Visualization of Phagosomal Hydrogen Peroxide Production by a Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling

Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O6-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence...