Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions

The photoreduction of crystal violet to a carbon-centered radical was detected directly by electron spin resonance (ESR) spectroscopy under anaerobic conditions. The linewidth (0.9 G) of this radical was less broad than the linewidth (11.0 G) of the free radical obtained in Trypanosoma cruzi incubations. No crystal violet radical could be detected under aerobic conditions. However, crystal violet was found to convert oxygen to superoxide anion and hydrogen peroxide in the presence of light. This superoxide anion and hydrogen peroxide formation was greatly enhanced by reducing agents such as NAD(P)H. In addition, irradiation of crystal violet did not generate detectable amounts of singlet oxygen.     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators

Glucose oxidase enzymes were used to produce hydrogen peroxide from glucose and oxygen in aqueous solutions. Different working conditions, that is, temperature, aeration with liquefied air, presence of cotton fibre and time of enzyme activity, were tested in order to obtain a solution with the highest possible concentration of hydrogen peroxide. The hydrogen peroxide produced was transformed into different peracids which could bleach the cotton fabric under mild conditions, at a pH between 7 and 8 and at a temperature of around 60°C. The conversion or activation of hydrogen peroxide was conducted with the bleach activators TAED, NOBS and TBBC. The concentrations of hydrogen peroxide and peracids in the solutions were measured with sodium thiosulphate titrations.

The results indicated that the formation of hydrogen peroxide with glucose oxidase was effective under optimal conditions, which are 50°C, pH 4.6 and aeration. Convenient activators for the conversion of hydrogen peroxide into peracids were TAED and TBBC, which enabled attainment of a relatively high degree of whiteness at pH 7.5 and temperature 50°C. Using the activator NOBS under these conditions did not provide enough peracid to markedly improve whiteness.     

Hydrogen peroxide triggers the proteolytic cleavage and the inactivation of calcineurin

Increases in the levels of reactive oxygen species (ROS) are correlated with a decrease in calcineurin (CN) activity under oxidative or neuropathological conditions. However, the molecular mechanism underlying this ROS-mediated CN inactivation remains unclear. Here, we describe a mechanism for the inactivation of CN by hydrogen peroxide. The treatment of mouse primary cortical neuron cells with Abeta(1-42) peptide and hydrogen peroxide triggered the proteolytic cleavage of CN and decreased its enzymatic activity. In addition, hydrogen peroxide was found to cleave CN in different types of cells. Calcium influx was not involved in CN inactivation during hydrogen peroxide-mediated cleavage, but CN cleavage was partially blocked by chloroquine, indicating that an unidentified lysosomal protease is probably involved in its hydrogen peroxide-mediated cleavage. Treatment with hydrogen peroxide triggered CN cleavage at a specific sequence within its catalytic domain, and the cleaved form of CN had no enzymatic ability to dephosphorylate nuclear factor in activated T cells. Thus, our findings suggest a molecular mechanism by which hydrogen peroxide inactivates CN by proteolysis in ROS-related diseases.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Bactericidal effect of cysteine exposed to atmospheric oxygen.

Peptostreptococcus anaerobius VPI 4330-1 was exposed to atmospheric oxygen in a dilution bland (0.2% gelatin, salts, resazurin) solution. The organisms were rapidly killed when the solution contained cysteine. The organisms were effectively protected by catalase and horseradish peroxidase as well as by the metal ion-chelating agents 8-hydroxyquinoline and 2,2'-bipyridine. Superoxide dismutase increased the rate of killing of the organisms, whereas singlet oxygen quenchers and scavengers of hydroxyl free radicals did not protect the organisms from the toxic effect of cysteine. Hydrogen peroxide was formed when cysteine was exposed to oxygen in the dilution blank solution, and the reaction was inhibited by metal ion-chelating agents. The organisms were rapidly killed by 20 microM hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide was completely abolished by catalase and metal ion-chelating agents. These results indicated that hydrogen peroxide was formed in the dilution blank solution in a metal ion-catalyzed autoxidation of cysteine and that hydrogen peroxide was toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions.     

Bactericidal effect of cysteine exposed to atmospheric oxygen.

Peptostreptococcus anaerobius VPI 4330-1 was exposed to atmospheric oxygen in a dilution bland (0.2% gelatin, salts, resazurin) solution. The organisms were rapidly killed when the solution contained cysteine. The organisms were effectively protected by catalase and horseradish peroxidase as well as by the metal ion-chelating agents 8-hydroxyquinoline and 2,2'-bipyridine. Superoxide dismutase increased the rate of killing of the organisms, whereas singlet oxygen quenchers and scavengers of hydroxyl free radicals did not protect the organisms from the toxic effect of cysteine. Hydrogen peroxide was formed when cysteine was exposed to oxygen in the dilution blank solution, and the reaction was inhibited by metal ion-chelating agents. The organisms were rapidly killed by 20 microM hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide was completely abolished by catalase and metal ion-chelating agents. These results indicated that hydrogen peroxide was formed in the dilution blank solution in a metal ion-catalyzed autoxidation of cysteine and that hydrogen peroxide was toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions.     

Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Generation of reactive oxygen species in water under exposure to visible or infrared irradiation at absorption bands of molecular oxygen

It is found that in bidistilled water saturated with oxygen, hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sources, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

The production of hydrogen peroxide by high oxygen pressures

Hydrogen peroxide is formed in solutions of glutathione exposed to oxygen. This hydrogen peroxide or its precursors will decrease the viscosity of polymers like desoxyribonucleic acid and sodium alginate. Further knowledge of the mechanism of these chemical effects of oxygen might further the understanding of the biological effects of oxygen. This study deals with the rate of solution of oxygen and with the decomposition of hydrogen peroxide in chemical systems exposed to high oxygen pressures. At 6 atmospheres, the absorption coefficient for oxygen into water was about 1 cm./hour and at 143 atmospheres, it was about 2 cm./hour; the difference probably being due to the modus operandi. The addition of cobalt (II), manganese (II), nickel (II), or zinc ions in glutathione (GSH) solutions exposed to high oxygen pressure decreased the net formation of hydrogen peroxide and also the reduced glutathione remaining in the solution. Studies on hydrogen peroxide decomposition indicated that these ions act probably by accelerating the hydrogen perioxide oxidation of glutathione. The chelating agent, ethylenediaminetetraacetic acid disodium salt, inhibited the oxidation of GSH exposed to high oxygen pressure for 14 hours. However, indication that oxidation still occurred, though at a much slower rate, was found in experiments lasting 10 weeks. Thiourea decomposed hydrogen peroxide very rapidly. When GSH solutions were exposed to high oxygen pressure, there was oxidation of the GSH, which became relatively smaller with increasing concentrations of GSH.     

Differential effect of l-histidine in human lymphocytes damaged by different oxygen radical producing systems

The effect of histidine on damage induced by oxygen radicals was studied in peripheral blood lymphocytes treated with free oxygen radical-inducing agents: hydrogen peroxide, xanthine oxidase plus hypoxanthine, bleumycin and γ-rays. l-Histidine, at a concentration of 1 mM, was found to potentiate both cell killing and inhibition of PHA-stimulated cell division brought about by hydrogen peroxide or xanthine oxidase plus hypoxanthine. In contrast, l-histidine did not affect γ-ray- or bleomycin-induced cell killing and inhibition of PHA-stimulated cell division. We suggest that l-histidine potentiation of cell damage is mainly mediated by interaction of the amino acid with hydrogen peroxide and/or iron rather than with other reactive oxygen species. In addition, these results also indicate that hydrogen peroxide produced by γ-radiation- or bleomycin-treated cells plays no role in the toxic effects elicited by these agents.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941?mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8?μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.