Protein Sensors and Reactive Oxygen Species - Part B: Thiol Enzymes and Proteins

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Reactions typically occur as a chain reaction where a free radical will capture a hydrogen moiety from an unsaturated carbon to form water. This leaves an unpaired electron on the fatty acid that is then capable of capturing oxygen, forming a peroxy radical Figure 5. Lipid peroxides are unstable and decompose to form a complex series of compounds, which include reactive carbonyl compounds, such as malondialdehyde MDA. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid TBA reactive compounds such as malondialdehyde generated from the decomposition of lipid peroxidation products [25].

While this method is controversial in that it is quite sensitive, but not necessarily specific to MDA, it remains the most widely used means to determine lipid peroxidation. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes IsoP has been shown to be specific for lipid peroxidation [30].

Unlike the TBA assay, measurement of IsoP appears to be specific to lipid peroxides, they are stable and are not produced by any enzymatic pathway making interpretation easier. There have been a number of commercial ELISA kits developed for IsoPs, but interfering agents in samples requires partial purification of samples prior to running the assay.

Lipid peroxidation in live cells can be visualized using fluorescent derivatives that localize to membranes. The ratio of red fluorescence to green fluorescence provides a measure of lipid peroxidation that is independent of factors such as lipid density that may influence measurement with singleemission probes.

Protein Sensors And Reactive Oxygen Species Part B Thiol Enzymes And Proteins

Because this reagent is compatible with live cells, measurements can take place in real time without fixation and staining. This reagent has also been used for demonstrating the antioxidant capacity of plasma [35] and lipid vesicles [36]. Linoleic acid is the most abundant polyunsaturated fatty acid found in mammals and its lipid peroxidation products likely account for the majority of lipid-derived protein carbonyls [37]. When incubated with live cells, LAA incorporates into cellular membranes.

Superoxide detection is based on the interaction of superoxide with some other compound to create a measurable result. The reduction of ferricytochrome c to ferrocytochrome c has been used in a number of situations to assess the rate of superoxide formation [38]. While not completely specific for superoxide this reaction can be monitored colorimetrically at nm. The addition of enzyme inhibitors such as CN- or scavengers of reactive species such as catalase can minimize any reoxidation. Aconitase catalyzes the conversion of citrate to isocitrate.

Thus superoxide concentrations can be estimated by the degree of enzyme inactivation. The activity of the enzyme can be monitored by following the conversion of 20 mM isocitrate to cicaconitate using absorbance at nm. Chemiluminescent reactions have been used for their potential increase in sensitivity over absorbance-based detection methods. The most widely used chemiluminescent substrate is Lucigenin, but this compound has a propensity for redox cycling, which has raised doubts about its use in determining quantitative rates of superoxide production [39].

The use of low concentrations of this compound has been suggested as a means to minimize this problem. Coelenterazine has also been used as a chemiluminescent substrate.

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This lypophilic compound does not redox cycle and is brighter than Lucigenin. It is not however completely specific towards superoxide, as the presence of peroxynitrite will result in chemiluminescence [40]. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical. These dyes are synthesized by reducing the iminium cation of the cyanine Cy dyes with sodium borohydride.

While weakly fluorescent, upon oxidation their fluorescence intensity increases fold.

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In addition to being fluorescent, oxidation also converts the molecule from being membrane permeable to an ionic impermeable moiety [41]. The most characterized of these probes are Hydro- Cy3 and Hydro-Cy5. Cellular production of superoxide can be visualized by dihydroethidium, also referred to as hydroethidine. This compound exhibits a blue fluorescence in the cytosol until oxidized primarily by superoxide and to a much lesser extent other reactive oxygen or reactive nitrogen species. Oxidation of dihydroethidium results in hydroxylation at the 2-position forming 2-hydroxyethidium Figure 9.

With oxidation the compound intercalated with cellular DNA, staining the nucleus a bright fluorescent red with reported excitation and emission wavelengths of nm and nm respectively [42]. Figure 9. Oxidation of Dihydroethidium to 2-Hydroxyethidium by Superoxide. The cationic triphenylphosphonium substituent of MitoSOX Red indicator is responsible for the electrophoretically driven uptake of the probe in actively respiring mitochondria Figure As with dihydroethidium, this compound intercalates with mitochondrial DNA resulting in red fluorescence. Figure As an example, the non-steroidal anti-inflammatory drug Diclofenac has been associated with hepatotoxicity through the induction of reactive oxygen species [43].

Autofocus performed on DAPI stained cells [44]. These cell-permeant dyes are weakly fluorescent while in a reduced state and exhibits photostable fluorescence upon oxidation by reactive oxygen species ROS. This compound has an excitation wavelength of nm and an emission wavelength of nm, making it amenable for imaging using the Green GFP filter sets.

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  2. Protein Sensors And Reactive Oxygen Species Part B Thiol Enzymes And Proteins!
  3. Metabolism of reactive species;

This reagent can be formaldehyde-fixed and its signal survives detergent treatment, allowing it to be it multiplexed with other compatible dyes and antibodies. Hydrogen peroxide H 2 O 2 is the most important ROS in regards to mitogenic stimulation or cell cycle regulation.

There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase HRP enzyme to produce intensely fluorescent products [21]. In these examples, increasing amounts of H 2 O 2 form increasing amounts of fluorescent product. For example, Amplex Red is oxidized by hydrogen peroxide in the presence of HRP and in doing so converted to resorufin [47].

Unlike Amplex Red, resorufin is a highly colored compound that can be detected colorimetrically at nm or by fluorescence using excitation of nm and emission of nm [48] Figure Homovannilic acid dimerizes when oxidized by hydrogen peroxide through horseradish peroxidase catalysis. As with Amplex red, homovanillic acid monomer is non-fluorescent, but as a dimmer, it possesses a peak excitation wavelength of nm, with an emission wavelength of nm Figure Care should be taken when using this compound to assess hydrogen peroxide production.

The near UV nature of the excitation and emission wavelengths for fluorescence measurements make this compound prone to inordinate background signal, particularly when polystyrene microplates are used. Several peroxidase-like metalloporphoryns have been shown to be catalytic for the reaction in addition to HRP [49]. Dimerization of homovanillic acid by the action of HRP and hydrogen peroxide. A number of colorimetric substrates such as tetramethylbenzidine TMB and phenol red have also been used in conjunction with HRP to measure hydrogen peroxide concentrations.

In general colorimetric means are less sensitive than fluorescent detection methods, but instrumentation costs are significantly lower than those required for fluorescence based measurements when using tube based or microplate based detection methodologies. There are a number of issues that one should be aware of when using HRP catalyzed substrates to quantitate hydrogen peroxide.

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Cellular compounds such as thiols can serve as a substrate for HRP. Endogenous catalase activity can artificially reduce the amount of H 2 O 2 present.

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Cellular components can affect the fluorescent signal depending on the excitation and emission wavelengths, as with homovanillic dimer, while other wavelengths may suffer from signal quenching. Tarpley et. The reported wavelengths for the measurement of DCF fluorescence are nm for excitation and nm for emission. In addition, as H2DCF is no longer ionic it is not precluded from migrating out of the cell and accumulating in the media, where it is free to interact with oxidants. For example, the cytotoxic quinoline alkaloid camptothecin, which inhibits DNA topoisomerase I, causes oxidative stress with cultured primary hepatocytes [51].

Oxidized DCF fluorescence in hepatocytes. Images of cultured hepatocytes captured after 0 and 30 minute treatments with nM camptothecin. In order to address the weaknesses of DCF fluorescence several new fluorescent probes have been developed. These boronate-based H 2 O 2 probes have been reported to have high selectivity, membrane permeability, along with visible-wavelength excitation and emission wavelengths [50]. Reacting with hydrogen peroxide, results in a fold and fold increase in fluorescence for PG1 and PC1, respectively. PG1 features an excitation wavelength of nm with emission maxima at nm Figure PC1 demonstrates improved characteristics of red-shifted excitation and larger stokes shift which reduces autofluorescence excitation: nm; emission: nm [52].


Structure of Peroxy Green 1 and Peroxy Crimson 1. These molecules exhibit a direct reaction with hydrogen peroxide not observed with H 2 DCF. In addition these molecules are unreactive towards high-valent metal-oxo species derived from heme-proteins and H 2 O 2. Calcein-acetoxymethylester Calcein-AM has also been reported as a detector to intracellular oxidative activity [53].

Calcein-AM is a fluorogenic cell permeable compound that is converted by intracellular esterases into the cell impermeant anion calcein, which is fluorescent Figure Historically intracellular calcein production has been used in both microscopy and fluorometery as an indicator of viable cells. In regards to the detection of ROS, the kinetics of ROS reaction are favorable relative to esterease conversion to calcein. The ROS-oxidized product of Calcein AM has been shown to be chemically distinct from Calcein-AM through thin layer chromatography [53], but it still retains the ability to cross cell membranes and has similar spectral properties to the fluorescent calcein.