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

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 (O-1(2)), the contribution of superoxide (O-2(center dot-)) to miniSOG-generated ROS remains unclear. We measured the light-dependent O-2(center dot-) production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to O-1(2) and establish that DHE is a suitable indicator to measure O-2(center dot-) production in a system that produces both O-1(2) and O-2(center dot-). We report that miniSOG produces both O-1(2) and O-2(center dot-), as can its free chromophore, flavin mononucleotide. miniSOG produced O-2(center dot-) at a rate of similar to 4.0 mu mol O-2(center dot-)/min/mu mol photosensitizer for an excitation fluence rate of 5.9 mW/mm(2) at 470 +/- 20 nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O-2(center dot-) to miniSOG phenotypes should be considered.