scholarly journals Formation of peroxide- and globin-derived radicals from the reaction of methaemoglobin and metmyoglobin with t-butyl hydroperoxide: an ESR spin-trapping investigation

1997 ◽  
Vol 322 (2) ◽  
pp. 633-639 ◽  
Author(s):  
Jolanda Van der ZEE
Keyword(s):  
Spin Trap ◽  
Hydrogen Abstraction ◽  
Spin Trapping ◽  
Tyrosyl Radical ◽  
Alkoxyl Radical ◽  
Butyl Hydroperoxide ◽  
Heterolytic Cleavage ◽  
Esr Spin Trapping ◽  
Radical Adduct ◽  
Protein Radicals

The reaction of human methaemoglobin and horse metmyoglobin with t-butyl hydroperoxide (t-BuOOH) was investigated with the ESR spin-trapping technique. With the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) the formation of peroxyl, alkoxyl and methyl radicals derived from t-BuOOH could be detected. The relative contributions of these radicals were determined at various DMPO concentrations by computer simulation. From these data it could be concluded that the alkoxyl radical was the initial radical produced, which indicates that the hydroperoxide is cleaved homolytically. Further investigations, with the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), showed the formation of globin-centred radicals. Non-specific proteolysis of the MNP adducts revealed isotropic three-line spectra, which means that the radical adducts were centred on a tertiary carbon with no bonds to a hydrogen or nitrogen. Comparison with MNP adducts of several amino acids indicated that in methaemoglobin the radical adduct was most probably located on a valine residue. With metmyoglobin the same adduct was obtained, whereas an additional adduct could be assigned to a tyrosyl radical. These protein radicals most probably resulted from hydrogen abstraction by the metal–oxo species, formed by heterolytic cleavage of the hydroperoxide. These results therefore show that homolytic cleavage of the hydroperoxide leads to the formation of peroxide-derived radicals, whereas concurrent heterolytic cleavage results in protein-derived radicals.

scholarly journals Mechanism in the reaction of cytochrome c oxidase with organic hydroperoxides: an ESR spin-trapping investigation

2002 ◽  
Vol 365 (2) ◽  
pp. 461-469 ◽  
Author(s):  
Yeong-Renn CHEN ◽  
Ronald P. MASON
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Spin Trap ◽  
Spin Trapping ◽  
Initial Reaction ◽  
Radical Species ◽  
Alkoxyl Radical ◽  
Tyrosine Residues ◽  
Esr Spin Trapping ◽  
Organic Hydroperoxides

Organic hydroperoxides are of great utility in probing the reaction mechanism and the toxicological consequences of lipid peroxidation. In the present study, ESR spin-trapping was employed to investigate the peroxidation of mitochondrial cytochrome c oxidase (CcO) with t-butyl hydroperoxide (t-BuOOH) and cumene hydroperoxide (CumOOH). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect the radical species formed from the reaction of CcO with t-BuOOH. The presence of t-BuOOH-derived alkoxyl radical (t-BuO˙) as the primary radical indicates reductive scission of the O—O bond by CcO. The ESR signal of DMPO/˙Ot-Bu can be partially abolished by cyanide, implying that the reductive cleavage involved the haem a3CuB binuclear site of CcO. A nitroso spin trap, 2-methyl-2-nitrosopropane (MNP), was used to detect and identify radical species from the reaction of CcO with CumOOH. In addition to the t-BuOOH-derived methyl, hydroxylmethyl and tertiary carbon-centred radicals, a protein-derived radical was detected. The intensity of the ESR signal from the protein radical increased with the CumOOH concentration at low CumOOH/CcO ratios, with maximal intensity at a ratio of 100mol of CumOOH/mol of CcO. The immobilized protein radical adduct of MNP was stable and persistent after dialysis; it was also resistant to proteolytic digestion, suggesting that it was formed in the transmembrane region, a region that is not accessible to proteases. Its signal was greatly enhanced when CcO cysteine residues were chemically modified by N-ethylmaleimide, when the tryptophan residues in CcO were oxidized by N-bromosuccimide, and when tyrosine residues on the surface of CcO were iodinated, showing that a radical equilibrium was established among the cysteine, tryptophan and tyrosine residues of the protein-centred radical. Pre-treatment of CcO with cyanide prevented detectable MNP adduct formation, confirming that the haem a3-CuB binuclear centre was the initial reaction site. When the CcO was pre-treated with 10mM (100 equivalents) of CumOOH, the enzyme activity decreased by more than 20%. This inhibition was persistent after dialysis, suggesting that the detected protein-centred radical was, in part, involved in the irreversible inactivation by CumOOH. Visible spectroscopic analysis revealed that the haem a of CcO was not affected during the reaction. However, the addition of pyridine to the reaction mixture under alkaline conditions resulted in the destruction of the haem centre of CcO, suggesting that its protein matrix rather than its haem a is the target of oxidative damage by the organic hydroperoxide.

scholarly journals Spin trapping studies of essential oils in lipid systems

Nukleonika ◽  
2015 ◽  
Vol 60 (3) ◽  
pp. 461-468 ◽  
Author(s):  
Katerina Makarova ◽  
Kinga Drązikowska ◽  
Beata Suska ◽  
Katarzyna Zawada ◽  
Iwona Wawer
Keyword(s):  
Essential Oils ◽  
Fenton Reaction ◽  
Spin Trap ◽  
Spin Trapping ◽  
Oh Radicals ◽  
Bath Salts ◽  
Radical Adduct ◽  
Hyperfine Splittings ◽  
Skin Care Products ◽  
Fenton System

Abstract In the present work, we report the results of a spin trapping ESR study of four essential oils widely used for skin care products such as creams and bath salts. The studied essential oils are Rosmarini aetheroleum (rosemary), Menthae piperitae aetheroleum (mint), Lavandulae aetheroleum (lavender), and Thymi aetheroleum (thyme). Fenton reaction in the presence of ethanol was used to generate free radicals. The N-tert-butyl-α-phenylnitrone (PBN) was used as a spin trap. In the Fenton reaction, the rosemary oil had the lowest effect on radical adduct formation as compared to the reference Fenton system. Since essential oils are known to be lipid soluble, we also conducted studies of essential oils in Fenton reaction in the presence of lipids. Two model lipids were used, namely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The obtained results suggested that in the presence of DOPC lipids, the •OH and PBN/•CHCH3(OH) radicals are formed in both phases, that is, water and lipids, and all the studied essential oils affected the Fenton reaction in a similar way. Whereas, in the DPPC system, the additional type of PBN/X (aN = 16.1 G, aH = 2.9 G) radical adduct was generated. DFT calculations of hyperfine splittings were performed at B3LYP/6-311+G(d,p)/EPR-II level of theory for the set of c-centered PBN adducts in order to identify PBN/X radical.

scholarly journals Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS

2002 ◽  
Vol 363 (2) ◽  
pp. 281 ◽  
Author(s):  
Steven Yue QIAN ◽  
Yeong-Renn CHEN ◽  
Leesa J. DETERDING ◽  
Yang C. FANN ◽  
Colin F. CHIGNELL ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome C ◽  
Spin Trapping ◽  
Tyrosyl Radical ◽  
Esr Spin Trapping

scholarly journals Investigation into the Direct Photolysis Process of Photo-Induced RAFT Polymerization by ESR Spin Trapping

Polymers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1722
Author(s):  
Jiajia Li ◽  
Mengmeng Zhang ◽  
Jian Zhu ◽  
Xiulin Zhu
Keyword(s):  
Vinyl Acetate ◽  
Hyperfine Coupling ◽  
Spin Trap ◽  
Raft Polymerization ◽  
Spin Trapping ◽  
Direct Photolysis ◽  
Esr Spin Trapping ◽  
Reversible Addition ◽  
Spin Resonance ◽  
Successful Capture

The direct photolysis of reversible addition fragmentation chain transfer (RAFT) agents under visible light was demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline N-oxide as a typical spin trap. The hyperfine coupling lines obtained by ESR spectroscopy showed the successful capture of the carbon-centered and the sulfur-centered radical. Photo-polymerization of vinyl acetate under different wavelengths was performed to verify the effects of wavelength on the process. The effect of the R group of RAFT agents on the photolysis was investigated by spin-trapping experiments using poly (butyl acrylate) and poly (vinyl acetate) as macroRAFT agents. The quantitative experiment showed the yield of photolysis of a xanthate to be only 0.023% under λ > 440 nm.

scholarly journals Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Spin trapping of alkoxyl and peroxyl radicals in organic solvents

1986 ◽  
Vol 240 (3) ◽  
pp. 789-795 ◽  
Author(s):  
M J Davies ◽  
T F Slater
Keyword(s):  
Fatty Acids ◽  
Spin Trap ◽  
Hydrogen Abstraction ◽  
Spin Trapping ◽  
Ring Closure ◽  
Peroxyl Radicals ◽  
Resonance Spectroscopy ◽  
Oxygen Oxygen ◽  
Low Levels ◽  
Trap Radical

Spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to detect and distinguish between the carbon-centred, alkoxyl, and peroxyl radicals produced during the photolytic decomposition of hydroperoxides. Photolysis of tert-butyl and cumene hydroperoxides, and peroxidized fatty acids, in toluene, with low levels of u.v. light, is shown to lead to the initial production of alkoxyl radicals by homolysis of the oxygen-oxygen bond. Subsequent reaction of these radicals with excess hydroperoxide leads, by hydrogen abstraction, to the production of peroxyl radicals that can be detected as their corresponding adducts with the spin trap. Subsequent breakdown of these adducts produces alkoxyl radicals and a further species that is believed to be the oxidized spin-trap radical 5,5-dimethyl-1-pyrrolidone-2-oxyl. No evidence was obtained at low hydroperoxide concentrations, with either the cumene or lipid alkoxyl radicals, for the occurrence of beta-scission reactions; the production of low levels of carbon-centred radicals is believed to be due to the alternative reactions of hydrogen abstraction, ring closure, and/or 1,2 hydrogen shifts. Analogous experiments with 3,3,5,5-tetramethyl-1-pyrroline N-oxide (TMPO) led only to the trapping of alkoxyl radicals with no evidence for peroxyl radical adducts, this is presumably due to a decreased rate of radical addition because of increased steric hindrance.

scholarly journals Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide

1998 ◽  
Vol 330 (3) ◽  
pp. 1293-1299 ◽  
Author(s):  
R. Michael GUNTHER ◽  
Richard A. TSCHIRRET-GUTH ◽  
H. Ewa WITKOWSKA ◽  
C. Yang FANN ◽  
P. David BARR ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Spin Trap ◽  
Sperm Whale ◽  
Spin Trapping ◽  
Tryptophan Residue ◽  
Radical Adduct ◽  
Unpaired Electron Density ◽  
Trapping Agent ◽  
Tyrosine Radical ◽  
Radical Yield

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

scholarly journals Studies on the metal-ion and lipoxygenase-catalysed breakdown of hydroperoxides using electron-spin-resonance spectroscopy

1987 ◽  
Vol 245 (1) ◽  
pp. 167-173 ◽  
Author(s):  
M J Davies ◽  
T F Slater
Keyword(s):  
Metal Ion ◽  
Spin Trap ◽  
Cumene Hydroperoxide ◽  
Initial Step ◽  
Peroxyl Radicals ◽  
Resonance Spectroscopy ◽  
Alkoxy Radical ◽  
Alkoxyl Radical ◽  
Radical Adduct ◽  
Chemical Studies

The breakdown of cumene hydroperoxide and peroxidized fatty acids by iron is shown, by use of the spin trap 5,5-dimethyl-l-pyrroline-N-oxide, to be sensitive to (a) the oxidation state of the metal and (b) the nature of the chelating ligands. The initial step in the Fe2+-catalysed breakdown is the production of an alkoxyl radical by one-electron reduction, and this type of radical has been successfully trapped from each substrate. Subsequent reactions of this alkoxyl species produce both carbon-centred and peroxyl radicals, depending on the concentrations of the reagents present. The use of the same spin trap in microsomal systems undergoing either NADPH-supported or Fe2+-induced peroxidation led to the detection of low concentrations of radical adducts, among which are signals that are believed to be due to lipid alkoxyl radicals. Reaction of polyunsaturated fatty acid hydroperoxides with both Fe2+ and lipoxygenase under anaerobic conditions gives rise to signals not only from the alkoxy-radical adduct, but also from a further species which is tentatively identified as being due to an acyl [RC(O).]-radical adduct; chemical studies lend support to this assignment.

scholarly journals ESR Spin-trapping of a Protein-derived Tyrosyl Radical from the Reaction of Cytochromecwith Hydrogen Peroxide

1996 ◽  
Vol 271 (26) ◽  
pp. 15498-15503 ◽  
Author(s):  
David P. Barr ◽  
Michael R. Gunther ◽  
Leesa J. Deterding ◽  
Kenneth B. Tomer ◽  
Ronald P. Mason
Keyword(s):  
Hydrogen Peroxide ◽  
Spin Trapping ◽  
Tyrosyl Radical ◽  
Esr Spin Trapping
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