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

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.

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Spin trapping studies of essential oils in lipid systems

Nukleonika ◽

10.1515/nuka-2015-0082 ◽

2015 ◽

Vol 60 (3) ◽

pp. 461-468 ◽

Cited By ~ 1

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.

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Tryptophan-14 Is the Preferred Site of DBNBS Spin Trapping in the Self-Peroxidation Reaction of Sperm Whale Metmyoglobin with a Single Equivalent of Hydrogen Peroxide

Chemical Research in Toxicology ◽

10.1021/tx0256580 ◽

2003 ◽

Vol 16 (5) ◽

pp. 652-660 ◽

Cited By ~ 16

Author(s):

Michael R. Gunther ◽

Richard A. Tschirret-Guth ◽

Olivier M. Lardinois ◽

Paul R. Ortiz de Montellano

Keyword(s):

Hydrogen Peroxide ◽

Sperm Whale ◽

Spin Trapping ◽

The Self

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

Biochemical Journal ◽

10.1042/bj3220633 ◽

1997 ◽

Vol 322 (2) ◽

pp. 633-639 ◽

Cited By ~ 23

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.

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EPR detection of the unstable tert-butylperoxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline N-oxide: a combined spin-trapping and continuous-flow investigation

Journal of the Chemical Society Perkin Transactions 2 ◽

10.1039/b207709b ◽

2002 ◽

pp. 2044-2051 ◽

Cited By ~ 20

Author(s):

Clare M. Jones ◽

Mark J. Burkitt

Keyword(s):

Continuous Flow ◽

Spin Trap ◽

Spin Trapping ◽

Radical Adduct ◽

Flow Investigation

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The spin trapping agent PBN stimulates H2O2-induced Erk and Src kinase activity in human neuroblastoma cells

Neuroreport ◽

10.1097/00001756-200206120-00016 ◽

2002 ◽

Vol 13 (8) ◽

pp. 1057-1061 ◽

Cited By ~ 16

Author(s):

Pelin Kelicen ◽

Ippolita Cantuti-Castelvetri ◽

Can Pekiner ◽

K. Eric Paulson

Keyword(s):

Kinase Activity ◽

Neuroblastoma Cells ◽

Src Kinase ◽

Spin Trapping ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Trapping Agent

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Mechanism of photodegradation of aqueous organic pollutants. 2. Measurement of the primary rate constants for reaction of hydroxyl radicals with benzene and some halobenzenes using an EPR spin-trapping method following the photolysis of hydrogen peroxide

Environmental Science & Technology ◽

10.1021/es00026a004 ◽

1992 ◽

Vol 26 (2) ◽

pp. 262-265 ◽

Cited By ~ 98

Author(s):

Jan Kochany ◽

James R. Bolton

Keyword(s):

Hydrogen Peroxide ◽

Organic Pollutants ◽

Hydroxyl Radicals ◽

Rate Constants ◽

Spin Trapping ◽

Trapping Method ◽

Epr Spin Trapping

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Effect of Superoxide Dismutase Mimics on Radical Adduct Formation during the Reaction between Peroxynitrite and Thiols—An ESR-Spin Trapping Study

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1996.0232 ◽

1996 ◽

Vol 330 (1) ◽

pp. 115-124 ◽

Cited By ~ 9

Author(s):

Hakim Karoui ◽

Neil Hogg ◽

Joy Joseph ◽

B. Kalyanaraman

Keyword(s):

Superoxide Dismutase ◽

Spin Trapping ◽

Adduct Formation ◽

Esr Spin Trapping ◽

Radical Adduct ◽

Superoxide Dismutase Mimics

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Activation of ENaC in collecting duct cells by prorenin and its receptor PRR: involvement of Nox4-derived hydrogen peroxide

AJP Renal Physiology ◽

10.1152/ajprenal.00492.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. F1243-F1250 ◽

Cited By ~ 40

Author(s):

Xiaohan Lu ◽

Fei Wang ◽

Mi Liu ◽

Kevin T. Yang ◽

Adam Nau ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Spin Trap ◽

Collecting Duct ◽

Reactive Oxygen ◽

Oxygen Species ◽

Equivalent Current ◽

Duct Cells ◽

Collecting Duct Cells ◽

Nanomolar Range

The collecting duct (CD) has been recognized as an important source of prorenin/renin, and it also expresses (pro)renin receptor (PRR). The goal of this study was to examine the hypothesis that prorenin or renin via PRR regulates epithelial Na+ channel (ENaC) activity in mpkCCD cells. Transepithelial Na+ transport was measured by using a conventional epithelial volt-ohmmeter and was expressed as the calculated equivalent current ( Ieq). Amiloride-inhibitable Ieq was used as a reflection of ENaC activity. Administration of prorenin in the nanomolar range induced a significant increase in Ieq that was detectable as early as 1 min, peaked at 5 min, and gradually returned to baseline within 15 min. These changes in Ieq were completely prevented by a newly developed PRR decoy inhibitor, PRO20. Prorenin-induced Ieq was inhibitable by amiloride. Compared with prorenin, renin was less effective in stimulating Ieq. Prorenin-induced Ieq was attenuated by apocynin but enhanced by tempol, the latter effect being prevented by catalase. In response to prorenin treatment, the levels of total reactive oxygen species and H2O2 were both increased, as detected by spin-trap analysis and reactive oxygen species (ROS)-Glo H2O2 assay, respectively. Both siRNA-mediated Nox4 knockdown and the dual Nox1/4 inhibitor GKT137892 attenuated prorenin-induced Ieq. Overall, our results demonstrate that activation of PRR by prorenin stimulates ENaC activity in CD cells via Nox4-derived H2O2.

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