors: Ronald P Mason
    Abstract:

    The accurate and sensitive detection of biological free radicals in a reliable manner is required to define the mechanistic roles of such species in biochemistry, medicine and toxicology. Most of the techniques currently available are either not appropriate to detect free radicals in cells and tissues due to sensitivity limitations (electron Spin resonance, ESR) or subject to artifacts that make the validity of the results questionable (fluorescent probe-based analysis). The development of the immuno-Spin Trapping technique overcomes all these difficulties. This technique is based on the reaction of amino acid- and DNA base-derived radicals with the Spin trap 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) to form protein- and DNA-DMPO nitroxide radical adducts, respectively. These adducts have limited stability and decay to produce the very stable macromolecule-DMPO-nitrone product. This stable product can be detected by mass spectrometry, NMR or immunochemistry by the use of anti-DMPO nitrone antibodies. The formation of macromolecule-DMPO-nitrone adducts is based on the selective reaction of free radical addition to the Spin trap and is thus not subject to artifacts frequently encountered with other methods for free radical detection. The selectivity of Spin Trapping for free radicals in biological systems has been proven by ESR. Immuno-Spin Trapping is proving to be a potent, sensitive (a million times higher sensitivity than ESR), and easy (not quantum mechanical) method to detect low levels of macromolecule-derived radicals produced in vitro and in vivo. Anti-DMPO antibodies have been used to determine the distribution of free radicals in cells and tissues and even in living animals. In summary, the invention of the immuno-Spin Trapping technique has had a major impact on the ability to accurately and sensitively detect biological free radicals and, subsequently, on our understanding of the role of free radicals in biochemistry, medicine and toxicology.

  • oxidation of α lactalbumin after a lactoperoxidase catalysed reaction an oxidomics approach applying immuno Spin Trapping and mass spectrometry
    International Dairy Journal, 2014
    Co-Authors: Trine Kastrup Dalsgaard, Leesa J Deterding, Mathilde Triquigneaux, Fiona A Summers, Grith Mortensen, Ronald P Mason
    Abstract:

    Free radicals were characterised by mass spectrometry (MS) and immuno-Spin Trapping on α-lactalbumin (α-lac) after a lactoperoxidase (LPO)-catalysed reaction. Although Spin traps have historically been used for electron Spin resonance (ESR) studies, here free radicals were detected by immuno-Spin Trapping using the Spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), Western blotting, and MS; the latter yields information about the location of radical formation in the protein structure. Radical formation was highly specific towards Tyr residues; DMPO-trapped radicals were detected at Tyr18 and Tyr50, and the results also indicated a radical on Tyr36. These three oxidation sites on α-lac are all exposed on the surface of the protein, indicating that accessibility for LPO or DMPO was important. Applying immuno-Spin Trapping in combination with MS is a good approach to understand how different oxidation mechanisms affect the generation of radicals on proteins, and this oxidomics approach has future potential in food science.

  • dna cleavage and detection of dna radicals formed from hydralazine and copper ii by esr and immuno Spin Trapping
    Chemical Research in Toxicology, 2014
    Co-Authors: Birandra K Sinha, Fabian Leinisch, Suchandra Bhattacharjee, Ronald P Mason
    Abstract:

    Metal ion-catalyzed oxidation of hydrazine and its derivatives leads to the formation of the hydrazyl radical and subsequently to oxy-radicals in the presence of molecular oxygen. Here, we have examined the role of Cu2+-catalyzed oxidation of hydralazine in the induction of DNA damage. Neither 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) nor dimethyl sulfoxide (DMSO) was effective in inhibiting hydralazine-Cu2+-induced DNA damage. Singlet oxygen did not appear to participate in this DNA cleavage. The one-electron oxidation of hydralazine also leads to the formation of DNA radicals as confirmed by immuno-Spin Trapping with 5,5-dimethyl-1-pyrroline-N-oxide. Electron Spin resonance (ESR) and Spin-Trapping studies further confirmed the formation of DNA radicals; predominantly, 2′-deoxyadenosine radical adducts were detected, while some radicals were also detected with other nucleosides. Our results suggest that free hydroxyl radicals may not be the main damaging species causing DNA cleavage and that possibly Cu-pe...

  • detection of ras gtpase protein radicals through immuno Spin Trapping
    Free Radical Biology and Medicine, 2012
    Co-Authors: Michael F Davis, Ronald P Mason, Kalina Ranguelova, Li Zhou, Marilyn Ehrenshaft, Harsha P Gunawardena, Xian Chen, Marcelo G Bonini, Sharon L Campbell
    Abstract:

    Abstract Over the past decade immuno-Spin Trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of • NO-mediated protein radical formation in the HRas GTPase. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.

Jean-luc Parrain - One of the best experts on this subject based on the ideXlab platform.

Béatrice Tuccio - One of the best experts on this subject based on the ideXlab platform.

  • Spin Trapping : la chasse aux intermédiaires radicalaires
    L'Actualité Chimique, 2019
    Co-Authors: Christiane André-barrès, Jacques Lalevée, Elsa Anselmi, Guillaume Dagousset, Emmanuel Magnier, Didier Gigmes, Béatrice Tuccio
    Abstract:

    Le Spin Trapping couplé à la RPE est une méthode de détection des radicaux à courte durée de vie. Son efficacité dans la caractérisation d'intermédiaires radicalaires est illustrée par trois exemples choisis dans les domaines de la chimie organique et de la polymérisation. Mots-clés Spin Trapping, RPE, intermédiaire radicalaire, adduit de Spin, mécanisme réactionnel. Abstract Spin Trapping: running after the radical intermediates The EPR/Spin Trapping method allows the detection of short-lived radicals. Three examples of its use are given to illustrate its efficiency in radical intermediate characterization in the fields of organic chemistry and polymerization. Keywords Spin Trapping, EPR, radical intermediate, Spin adduct, reaction mechanism. a RPE est la technique de choix pour l'étude des espèces radicalaires, à condition toutefois que ces composés soient suffisamment stables pour s'accumuler dans le milieu et atteindre une concentration stationnaire permettant leur détection. Mais comment faire lorsqu'un radical a une durée de vie excessivement courte et ne peut pas être détecté par RPE de façon conventionnelle ? Ce cas se rencontre fréquem-ment dans de très nombreux processus radicalaires en biochi-mie, chimie organique, polymérisation ou encore en catalyse. Le « Spin Trapping » (piégeage de Spin), décrit il y a cinq décen-nies [1-3], offre alors un moyen de résoudre le problème. Un rapide examen de la bibliographie montre que l'immense majorité des applications de cette technique se situent dans le domaine de la biologie et de la recherche biomédicale [4]. Ceci résulte de l'observation que la production d'espèces oxygé-nées ou azotées réactives, notamment radicalaires, est asso-ciée au développement de nombreuses pathologies [5]. Mais la méthode du Spin Trapping peut aussi s'avérer très efficace pour l'étude de processus radicalaires dans de nombreux autres domaines de la chimie, par exemple en vue de l'amélio-ration de procédés catalytiques, de l'identification de pol-luants atmosphériques, de l'étude de matériaux inorganiques, de la détermination de mécanismes en chimie organique, ou encore de l'élaboration de nouveaux polymères, pour n'en citer que quelques-uns [6]. Néanmoins, force est de constater qu'en dehors des études biochimiques, peu de chimistes recourent à cette technique analytique pourtant utile dès lors que l'on soupçonne la présence d'intermédiaires radicalaires. Dans l'optique de montrer les potentialités de cette approche en chimie, nous avons choisi de décrire son utilisation dans trois exemples d'études mécanistiques réalisées dans nos équipes, dans les domaines de la synthèse organique et de la polymérisation.

  • trans-1,2-Disiloxybenzocyclobutene, an adequate partner for the auto-oxidation: EPR/Spin Trapping and theoretical studies.
    Physical chemistry chemical physics : PCCP, 2014
    Co-Authors: Jean Drujon, Raphaël Rahmani, Virginie Héran, Romain Blanc, Yannick Carissan, Béatrice Tuccio, Laurent Commeiras, Jean-luc Parrain
    Abstract:

    The auto-oxidation of trans-1,2-disiloxybenzocyclobutene 1 was found to be very efficient, giving endo-peroxide 7 in quantitative yield. Each step of the mechanism of Spin-forbidden addition of triplet oxygen O2(3Σg) was studied by both EPR/Spin Trapping and theoretical studies.

  • Trans-1,2-disiloxybenzocyclobutene, an adequate partner for the auto-oxisation: EPR/Spin Trapping and theoretical studies
    Physical Chemistry Chemical Physics, 2014
    Co-Authors: Jean Drujon, Raphaël Rahmani, Virginie Héran, Romain Blanc, Yannick Carissan, Béatrice Tuccio, Laurent Commeiras, Jean-luc Parrain
    Abstract:

    The auto-oxidation of trans-1,2-disiloxybenzocyclobutene was found to be very efficient, giving endoperoxide in quantitative yield. Each step of the mechanism of Spin-forbidden addition of triplet oxygen O2 was studied by both EPR/Spin Trapping and theoretical studies.

  • Spin Trapping of superoxide by diester-nitrones
    Organic & biomolecular chemistry, 2005
    Co-Authors: Ahmad Allouch, Valérie Roubaud, Robert Lauricella, Jean-claude Bouteiller, Béatrice Tuccio
    Abstract:

    The nitrone N-[(1-oxidopyridin-1-ium-4-yl)-methylidene]-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPyON) was synthesized and used as a Spin Trapping agent. The kinetic aspects of the superoxide detection by this new Spin trap and by two other diester-nitrones, i.e. 2,2-diethoxycarbonyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPO) and N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN), were examined by determining the rate constants for the Trapping reaction and for the Spin adduct decay at pH 7.2. Comparing the results obtained to those given by analogous monoester-nitrones showed that both the Spin Trapping and the adduct decay reactions were faster in the presence of a second ester group in the cyclic nitrone series, while the superoxide Trapping capacities of linear diester-nitrones were found to be dramatically weak. It follows from this study that DEPO and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) are superior when it comes to superoxide detection. Below 0.005 mol dm−3, DEPO is to date the only nitrone capable of clearly detecting superoxide, while EMPO should be preferred at higher Spin trap concentration.

  • EPR/Spin Trapping study of the spontaneous addition of dioxygen on a dienol
    Tetrahedron Letters, 2005
    Co-Authors: Fadia Najjar, Robert Lauricella, Christiane André-barrès, Liliane Gorrichon, Béatrice Tuccio
    Abstract:

    The spontaneous addition of triplet oxygen on dienol 1, yielding endoperoxide 2, was followed by EPR/Spin Trapping. The use of nitroso and nitrone Spin traps allowed the detection of two radical centers, showing that this reaction could likely follow a radical pathway.

Jean Drujon - One of the best experts on this subject based on the ideXlab platform.

Gerald M. Rosen - One of the best experts on this subject based on the ideXlab platform.

  • In vivo Spin Trapping of nitric oxide from animal tumors.
    Nitric oxide : biology and chemistry, 2006
    Co-Authors: Katarzyna Pustelny, Gerald M. Rosen, Joanna Bielanska, Przemyslaw M. Plonka, Martyna Elas
    Abstract:

    Abstract Spin Trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO ) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive Spin Trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts—DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)2 was used as the Spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO–Fe(DTCS)2 (AN = 13 G) was observed (n = 4, out of total n = 6) in non-treated tumors and in tumors of animals treated with l -arginine. Substrate availability did not limit the detection of NO by Spin Trapping. Half-life time of the NO–Fe(DTCS)2 in tumor tissue was about 60 min. The feasibility of non-invasive Spin Trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.

  • superoxide dismutase versus ferricytochrome c determining rate constants for the Spin Trapping of superoxide by cyclic nitrones
    Journal of Organic Chemistry, 2004
    Co-Authors: John Weaver, Pei Tsai, Sovitj Pou, Gerald M. Rosen
    Abstract:

    Given that Spin Trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific Spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for Spin Trapping O(2)(*-) compared to the well-studied Spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a Spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for Spin Trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with Spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known Spin traps were also undertaken.

  • Differential effect of buffer on the Spin Trapping of nitric oxide by iron chelates
    Analytical biochemistry, 2001
    Co-Authors: Supatra Porasuphatana, John Weaver, Theodore A. Budzichowski, Pei Tsai, Gerald M. Rosen
    Abstract:

    Nitric oxide synthase (NOS) generates nitric oxide (NO*) by the oxidation of l-arginine. Spin Trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO* in real time and at its site of generation. The Spin Trapping of NO* from isolated NOS I oxidation of L-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO-Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO* and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO-Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at Spin Trapping NO* from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at Spin Trapping NO* but their maximal efficiency may be affected by experimental conditions.

  • free radicals biology and detection by Spin Trapping
    1999
    Co-Authors: Gerald M. Rosen
    Abstract:

    1. In the Beginning 2. The Oxygen Paradox 3. Nitric Oxide: A Simple Molecule with Complex Biological functions 4. Methods of Free Radical Detection 5. Spin Trapping Free Radicals: Historical Perspective 6. The Synthesis of Spin Traps 7. Electron Paramagnetic Resonance 8. Spectroscopy: Principles and practical Aspects 9. Kinetics of Spin Trapping Free Radicals 10. The Chemistry of Spin Traps and Spin trapped Adducts 11. Spin Trapping Free Radicals in Biological Systems 12. The Pharmacological Activity of Spin Traps 13. Future Directions

  • A Kinetic Approach to the Selection of a Sensitive Spin Trapping System for the Detection of Hydroxyl Radical
    Analytical biochemistry, 1994
    Co-Authors: Sovitj Pou, Myron S. Cohen, Carroll L. Ramos, T. Gladwell, E. Renks, M. Centra, D. Young, Gerald M. Rosen
    Abstract:

    The Spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) alone, as well as DMPO or N-tert-butyl-alpha-phenylnitrone (PBN) in the presence of excess dimethyl sulfoxide (Me2SO), have been used as Spin Trapping systems for the detection of hydroxyl radical. However, the instability of DMPO and many of its corresponding Spin-trapped adducts has limited the usefulness of this Spin trap, particularly in biological systems. Spin Trapping of multiple free radicals by the PBN/Me2SO system may undermine the sensitivity of this method to detect small, yet biologically significant amounts of hydroxyl radical. The present study was undertaken to select a Spin Trapping system with greater sensitivity and selectivity toward.OH than DMPO, DMPO/Me2SO, or PBN/Me2SO. We report that alpha-hydroxyethyl radical, resulting from the reaction of photolytically generated.OH with excess ethanol is Spin trapped by 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) with a second-order rate constant nearly 10-fold greater than that for DMPO or PBN. In contrast to DMPO Spin-trapped adducts, the alpha-hydroxyethyl radical adduct of 4-POBN, 4-POBN-CH(CH3)OH, is resistant to reduction by superoxide, even in the presence of cysteine. The efficiency of Spin Trapping and the marked stability of the resulting Spin-trapped adduct confer a high degree of sensitivity and demonstrate the potential application of 4-POBN/ETOH toward the detection of hydroxyl radical in biological systems.

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