Significance: Since the discovery of the superoxide dismutase enzyme, the generation and fate of short-lived oxidizing, nitrosating, nitrating, and halogenating species in biological systems has been of great interest. Despite the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in numerous diseases and intracellular signaling, the rigorous detection of ROS and RNS has remained a challenge. Recent Advances: Chemical characterization of the reactions of selected ROS and RNS with electron paramagnetic resonance (EPR) spin traps and fluorescent probes led to the establishment of species-specific products, which can be used for specific detection of several forms of ROS and RNS in cell-free systems and in cultured cells in vitro and in animals in vivo . Profiling oxidation products from the ROS and RNS probes provides a rigorous method for detection of those species in biological systems. Critical Issues: Formation and detection of species-specific products from the probes enables accurate characterization of the oxidative environment in cells. Measurement of the total signal (fluorescence, chemiluminescence, etc .) intensity does not allow for identification of the ROS/RNS formed. It is critical to identify the products formed by using chromatographic or other rigorous techniques. Product analyses should be accompanied by monitoring of the intracellular probe level, another factor controlling the yield of the product(s) formed. Future Directions: More work is required to characterize the chemical reactivity of the ROS/RNS probes, and to develop new probes/detection approaches enabling real-time, selective monitoring of the specific products formed from the probes. Antioxid. Redox Signal . 28, 1416–1432.

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m⁻¹ s⁻¹) were significantly higher than that measured for H₂O₂ (1.5 m⁻¹ s⁻¹). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 10³ m⁻¹ s⁻¹. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.