A Kinetic Approach to the Selection of a Sensitive Spin Trapping System for the Detection of Hydroxyl Radical

Pou, Sovitj; Ramos, Carroll L.; Gladwell, Tim; Renks, E.; Centra, Michelle; Young, David H.; Cohen, Marc; Rosen, Gerald M.

doi:10.1006/abio.1994.1085

Cited by 115 publications

(93 citation statements)

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“…Upon illumination (Fig. 2C), the EPR spectrum exhibited the six lines with three equivalent doublets reported for the POBN-CH(CH 3 )OH adduct spectrum (22). (24).…”

Section: Resultsmentioning

confidence: 63%

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

Kale

Hebert

Frankel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

134

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The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O 2 . The identification of specific amino acid residues oxidized by reactive oxygen species provides insights into the mechanism of damage to the D1 and D2 proteins under light stress.photosynthesis | Photosystem II | reactive oxygen species | photo inhibition | mass spectrometry P hotosystem II (PSII) functions as a water-plastoquinone oxidoreductase (1, 2) and is a thylakoid membrane pigmentprotein complex present in all oxygenic photosynthetic organisms (cyanobacteria, algae, and higher plants). Current high-resolution structures of thermophilic cyanobacterial PSII (3, 4), and lower resolution structures of the red algal (5) and higher plant photosystems (6), have been critically important in furthering our understanding of the molecular organization of PSII. Structurally, the PSII reaction center core is composed of five proteins: D1, D2, the α-and β-subunits of cytochrome b 559 , and PsbI. These components bind all of the redox-active cofactors of PSII.Excitation energy transfer and electron transport within PSII are unavoidably associated with production of reactive oxygen species (ROS) when the absorption of light by the chlorophyll antenna exceeds the capacity for energy utilization. Many mechanisms for ROS production have been proposed (for reviews, see refs. 7 and 8). Briefly, singlet oxygen ( 1 O 2 ) may be formed by excitation energy transfer from triplet chlorophylls (formed either by the change in orientation of the spin of an excited electron in the PSII antenna complex, or via charge recombination of the primary radical pair 3 [P 680 •+ Pheo •− ]) to O 2 (9, 10). ROS production by electron transport involves either the two-electron oxidation of water or the one-electron reduction of O 2 on the PSII electron donor and acceptor sides, respectively. On the PSII electron donor side, a twoelectron oxidation of water leads to the formation of hydrogen peroxide (H 2 O 2 ), which may be oxidized to the superoxide anion radical (O 2•−

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“…Upon illumination (Fig. 2C), the EPR spectrum exhibited the six lines with three equivalent doublets reported for the POBN-CH(CH 3 )OH adduct spectrum (22). (24).…”

Section: Resultsmentioning

confidence: 63%

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

Kale

Hebert

Frankel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

134

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“…For our study, the spin traps DMPO and 4-POBN were employed. With DMPO, we can detect both superoxide and hydroxyl radical, whereas with 4-POBN in combination with EtOH, we can only identify hydroxyl radical [5,40,41]. Experiments were conducted by substituting either DMPO or 4-POBN/EtOH in place of cytochrome c in the above experiments.…”

Section: Resultsmentioning

confidence: 99%

“…Spin trapping of superoxide and hydroxyl radical was performed as described previously [5,40,41]. Spin trapping of superoxide was undertaken by mixing neutrophils (5 to 10 ϫ 10 6 /mL), DMPO (100 mM), PMA (100 ng/mL in DMSO), and sufficient buffer to reach a final volume of 0.5 mL.…”

Section: Esr/spin Trapping Of Superoxide and Hydroxyl Radicalmentioning

confidence: 99%

“…Spin trapping of superoxide was undertaken by mixing neutrophils (5 to 10 ϫ 10 6 /mL), DMPO (100 mM), PMA (100 ng/mL in DMSO), and sufficient buffer to reach a final volume of 0.5 mL. Spin trapping of hydroxyl radicals [41] was conducted by mixing neutrophils (10 to 20 ϫ 10 6 /mL), 4-POBN (10 mM), ethanol (170 mM), PMA (100 ng/mL in ethanol), and sufficient buffer to achieve a final volume of 0. Assay of elastase enzyme activity and effect of superoxide, hydroxyl radical, hydrogen peroxide, hypochlorous acid, and peroxynitrite on ICI200355 and ZD0892 Each elastase inhibitor (20 µM) was incubated at 37°C for 2 h in the presence of each oxidant.…”

Section: Esr/spin Trapping Of Superoxide and Hydroxyl Radicalmentioning

confidence: 99%

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Effect of trifluoromethyl ketone-based elastase inhibitors on neutrophil function in vitro

Huang

Surichamorn

Cao

et al. 1998

Journal of Leukocyte Biology

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Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous ␣ 1 -proteinase inhibitor (␣ 1 Pi). Nevertheless, under pathological conditions, ␣ 1 Pi is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/␣ 1 Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria. J. Leukoc. Biol. 64: 322-330; 1998.

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“…Here, we have monitored intracellular and extracellular free radical formation in S. aureus cells employing the lipophilic spin trap probes PBN (N-tert-butyl-␣-phenylnitrone), which mainly reacts with radicals buried in cell membrane, and DMPO (5,5-dimethyl-1-pyrroline N-oxide), which is relatively hydrophilic and applicable for detecting radicals in the water phase (32). For the PBN probe, sensitivity is significantly enhanced by ethanol, which reacts with hydroxyl and alkoxyl radicals to form relatively stable 1-hydroxyethyl radicals, which give highly stable spin adducts (33)(34)(35). Our results suggest that bactericidal antibiotics enhance extracellular free radical formation through repression of catalase expression, and they highlight the role of hydrogen peroxide-scavenging enzymes in the monitoring of free radical formation in bacterial cells.…”

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confidence: 99%

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Wang

Hougaard

Paulander

et al. 2015

Appl Environ Microbiol

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bDetection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-␣-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.

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A Kinetic Approach to the Selection of a Sensitive Spin Trapping System for the Detection of Hydroxyl Radical

Cited by 115 publications

References 0 publications

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

Effect of trifluoromethyl ketone-based elastase inhibitors on neutrophil function in vitro

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

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