Enzymatic Recording of Local Hydrogen Peroxide Generation Using Genetically Encodable Enzyme.

Reactive oxygen species (ROS) are endogenously generated in live cells and essential for cell signaling. However, excess ROS generation can cause oxidative damage to biomolecules, which are implicated in various human diseases, including aging. Here, we developed an in vivo hydrogen peroxide monitoring method using a genetically encodable peroxidase (APEX2)-based system. We confirmed that APEX2 is activated by endogenous HO and generates phenoxyl radicals to produce biotinylated signals (i.e., biotin-phenol) and fluorescent signals (i.e., AmplexRed), which can be detected using a fluorescence microscope. We observed that all subcellular targeted APEX2s were activated by local HO generation by menadione treatment. Among them, the endoplasmic reticulum lumen and lysosome-targeted APEX2 showed the highest response upon addition of menadione which implies that local HO levels in those spaces are highly increased by menadione treatment. Using APEX2, we also found that a minimum amount of menadione (>10 μM) is required to generate detectable levels of HO in all subcellular compartments. We also checked the local HO-quenching effect of -acetylcysteine using our system. As APEX2 can be genetically expressed in diverse live organisms (e.g., cancer cell lines, mice, fly, worm, and yeast), our method can be effectively used to detect local generation of endogenously produced HO in diverse live models.