Since the discovery of reactive oxygen species (ROS) and their role in cell physiology and pathology (1), a number of accurate techniques for the measurement of ROS in vitro have

John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_6

been developed and are now widely used. Most of them can be applied to model systems such as isolated proteins, organelles, cell or tissue extracts, or permeabilized cells. However, most attempts to directly measure ROS in intact cells were not very successful. Often, information about ROS participation in biochemical processes is mostly based on indirect evidence, for example, the level of DNA/lipids/proteins oxidation. Also, the significance of the data obtained with optical fluorescent probes for ROS detection is now not clear because of the high risk of artifacts. The most known dichlorofluorescein (DCF) derivatives fluoresce after oxidation by ROS. However, these dyes could be oxidized by different types of ROS and reactive nitrogen species (2). Moreover, DCF derivatives are able to produce ROS upon exposure to light (3, 4), which leads to an artifactual ROS generation and signal amplification. Like most chemical probes, these dyes cannot be targeted to various intracellular compartments known to be the major ROS sources-such as plasma membrane and mitochondria.

Recently, we developed the first genetically encoded fluorescent indicator for intracellular hydrogen peroxide detection, named HyPer (5). It consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the Escherichia coli H2O2-sensing protein, OxyR. OxyR contains an H2 O2-sensitive regulatory domain (amino acids 80310) and a DNA-binding domain (amino acids 1-79). Upon oxidation by H2O2, the reduced form of OxyR is converted into an oxidized DNA-binding form. The key residues of OxyR in this regard are Cys199 and Cys208 (6). Cys199 resides within a hydrophobic pocket, and exposure of OxyR to H2 O2 converts Cys199 to a charged intermediate that is released from the hydrophobic surround and forms a disulfide bridge with Cys208. As a result, a dramatic conformational change occurs in the regulatory domain of OxyR, especially within a flexible region located at residues 205-222 (7).

We produced HyPer by inserting cpYFP into the regulatory domain of OxyR between residues 205 and 206. HyPer demonstrates a submicromolar affinity to H2 O2, and at the same time it is insensitive to other oxidants. HyPer has two excitation peaks at 420 and 500 nm and one emission peak at 516 nm. Upon exposure to H2O2, the excitation peak at 420 nm decreases proportionally to the increase in the peak at 500 nm in a ratio-metric manner.

In contrast to DCF derivatives, HyPer is genetically encoded and therefore could be targeted to various subcellular compartments. HyPer is able to detect relatively high concentrations of H2O2 produced during apoptosis as well as low-level H2O2 produced in cells upon physiological stimulation by growth factors. HyPer can be rapidly oxidized by submicromolar H2O2 even in a highly reduced intracellular environment. Thus, HyPer lacks disadvantages of chemical probes for ROS detection. The use of HyPer significantly simplifies both monitoring of changes in H2O2 levels and in the interpretation of the results.

In this chapter, we describe a protocol for fluorescent confocal HyPer imaging of changes in H2O2 level in the cytoplasm of HeLa cells stimulated with epidermal growth factor (EGF). The protocol is short and simple and can be applied with minimal variations for other cell lines upon different conditions where a change in the level of H2O2 inside the cell is suspected.

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