On the use of peroxy-caged luciferin (PCL-1) probe for bioluminescent detection of inflammatory oxidants in vitro and in vivo – Identification of reaction intermediates and oxidant-specific minor products

Abstract

Peroxy-caged luciferin (PCL-1) probe was first used to image hydrogen peroxide in living systems (Van de Bittner GC et al. Proc Natl Acad Sci U S A. 2010; 107:21316–21). Recently this probe was shown to react with peroxynitrite more potently than with hydrogen peroxide (Sieracki et al. Free Radic Biol Med. 2013; 61:40–50) and was suggested to be a more suitable probe for detecting peroxynitrite under in vivo conditions. In this work, we investigated in detail the products formed from the reaction between PCL-1 and hydrogen peroxide, hypochlorite, and peroxynitrite. HPLC analysis showed that hydrogen peroxide reacts slowly with PCL-1, forming luciferin as the only product. Hypochlorite reaction with PCL-1 yielded significantly less luciferin, as hypochlorite oxidized luciferin to form a chlorinated luciferin. Reaction between PCL-1 and peroxynitrite consists of a major and minor pathway. The major pathway results in luciferin and the minor pathway produces a radical-mediated nitrated luciferin. Radical intermediate was characterized by spin trapping. We conclude that monitoring of chlorinated and nitrated products in addition to bioluminescence in vivo will help identify the nature of oxidant responsible for bioluminescence derived from PCL-1.

Tracking isotopically labeled oxidants using boronate-based redox probes

“…Boronates are oxidized more than a thousand times faster by HOCl than by H2O2 at neutral pH (44). The product of the reaction is a phenol (or alcohol), which may undergo chlorination in the presence of excess HOCl (47,51,57). To determine the source of the oxygen atom during the conversion of oMitoPhB(OH)2 into oMitoPhOH, we generated H 16 OCl and H 18 OCl in situ from myeloperoxidase (MPO)-catalyzed oxidation of chloride anions by H2 16 O2 and H2 18 O2, respectively (Fig.…”

“…The omission of KCl or MPO resulted in a significantly lower yield of the product. Also, addition of small amounts of dimethyl sulfoxide (DMSO), known to rapidly scavenge HOCl (47,58), led to a significant attenuation of the formation of the phenolic product. Formation of HOCl was further confirmed by the detection of 2-Cl-E + in analogous systems, using the HE probe instead of the boronate (Fig.…”

“…8), formed in 10% and 0.5% yields, respectively (51). Although the mechanism of the oxidation of boronates by ONOOhas been extensively studied, both experimentally and using theoretical calculations (44,45,47,49), the isotopelabeling studies have not been performed. We decided to test the proposed reaction mechanism by reacting oMitoPhB(OH)2 with 18 O-labeled ONOO -, produced in situ from co-generated fluxes of nitric oxide ( • NO) and 18 O2 •-(7,8).…”

“…In the presence of excess HOCl or ONOO -, the phenolic product may undergo chlorination or nitration, respectively, providing an opportunity to identify the oxidant by profiling the products formed (14,44). As an example, in the presence of HOCl, the peroxy-caged luciferin probe is converted not only to luciferin but also to chloroluciferin (47). The reaction of boronate probes with ONOOis of special interest, as this reaction typically proceeds via two pathways of the decomposition of peroxynitrite adduct to the boronate: (i) major pathway (~85%-90%) involving heterolytic cleavage of the peroxyl bond, leading to the formation the phenolic product; and (ii) minor pathway (10%-15%), involving a homolytic cleavage of the peroxyl bond, with the formation of phenyl-type radical and • NO2, which upon recombination form nitrobenzene-type product (Fig.…”

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future

“…5-40 K) is eminently suitable for detecting and investigating redox-active mitochondrial iron-sulfur proteins (aconitase and mitochondrial respiratory chain complexes) (7)(8)(9). During the last decade, much progress has been made with respect to understanding the mechanisms of ROS-induced oxidation of fluorescent, chemiluminescent, and bioluminescent probes (10,11). A comprehensive understanding of the kinetics, stoichiometry, and intermediate and product analyses of several ROS probes in various ROS-generating systems makes it possible to investigate these species in cells and tissues (12)(13)(14)(15).…”