Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide.

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO2 reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of 18O2 during reduction of CO2 was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO2 assimilation resulted in the formation of H2O2. This was indicated by a large stimulation of CO2 reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H2O2 was shown to be at the level of phosphoglycerate formation. Evidence for electron transport of oxygen was also obtained from 14CO2 experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO2 reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO2 and oxygen during photosynthesis.     

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of "active oxygen" species, including H2O2, free radicals, such as superoxide (O2-.) and hydroxyl (HO.), and singlet molecular oxygen (1O2). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS from Escherichia coli strains 026:B6 and 055:B5, as well as Salmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.     

Inhibition of myeloperoxidase by salicylhydroxamic acid.

Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events.     

Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.     

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol–H₂O₂–horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO^•/PhOH) vs. luminol radicals (L^•/LH⁻) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (σ) for their substituents similar or greater than 0.20 are enhancers of the luminol–H₂O₂–horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (σ) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted. © 1998 John Wiley & Sons, Ltd.     

Effect of oxygen abstraction on the peroxidase–luminol–perborate system: Relevance to the HRP enhanced chemiluminescence mechanism

Abstraction of oxygen from the HRP enhanced chemiluminescence system has no significant effect on the chemiluminescence generated. It is, therefore, proposed that in the peroxidase-luminol-perborate system at pH 7.3, chemiluminescence is generated by a direct reaction of diazaquinones with hydrogen peroxide and not, as generally assumed, from the reaction of luminol radicals with the molecular oxygen.     

Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture

Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration. Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS. The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction. The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected. Similar results were obtained using HRP. It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro. The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals. The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2. In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions. The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.     

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.     

Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa

Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence of the luminol–AgNO3 system by Ag nanoparticles

The oxidation reaction of luminol with AgNO₃ can produce chemiluminescence (CL) in the presence of silver nanoparticles (NPs) in alkaline solution. Based on the studies of UV‐vis absorption spectra, photoluminescence (PL) spectra and CL spectra, a CL enhancement mechanism is proposed. The CL emission spectrum of the luminol–AgNO₃–Ag NPs system indicated that the luminophore was still 3‐aminophthalate. On injection of silver nanoparticles into the mixture of luminol and AgNO₃, they catalysed the reduction of AgNO₃ by luminol. The product luminol radicals reacted with the dissolved oxygen, to produce a strong CL emission. As a result, the CL intensity was substantially increased. Moreover, the influences of 18 amino acids, e.g. cystine, tyrosine and asparagine, and 25 organic compounds, including gallic acid, tannic acid and hydroquinone, on the luminol–AgNO₃–Ag NPs CL system were studied by a flow‐injection procedure, which led to an effective method for detecting these compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils

Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H(2)O(2) in Earl's solution (pH=7.2) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H(2)O(2) system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN(3)), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.     

In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: The nature of oxidants that directly cause luminol oxidation

In this study, we investigated the pathways (including the formation of hydroxyl radicals and chloramines) leading to luminol chemiluminescence induced by hypochlorite generated in a suspension of stimulated rabbit polymorphonuclear leukocytes. Chemiluminescence of leukocytes stimulated by phorbol myristate acetate, which was enhanced by luminol (0.02 mM), did not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02–2.6 mM), under which the latter should manifest the specific ability to scavenge hydroxyl radicals. This indicates that stimulation of polymorphonuclear leukocytes is not accompanied by the generation of hydroxyl radicals with the involvement of superoxide anion and hypochlorite synthesized by myeloperoxidase. At high concentrations of dimethyl sulfoxide (260 mM), chemiluminescence markedly declined because dimethyl sulfoxide directly reacts with hypochlorite. The luminol emission intensity considerably increased after its addition to a suspension of leukocytes that were preliminarily stimulated for 10 min. This effect was caused by the accumulation of hydrogen peroxide rather than chloramines. Exogenous amino acids and taurine at high concentrations (3–15 mM) quench chemiluminescence. All these data indicate that chemiluminescence in the system studied is largely determined by the direct initial reaction of hypochlorite with luminol, the emission intensity increasing as a result of oxidation of luminol transformation products by hydrogen peroxide.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.     

Effect of psoralens on Fenton-like reaction generating reactive oxygen species

Psoralens (psoralen, 5-methoxypsoralen, 8-methoxypsoralen, khellin, and visnagin) in 1 mM doses were shown to enhance the generation of reactive oxygen species, such as the hydroxyl radical (HO*), the superoxide anion radical (O2(-)), and singlet oxygen ((1)O(2)), from the system generating chemiluminescence (CL), as well as free radicals in the absence of light. The system that generated CL was made up of CoCl(2) and H(2)O(2). Incubation of psoralens in 0.2 mM doses with the generating system showed that only 8-methoxypsoralen and khellin have antioxidative effects. Antioxidative effects were also observed in the case of visnagin but in low concentration (0.05 mM). High doses of psoralens (1 mM) showed prooxidative effects. Measurements were done using a deoxyribose assay, the CL method, and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy and spectrophotometry methods.     

Hydroxyl radical involvement in the luminol chemiluminescence from the reaction of arachidonic acid with sheep vesicular gland microsomes

The luminol chemiluminescence associated with the reaction between vesicular gland microsomes and arachidonic acid was found to be inhibited by superoxide dismutase, catalase and hydroxyl radical scavengers. It was also found that diethylenetriamine pentaacetic acid caused only slight inhibition. This is interpreted as showing the formation of hydroxyl radicals in a Haber-Weiss reaction and the interaction of luminol with the hydroxyl radicals. The Haber-Weiss catalyst is taken to be heme iron as the luminescence was not inhibited by diethylenetriamine pentaacetic acid. A model system involving lipoxygenase, haematin and arachidonic acid was shown to be similar, the hydroxyl radicals coming from the interaction of haematin with the hydroperoxides, produced from the lipoxygenase-arachidonic acid reaction.