Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines

It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.     

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.     

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Physico-chemical modeling of the role of free radicals in photodynamic therapy. IV. Quantitative aspects of photodynamic effects on free radicals generated in cell cultures

Production and the mechanism of the interactions of free radicals generated by stimulated macrophages in the presence of luminol and a free radical inhibitor was investigated to determine the possibility of using luminol-dependent chemiluminescence for studying photodynamic effects in biology. Earlier measurements have been revisited and additional experiments performed indicating that oxidation products of luminol neither inhibit the in vitro formation of radicals nor quench CL. Simulation based on the mechanism suggested revealed that the likely value for the rate constant of the primary step between luminol and superoxide anion radicals producing luminol radicals is 5x10(2)-1x10(3) M-1s-1. It has been established that the ratio of the concentration of radicals generated by the biological system to that formed by oxidation of luminol exceeds 10(3); that is, the contribution of the latter is negligible and the system is appropriate to measure quantitatively the effect of excited photosensitizers on free radicals.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

The effect of exogenous peroxidase on the intensity of luminol-dependent macrophage chemiluminescence

The influence of exogenous horse-radish peroxidase on the capacity of mouse peritoneal macrophages to luminol-dependent chemiluminescence induced by zymosan was investigated. It was revealed that peroxidase (1-50 mg/ml) increased the chemiluminescence in a dose-dependent manner. The maximum increase of the response (4-6 times) was obtained with the enzyme concentration being 10 mg/ml. It is found that peroxidase acts as a co-oxidant of the peroxide-dependent extracellular luminol oxidation. The including of the enzyme into macrophages makes it possible to register the intracellular chemiluminescence.     

Chemiluminescence in oxidation of luminol by chloramine derivatives of biogenic compounds

Chloramine derivatives of amino acids induce chemiluminescence of a luminol solution. The chemiluminescence is more prolonged than the emission of luminol produced by hypochlorite. Persistent chemiluminescence also appears under the action of hypochlorite on a mixture of luminol and amino acids. It is assumed that the chemiluminescence of luminol in suspensions of stimulated phagocytes may be associated with its oxidation by chloramines.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.     

Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol–H₂O₂–horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intesity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects. © 1997 John Wiley & Sons, Ltd.     

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.     

Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils.

The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in the absence of the apoprotein, was triggered by superoxide anion. The production of reactive oxygen metabolites by fMet-Leu-Phe- and 4b-phorbol 12b-myristate 13a-acetate-stimulated neutrophils was studied by means of the luminescence of synthetic coelenterazine. The features of this chemiluminescent probe were compared with those of luminol and are summarized as follows: (a) coelenterazine-dependent chemiluminescence was inhibited by superoxide dismutase; (b) coelenterazine was as sensitive as luminol in detecting the oxidative burst of neutrophils; (c) azide failed to inhibit coelenterazine chemiluminescence; (d) in contrast with luminol, which requires the catalytic removal of hydrogen peroxide, coelenterazine chemiluminescence did not depend on the activity of cell-derived myeloperoxidase. These results indicate the usefulness of coelenterazine as a very sensitive and specific chemiluminescence probe of superoxide anion.     

Kinetics of luminol sonochemiluminescence quenched by purines

A homogeneous chemiluminescence (CL) reaction was initiated by ultrasound irradiation. Luminol sonochemiluminescence (SCL) reaction kinetics were determined under pseudo‐first‐order conditions, and the reaction followed the model for simple rise–fall kinetics. In addition, SCL quenching reactions induced by purines were also investigated in which the interactions between luminol and purines were analysed using the Stern–Volmer (S‐V) mechanism. The results implied that the high rate constant of luminol CL quenched by purines may be attributed to ground state interactions originating from hydrogen bonding. Copyright © 2012 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.     

Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

Potential of the luminol reaction in the sensitive detection of pesticide residues by flow injection analysis.

This study presents the first analytical application of the luminol chemiluminescence (CL) reaction for the sensitive detection of carbamate residues. Some experiments have been carried out to check the influence of the presence of traces of a N-methylcarbamate (carbaryl) on the CL emission produced from the oxidation of luminol using different oxidants, showing a significant enhancing effect on the CL emission when the oxidation of luminol is produced by potassium permanganate in alkaline medium, this enhancement being proportional to the carbaryl concentration. This fact has permitted the establishment of a sensitive chemiluminescence flow-injection (CL-FIA) method for the direct determination of carbaryl. The optimization of instrumental and chemical variables influencing the CL response has been carried out by applying experimental designs. Under the optimal conditions, the CL intensity was linear for a carbaryl concentration over the range 5-100 ng/mL with a detection limit of 4.9 ng/mL. This luminol-KMnO4-based FIA-CL system in basic medium shows an easy, fast and cheap alternative detection mode for the analysis of carbaryl residues in environmental water samples.     

Variables in xanthine oxidase-initiated luminol chemiluminescence: Implications for chemiluminescence measurements in biological systems

We tested the effects of generally used chemiluminescence inhibitors on an example of luminol chemiluminescence elicited by xanthine oxidase/hypoxanthine system, and attempted to assess their capabilities in discovering the reaction pathways leading to chemiluminescence. Luminol itself is a xanthine oxidase inhibitor and its concentration affects the reaction mechanism. Maximal chemiluminescence response was observed at luminol concentration inhibiting urate production. Chemiluminescence was totally inhibited by superoxide dismutase, the inhibition by catalase depended on luminol concentration. Ferricytochrome c, a detector of superoxide, either stimulated or inhibited chemiluminescence in a concentration-dependent manner. Chemiluminescence was highly stimulated by peroxidases. A pronounced inhibition of chemiluminescence was caused by chelators; 1 mm desferal and 0.01 mm diethyldithiocarbamate. It is suggested that measurement of luminol chemiluminescence is not a suitable method for discrimination among individual reactive oxygen species and their quantitative determination in biological systems.