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. 281-288 ◽  
Author(s):  
Steven Yue QIAN ◽  
Yeong-Renn CHEN ◽  
Leesa J. DETERDING ◽  
Yang C. FANN ◽  
Colin F. CHIGNELL ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Uv Irradiation ◽  
Phenyl Ring ◽  
Esr Spectra ◽  
Trapping Site ◽  
Spectrometric Analysis ◽  
Tyrosyl Radical ◽  
Radical Trapping ◽  
Radical Adduct ◽  
Hplc Peak

The reaction of cytochrome c and H2O2 is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498–15503], we eliminated unreasonable structure models by ESR studies with a series of 13C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H2O2; (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H2O2. This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.

Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis

2004 ◽  
Vol 71 ◽  
pp. 97-106 ◽  
Author(s):  
Mark Burkitt ◽  
Clare Jones ◽  
Andrew Lawrence ◽  
Peter Wardman
Keyword(s):  
Cytochrome C ◽  
Hydroxyl Radical ◽  
Redox Regulation ◽  
Chemotherapeutic Agents ◽  
Tyrosyl Radical ◽  
Compound I ◽  
Definitive Evidence ◽  
Enhanced Production ◽  
Physico Chemical

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2′,7′-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2′,7′-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2′,7′-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.

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

1-Methyl-4-aryl-urazole (MAUra) labels tyrosine in proximity to ruthenium photocatalysts

2018 ◽  
Vol 54 (46) ◽  
pp. 5871-5874 ◽  
Author(s):  
Shinichi Sato ◽  
Kensuke Hatano ◽  
Michihiko Tsushima ◽  
Hiroyuki Nakamura
Keyword(s):  
Tyrosyl Radical ◽  
Tyrosine Residues ◽  
Radical Trapping ◽  
Trapping Agent

The 1-methyl-4-aryl-urazole (MAUra) structure was found to be a novel tyrosyl radical trapping agent to label tyrosine residues effectively in proximity to ruthenium photocatalysts.

scholarly journals A mass spectrometric analysis of 4-hydroxy-2-(E)-nonenal modificationof cytochrome c

2011 ◽  
Vol 46 (3) ◽  
pp. 290-297 ◽  
Author(s):  
Xiaoxia Tang ◽  
Lawrence M. Sayre ◽  
Gregory P. Tochtrop
Keyword(s):  
Cytochrome C ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis

scholarly journals Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper

Separations ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 66
Author(s):  
Eleonora Pizzi ◽  
Trine Grønhaug Halvorsen ◽  
Christian J. Koehler ◽  
Léon Reubsaet
Keyword(s):  
Cytochrome C ◽  
Protein Binding ◽  
Covalent Binding ◽  
Protein Analysis ◽  
Blue Staining ◽  
Spectrometric Analysis ◽  
Trypsin Activity ◽  
Potassium Periodate ◽  
Immobilize Trypsin ◽  
Fundamental Insight

This paper describes smart sampling paper to be used for bottom-up protein analysis. Four different manners to immobilize trypsin on cellulose were evaluated. Untreated paper, potassium-periodate-functionalized paper (with and without post-immobilization reduction) and 2-hydroxyethyl methacrylate (HEMA)/2-vinyl-4,4-dimethylazlactone (VDM)-functionalized paper were all used to immobilize trypsin. For the evaluation, Coomassie Brilliant Blue staining of proteins on paper and the BAEE trypsin activity assay needed to be modified. These methods allowed, together with data from mass spectrometric analysis of cytochrome C digestions, us to acquire fundamental insight into protein binding, and trypsin action and activity on paper. All functionalized discs bind more protein than the untreated discs. Protein binding to functionalized discs is based on both adsorption and covalent binding. Trypsin immobilized on potassium-periodate-functionalized discs exhibits the highest trypsin activity when using cytochrome C as substrate. It is proven that it is trypsin attached to paper (and not desorbed trypsin) which is responsible for the enzyme activity. The use of discs on complex biological samples shows that all functionalized discs are able to digest diluted serum; for the best-performing disc, HEMA-VDM functionalized, up to 200 high-confidence proteins are qualified, showing its potential.

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.

Grape skin extract prevents UV irradiation induced DNA damage of normal human epidermal keratinocytes cells

2020 ◽  
Vol 10 (4) ◽  
pp. 585-601
Author(s):  
Yutong Song ◽  
Serene Ezra Corpus Bondad ◽  
Hirotaka Tajima ◽  
Tomoyuki Sato ◽  
Nobutaka Wakamiya ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cytochrome C ◽  
Uv Irradiation ◽  
Epidermal Keratinocytes ◽  
Skin Extract ◽  
Gene Expressions ◽  
Human Epidermal Keratinocytes ◽  
Grape Skin ◽  
Normal Human Epidermal Keratinocytes ◽  
Normal Human

BACKGROUND: There is concern that DNA damage may occur in skin cells due to UV irradiation. In this study, we investigated whether extracts from grape skin, which should be discarded, can suppress DNA damage caused by UV radiation. OBJECTIVE: This study aims to investigate the effectivity of GSE in diminishing UV-induced cytotoxicity in normal human epidermal keratinocytes (NHEK) cells. METHODS: The polyphenol content in GSE was carried out using the HPLC instrument. UV dose was selected by measurement of cell viability, and ELISA results of DNA photoproduct. Optimum extracts condition of grape skin selected by DNA photoproduct content as well as at this condition; Bax/Bcl-2 ratio and cytochrome c gene expressions were evaluated by Western blotting. RESULTS: In this study, we confirmed that GSE protect against DNA damage-induced cell death. Condition for 80% EtOH for 24 h at 60 °C was suitable to extract for grape skin. Red grape skin (Zweigelt) was more effective than white grape skin (Niagara) in preventing (before irradiation) and repairing (after irradiation). UV-induced upregulation of Bax/Bcl-2 ratio and cytochrome c expression were reduced by GSE treatment. CONCLUSIONS: The study demonstrated a promising potential of GSEs in skin therapeutics application.

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