Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.
In this study, electron paramagnetic resonance spin-trapping spectroscopy was used to study the light-induced production of superoxide anion (O2 (•-)) and carbon-centered (R(•)) radicals by Photosystem II (PSII). It is evidenced here that exposure of PSII membranes to high light (2,000 μmol photons m(-2) s(-1)) or heat (47 °C) treatments prior to the illumination suppressed O2 (•-) production, while R(•) was formed. Formation of R(•) in the both high light- and heat-treated PSII membranes was enhanced by DCMU. Removal of molecular oxygen by glucose/glucose oxidase/catalase system and O2 (•-) scavenging by exogenous superoxide dismutase completely suppressed carbon-centered radical formation. It is proposed here that the oxidation of polyunsaturated fatty acids and amino acids by O2 (•-) on the electron acceptor side of PSII results in the formation of R(•), known to initiate a cascade reaction leading to the lipid peroxidation and protein degradation, respectively.
- MeSH
- elektronová paramagnetická rezonance MeSH
- fotochemické procesy MeSH
- fotosystém II - proteinový komplex chemie metabolismus MeSH
- kyslík chemie metabolismus MeSH
- oxidace-redukce MeSH
- spin trapping metody MeSH
- Spinacia oleracea MeSH
- superoxidy chemie metabolismus MeSH
- světlo MeSH
- volné radikály chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
When photosystem II (PSII) is exposed to excess light, singlet oxygen ((1)O(2)) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair (3)[P680(•+)Pheo(•-)] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of (1)O(2) in the donor side photoinhibition of PSII. Light-induced (1)O(2) production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R(•)) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R(•) by propyl gallate significantly suppressed (1)O(2). Based on our results, it is concluded that (1)O(2) formation correlates with R(•) formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680(•+)/TyrZ(•). It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R(•) with molecular oxygen is a plausible mechanism for (1)O(2) formation in the donor side photoinhibition of PSII.
- MeSH
- chemické modely MeSH
- elektronová paramagnetická rezonance metody MeSH
- fotochemie metody MeSH
- fotosystém II - proteinový komplex fyziologie MeSH
- koncentrace vodíkových iontů MeSH
- kyslík chemie MeSH
- propylgalan chemie MeSH
- singletový kyslík * MeSH
- spin trapping metody MeSH
- Spinacia oleracea MeSH
- světlo MeSH
- Synechococcus metabolismus MeSH
- uhlík chemie MeSH
- volné radikály MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydroxyl radical (HO•) production in photosystem II (PSII) was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that the exposure of PSII membranes to heat stress (40 °C) results in HO• formation, as monitored by the formation of EMPO-OH adduct EPR signal. The presence of different exogenous halides significantly suppressed the EMPO-OH adduct EPR signal in PSII membranes under heat stress. The addition of exogenous acetate and blocker of chloride channel suppressed the EMPO-OH adduct EPR signal, whereas the blocker of calcium channel did not affect the EMPO-OH adduct EPR signal. Heat-induced hydrogen peroxide (H₂O₂) production was studied by amplex red fluorescent assay. The presence of exogenous halides, acetate and chloride blocker showed the suppression of H₂O₂ production in PSII membranes under heat stress. Based on our results, it is proposed that the formation of HO• under heat stress is linked to uncontrolled accessibility of water to the water-splitting manganese complex caused by the release of chloride ion on the electron donor side of PSII. Uncontrolled water accessibility to the water-splitting manganese complex causes the formation of H₂O₂ due to improper water oxidation, which leads to the formation of HO• via the Fenton reaction under heat stress.
- MeSH
- chloridy metabolismus MeSH
- elektronová paramagnetická rezonance metody MeSH
- fotosystém II - proteinový komplex chemie metabolismus MeSH
- hydroxylový radikál metabolismus MeSH
- oxidace-redukce MeSH
- peroxid vodíku farmakologie MeSH
- spin trapping metody MeSH
- Spinacia oleracea chemie MeSH
- vytápění MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- krysa rodu rattus MeSH
- reperfuzní poškození myokardu metabolismus prevence a kontrola MeSH
- scavengery volných radikálů farmakologie MeSH
- spin trapping metody MeSH
- spinové značení MeSH
- techniky in vitro MeSH
- volné radikály analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
