Brief communicationAssays using horseradish peroxidase and phenolic substrates require superoxide dismutase for accurate determination of hydrogen peroxide production by neutrophils
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Superoxide radicals react with peptide-derived tryptophan radicals with very high rate constants to give hydroperoxides as major products
2018, Free Radical Biology and MedicineCitation Excerpt :These reactions of Trp• show some analogies to those of Tyr phenoxyl radicals, (Tyr•) which also react with O2•− to form hydroperoxides (e.g. [22,26,27]). The presence of SOD has been shown to inhibit the formation of peroxides formed on Tyr by O2•−, and to promote the formation of diTyr [22,26,68], suggesting that, as with Trp, reaction with O2•− competes with Tyr• dimerization to give the well-established cross-link, di-tyrosine. It should also be borne in mind that a contribution to the overall peroxide yields that might be detected in more complex systems, might also come from addition of O2 to Tyr• and Trp• to give peroxyl radicals and subsequently hydroperoxides by hydrogen atom abstractions.
Challenges and advances in quantum dot fluorescent probes to detect reactive oxygen and nitrogen species: A review
2015, Analytica Chimica ActaCitation Excerpt :We believe this difficulty has been hampered by the complexity involved in the design of such probe. For example, for intracellular detection, H2O2 is known not to react directly with fluorescent probes but normally requires the presence of an enzyme or metal catalyst bound to the fluorophore to influence the fluorescence signal of the fluorophore and also to provide specificity [14,92]. In addition, oxidation of H2O2-sensitive fluorescent probes by competitive radicals and other species usually leads to complications and false positive interpretations of experimental data.
Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases
2014, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Developed initially to measure NOX2 activity, all four probes have been used to measure extracellular H2O2 production by NOX4 [59,60,62] and DUOX [50,57,72–74,76]. Note that accurate detection of H2O2 by these assays requires addition of SOD, because O2― reacts with the probe, resulting in underestimation of H2O2 produced [94]. The oxidation of Amplex red is irreversible, has a stoichiometry of 1:1 [93], and is sensitive to low levels of H2O2, with a 5 pmol lower limit of detection and 30 pmol lower limit of quantitation [13,95].
The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells
2014, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Provided several important requirements are met, these assays can provide reliable quantitative data. Sufficient horseradish peroxidase must be present to scavenge all the peroxide as it is released, and SOD should be added to prevent the rapid reaction of the probe radical intermediate with superoxide [38]. Otherwise, this reaction can diminish the signal.
Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations
2012, Free Radical Biology and MedicineUncoupled respiration, ROS production, acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from UCP3-ablated mice
2011, Biochimica et Biophysica Acta - BioenergeticsCitation Excerpt :Mitochondrial H2O2 net production was determined fluorometrically by the use of the Amplex Red reagent (Invitrogen). Oxidation of Amplex Red coupled by horseradish peroxidase to reduction of H2O2 produces the red fluorescent oxidation product resorufin [17]. Mitochondria (0.1 mg of mitochondrial protein ml− 1) were incubated at 37 °C under magnetic stirring in the same buffer as that used for respiration studies.