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Free Radic Biol Med. 2018 Feb 20;116:134-140. doi: 10.1016/j.freeradbiomed.2018.01.014. Epub 2018 Jan 31.

Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.

Author information

1
University of Rochester Medical Center, Department of Pharmacology and Physiology, Rochester 14642, United States.
2
University of Rochester Medical Center, Department of Imaging Sciences, Rochester 14642, United States.
3
University of Rochester Medical Center, Department of Pharmacology and Physiology, Rochester 14642, United States; University of Rochester Medical Center, Department of Anesthesiology and Perioperative Medicine, Rochester 14642. United States. Electronic address: Andrew_Wojtovich@urmc.rochester.edu.

Abstract

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 ~4.0µmol O2•-/min/µmol photosensitizer for an excitation fluence rate of 5.9mW/mm2 at 470 ± 20nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•- to miniSOG phenotypes should be considered.

KEYWORDS:

Flavin mononucleotide; MiniSOG; Reactive oxygen species; Singlet oxygen; Superoxide

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