Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.
Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.
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DOI：
10.1016/j.freeradbiomed.2018.01.014
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发表时间：
2018-02-20
影响因子：
7.4
通讯作者：
Wojtovich AP
中科院分区：
文献类型：
--
作者：
Barnett ME;Baran TM;Foster TH;Wojtovich AP
基因编码的光敏剂响应光而产生活性氧 (ROS)。融合蛋白的转基因表达可以将光敏剂靶向特定的细胞区域，并允许在空间和时间上控制 ROS 的产生。这些 ROS 生成蛋白 (RGP) 广泛用于目标蛋白的细胞消融、诱变和发色团辅助光灭活。然而，由于间接测量和令人困惑的解释，RGP 产生的物种尚不清楚。最近，RGP 迷你“单线态氧气发生器”(miniSOG) 由拟南芥向光素 2 改造而成。虽然 miniSOG 产生单线态氧 (1O2)，但超氧化物 (O2•−) 对 miniSOG 生成的 ROS 的贡献仍不清楚。我们使用二氢乙锭 (DHE) 氧化产物的 HPLC 分离测量了纯化 miniSOG 的光依赖性 O2•− 产生。我们证明 DHE 对 1O2 不敏感，并确定 DHE 是测量同时产生 1O2 和 O2•− 的系统中 O2•− 产量的合适指标。我们报告说，miniSOG 既产生 1O2 又产生 O2•−，其游离发色团黄素单核苷酸也能产生这种情况。 miniSOG 产生 O2•− 的速率约为 4.0 μmol O2•−/min/μmol 光敏剂，在 470 ± 20 nm 处的激发注量率为 5.9 mW/mm2，并且该速率在各个注量（光剂量）之间保持一致。总体而言，应考虑 O2•− 对 miniSOG 表型的贡献。
Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini “Singlet Oxygen Generator” (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen (1O2), the contribution of superoxide (O2•−) to miniSOG-generated ROS remains unclear. We measured the light-dependent O2•− production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1O2 and establish that DHE is a suitable indicator to measure O2•− production in a system that produces both 1O2 and O2•−. We report that miniSOG produces both 1O2 and O2•−, as can its free chromophore, flavin mononucleotide. miniSOG produced O2•− at a rate of ~4.0 μmol O2•−/min/μmol photosensitizer for an excitation fluence rate of 5.9 mW/mm2 at 470 ± 20 nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•− to miniSOG phenotypes should be considered.
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