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 H₂O₂ 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 H₂O₂ 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 H₂O₂ 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 H₂O₂ in all subcellular compartments. We also checked the local H₂O₂-quenching effect of N-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 H₂O₂ in diverse live models.