Abstract
Hydrogen peroxide (H(2)O(2)) is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of its production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring local, subcellular changes in H(2)O(2) levels by fluorescence imaging. Specifically, we have exploited the versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface or interior of living cells with the use of boronate-capped dyes to selectively visualize H(2)O(2). The resulting SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to SNAP-tag fusion proteins. Spectroscopic measurements of the SNAP-PG constructs confirm their ability to detect H(2)O(2) with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence turn-on in response to changes in local H(2)O(2) levels. This work provides a general molecular imaging platform for assaying H(2)O(2) chemistry in living cells with subcellular resolution.