Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite

Two different mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite were found. Most substances investigated in these experiments acted by scavenging NaOCI. This mechanism was independent of the concentration of hydrogen peroxide and the incubation time between luminol and inhibitors. The most potent inhibitors were substances containing SH groups. Compounds with amino groups as a target for HOCI/OCI^? to yield chloramines were much less effective inhibitors. Another mechanism of inhibition was found for catalase. It depended on the presence of hydrogen peroxide in the incubation medium and the incubation time between luminol and catalase. The enzyme inhibited the luminescence by removing H₂O₂ at molar concentrations much smaller than those found for all other inhibitors. Our results confirm the present models of the mechanism of generation of luminescence in luminol oxidation.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.     

Chemiluminescence assay for tetrahydrobiopterin based on the generation of hydrogen peroxide using isoluminol-microperoxidase in the presence of 1-methoxy PMS.

We developed a novel highly sensitive chemiluminescence (CL) method for BH(4). The principle of the proposed method is based on active oxygen formation induced by 1-methoxy-5-methyl phenazinium methyl sulphate (1-methoxy PMS) in the presence of dissolved oxygen. Furthermore, active oxygen is determined by a CL assay involving the luminol reaction with microperoxidase. In this report, we examined the mechanism of formation and identified the reactive oxygen species derived from BH(4) employing 1-methoxy PMS. Additionally, optimum conditions for the CL assay of BH(4) were established.     

Natural catalyst for luminol chemiluminescence: application to validate peroxide levels in commercial hair dyes

Here, we report a hydrothermally treated green leaves (Moringa oleifera) extract exploited as an efficient and highly sensitive catalyst to catalyze the chemiluminescence (CL) reaction of luminol. In the absence of enhancer, this green and hydrothermally treated catalyst was found to significantly enhance the CL intensity ~3.5-fold compared with the traditionally used K₃Fe(CN)₆ catalyst. The structure and surface morphology of the catalyst was elucidated using X-ray photoelectron spectroscopy, scanning electron microscopy, X-ray diffraction, and Raman spectroscopy. The synergistic effect of the catalyst in the CL reaction was systematically investigated in the presence of hydrogen peroxide using ultraviolet–visible and CL spectroscopy. Studies showed that the sensitivity of the catalyst could be amplified by adjusting several parameters such as pH of the medium and concentrations of the base and luminol. The sensitivity of the novel-type catalyst was examined through the validation of hydrogen peroxide levels in commercial hair dye samples. Markedly, the catalyst displayed ultrasensitivity to hydrogen peroxide as the limit of detection of hydrogen peroxide using this catalyst was determined to be 0.02 μM under optimized conditions. In general, the proposed inexpensive, ecofriendly, and nontoxic catalyst could enable the determination of hydrogen peroxide for diverse analytical applications.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Regulation of morphological and functional properties of astrocytes by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H₂O₂ at 5 × 10^?7?1 × 10^?8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O ₂ ^?? ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10^?6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O ₂ ^?? under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H₂O₂ modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.     

An Improved Chemiluminescence Method for Hydrogen Peroxide Determination in Plant Tissues

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs. Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H₂O₂. The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H₂O₂ in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plant-extracts become unnecessary. To exemplified the high performance of the method, measurements of H₂O₂ were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H₂O₂ levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H₂O₂ scavenging enzyme in leaves of grapevine.     

Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. (c) 1993 Wiley & Sons, Inc.     

Amines for detection of dopamine by generation of hydrogen peroxide and peroxyoxalate chemiluminescence

A range of nitrogen-containing compounds (alkyl amines, piperazines, cyclohexylamines and nitrogen heterocyclics) were investigated for generation of hydrogen peroxide from dopamine and detection by peroxyoxalate chemiluminescence. Imidazole, ethyleneurea and allantoin among the nitrogen heterocyclic compounds tested generated hydrogen peroxide from dopamine following incubation at 60°C, pH 9.5–10.5, for 0–30 min. Imidazole was the most effective for generation of hydrogen peroxide, but imidazole derivatives with a primary amine side chain (histamine) or thiol (ethylenethiourea) were not effective. The presence of a ketone group (ethyleneurea, allantoin) did not hinder the reaction. Under optimal conditions (30 min incubation, 50 mmol/L imidazole) 10.5 nmol of dopamine could be detected. The cyclohexylamines tested produced low amounts of hydrogen peroxide (0.09–2.74% of light intensity with imidazole), and the piperazines and the alkyl amines tested produced no detectable hydrogen peroxide. Imidazole reacts with the phenolic groups of dopamine in a different manner from monoamine oxidase, and a reagent containing imidazole, ethyleneurea or allantoin was useful for non-enzymatic detection of dopamine by peroxyoxalate chemiluminescence.© John Wiley & Sons, Ltd.     

Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane-chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H₂O₂ and substrates, the pH value of the micelle polar phase and the H₂O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15-fold higher than for the ABI-COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.     

Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide

Chlorophyll is effectively bleached by H₂O₂ in the presence of certain phenols and peroxidase (EC 1.11.1.7) extracted from acetone powders of orange flavedo (Citrus sinensis). Optimal conditions for chlorophyll: hydrogen peroxide oxidoreductase include: pH, 5.9; [H₂O₂] 222 μM; ionic strength 0.11. A phenol is required and resorcinol is the most effective. Catechol and hydroquinone are inhibitory. Chlorophyll a, chlorophyllide a, and chlorophyll b all have similar V_max but K_m for chlorophyll a is about one-third that of chlorophyll b, while the K_m for chlorophyllide a is about one-half that of chlorophyll a. Pheophytin a was much less reactive than chlorophyll a, and Mg²⁺ included in the reaction system did not affect rates of pheophytin destruction.     

Enhancement and inhibition of luminol chemiluminescence by phenolic acids

We explored the behaviour of a series of phenolic acids used as enhancers or inhibitors of luminol chemiluminescence by three different methods to determine if behaviour was associated with phenolic acid structure and redox character. All the phenolic acids inhibited chemiluminescence when hexacyanoferrate(III) was reacted with the phenolic acids before adding luminol. The redox character of these compounds was clearly related to structure. When hexacyanoferrate(III)-luminol-O₂ chemiluminescence was initiated by phenolic acid-luminol mixtures some phenolic acids behaved as enhancers of chemiluminescence, and others as inhibitors. We propose a mechanism to explain these findings. We found direct relationships between the redox character of the phenolic acids and the enhancement or inhibition of the chemiluminescence of the luminol–H₂O₂–peroxidase system and we propose mechanism to explain these phenomena.     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Chemiluminescence of cereal products. II. Chemiluminescence spectra

Spectra of ultraweak chemiluminescence (CL) accompanying auto-oxidation and hydration of cereal products have been measured using single photon counting and cut-off filters. The spectra cover the 380–880 nm spectral range with maxima centred around 600 nm. Analytically pure air-dried carbohydrates like agar, cellulose and nitrocellulose give emission too weak for spectral measurements. The emission from water pure carbohydrates is on average 4–12 times higher and emission spectra are similar to those from cereal products. The effect of free radical scavengers, SOD and O*₂ (¹Δ_g)-quenchers on CL spectra indicates a contribution of radical reactions with the participation of excited carbonyls, O₂⁻ and excited molecular oxygen dimoles. Moreover, possible mechanisms of chemi-excitation due to a cooperative H-bond formation during the hydration of carbohydrates and/or recombination of trapped radicals and electron-holes are discussed. It is also postulated that the excitation energy transfer to natural sensitizers occuring in cereal products may account for non-specific broad spectra and differences in the intensity of CL. © 1998 John Wiley & Sons, Ltd.     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn²⁺ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H₂O₂). The analysis of H₂O₂ formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×10⁵?M^?1?s^?1.

The formation of H₂O₂ during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H₂O₂ production was found to be SOD-sensitive. The inhibition of H₂O₂ production by SOD was about 25% indicating that H₂O₂ is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with      

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Study on the generation mechanism of reactive oxygen species on calcium peroxide by chemiluminescence and UV-visible spectra.

In the present work, the generation mechanism of reactive oxygen species (ROS) on calcium peroxide (CaO(2)) was studied. A very intense chemiluminescence (CL) signal was observed when adding an aqueous solution of luminol or 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2alpha]-pyrazin-3-one hydrochloride (MCLA) to a suspension of CaO(2). The ROS released on CaO(2) were thought to be oxidizing agents leading to CL, and were characterized by CL, UV-visible (UV-vis) spectra and the effective scavengers of the special ROS. From experimental results, the hydroxyl (.OH) and superoxide (.O(2) (-)) radicals were suggested to exist on the surface of CaO(2). A reaction scheme for the formation of the ROS on CaO(2) was also proposed and discussed. Of more interest was the finding that the CaO(2) which released the .OH and .O(2) (-) on the surface exhibited good transition properties compared with alkaline-earth metal peroxides of the same group (MgO(2), BaO(2)).     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.