Kumar Aditya, Prasad Ankush, Sedlářová Michaela, Pospíšil Pavel
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University Olomouc, Olomouc, Czechia.
Department of Botany, Faculty of Science, Palacký University Olomouc, Olomouc, Czechia.
Front Physiol. 2019 Aug 13;10:958. doi: 10.3389/fphys.2019.00958. eCollection 2019.
Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.
几十年来，人们一直在研究暴露于高光下的光系统II（PSII）中蛋白质的氧化修饰，但蛋白质氧化形成的蛋白质自由基的特征在很大程度上尚不清楚。蛋白质氧化是由蛋白质与活性氧的直接反应诱导的，已知活性氧会形成高度反应性的蛋白质自由基，包括以碳为中心的（烷基）和以氧为中心的（过氧自由基和烷氧自由基）自由基。在本研究中，基于使用抗DMPO抗体检测5,5-二甲基-1-吡咯啉N-氧化物（DMPO）硝酮加合物，通过免疫自旋捕获技术监测了暴露于高光下的拟南芥中的蛋白质自由基。使用与抗DMPO抗体偶联的荧光素，通过共聚焦激光扫描显微镜对拟南芥叶片和叶绿体中的蛋白质自由基进行成像。使用PSII蛋白特异性抗体通过标准印迹技术对蛋白质自由基进行表征，结果表明蛋白质自由基在D1、D2、CP43、CP47和Lhcb3蛋白上形成。蛋白质条带的出现/消失所反映的蛋白质氧化表明，蛋白质自由基的形成与蛋白质片段化（D1肽键的断裂）和聚集（与另一个PSII亚基交联）有关。蛋白质自由基形成的表征对于更好地理解高光下PSII蛋白氧化修饰的机制很重要。
