Bilirubin chemiluminescence induced by the attack of active oxygen species

Ultraweak chemiluminescence (CL) from bilirubin occurs in the presence of triplet oxygen and is stimulated by the addition of aldehydes. Active oxygen species also enhance bilirubin CL, in the absence of aldehydes. An inhibitory effect of active oxygen scavengers on the CL indicated that active oxygens generated from the decomposition of added hydrogen peroxide or from the xanthine-xanthine oxidase reaction contributed to the CL from bilirubin molecules. However, the contribution of singlet oxygen to the CL disappeared in the presence of formaldehyde. This suggested that the scission of tetrapyrrole bonds via a dioxetane intermediate or the production of triplet carbonyls from the oxidation of aldehydes by singlet oxygen was not involved in the CL, at least in the presence of formaldehyde. The spectrum of CL induced by the generation of active oxygen was the same as that from the aldehyde-enhanced CL reaction. We propose that the formation of a hydroperoxide (and/or hydroxide) bilirubin intermediate, but not a dioxetane, may be involved in the excitation of bilirubin molecules for CL.     

Chemiluminescence in the crude extracts of soybean seedlings. Postulated mechanism on the formation of hydroperoxide intermediates.

It has been reported that weak chemiluminescence (CL) from crude extracts of soybean seedlings is remarkably enhanced with the addition of various aldehydes (Biochim. Biophys. Acta 1058, 209-216). The reactivity of certain emitter(s) with oxygen species was examined in the autoclaved extracts of seedlings. When samples were reduced by the addition of hydrosulfite, two different types of reactivities in CL were defined. One type showed an initial rapid increase and a subsequent fast decay in CL upon mixing with oxygen. This rapid increase in CL intensity was independent of the presence of aldehydes, and was significantly suppressed by SOD. However, the subsequent slow decay phase in CL was dependent on the presence of aldehydes. In the sample reduced more moderately by borohydride, the same slow decay of CL appeared upon mixing with acetaldehyde and oxygen. This second type of CL was not inhibited by active oxygen scavengers. Hydrogen peroxide added to unreduced (oxidized) samples also elicited CL. Three types of primary emitters may be oxidized to form transient hydroperoxide, and excited for light emission by slightly different ways: two of them are excited by abstraction of one atomic oxygen from the hydroperoxy intermediate with aldehyde or hydrogen peroxide, leading to formation of an excited hydroxide intermediate. The third is excited directly on the binding of superoxide anion to the reduced primary emitter.     

Chemiluminescence of the peroxomonosulphate-cobalt(II)-aliphatic monocarboxylic acids system.

A weak chemiluminescence (CL) emission was observed due to the production of singlet oxygen ((1)O(2)) during the decomposition of peroxomonosulphate (HSO(5)(-)) catalysed by cobalt(II). Low molecular mass aliphatic monocarboxylic acids, such as formic, acetic, propionic, butyric and valeric acids, influenced the CL emission, and the reaction of aliphatic monocarboxylic acids with HSO(5)(-)/Co(2+) solution was further investigated using a flow injection analysis (FIA) CL method. The results indicated that the CL intensities of aliphatic monocarboxylic acids were improved with increase in the carbon chain length in the potassium peroxomonosulphate-cobalt(II) sulphate system. Generation of singlet oxygen was confirmed by the fact that the CL emission of aliphatic monocarboxylic acids with the HSO(5)(-)/Co(2+) solution was quenched by NaN(3), and from the CL spectrum of the reaction system. Additionally, a possible mechanism of aliphatic monocarboxylic acids CL emission enhancement was proposed.     

Triplet state of 4-methoxybenzyl alcohol chemisorbed on silica nanoparticles

The knowledge of photochemical kinetics in colloidal systems is important in understanding environmental photochemistry on dispersed solid surfaces. As model materials for the chemically sorbed organic compounds present in natural environments, modified silica nanoparticles (NPs) were obtained here by condensation of the silanol groups of fumed silica nanoparticles with 4-methoxybenzyl alcohol. These particles were characterized by different techniques. To evaluate their toxicity, the inhibition of the natural luminescence emission of the marine bacterium Vibrio fischeri in suspensions of the particles was measured. Laser flash-photolysis experiments (λ(exc) = 266 nm) performed with NP suspensions in acetonitrile-aqueous phosphate buffer mixtures showed the formation of the lowest triplet excited state of the chemisorbed organic groups (λ(max) = 390 nm). DFT calculations of the absorption spectrum of this radical support the assignment. From the calculated triplet energy, a thermodynamically favorable energy transfer from these triplet states to oxygen to yield singlet molecular oxygen is predicted. A value of 0.09 was measured for the quantum yield of singlet molecular oxygen generation by air-saturated suspensions of the nanoparticles in the mixture of solvents acetonitrile-aqueous phosphate buffer. The quantum yield of singlet molecular oxygen generation by the free 4-methoxybenzyl alcohol in the same solvent is 0.31.     

Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO)

The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.     

Porphyrin-induced photooxidation of conjugated bilirubin

Visible light irradiation of 18 microM bilirubin ditaurate (BR-DT) at pH 7.0 for 30 min showed a 10% decrease in absorbance at 445 nm. When the reaction was carried out in the presence of a trace amount of uroporphyrin (UP), the spectrum of BR-DT disappeared without a concomitant formation of biliverdin. Photooxidation products were confirmed to be dipyrrole-containing compounds. Photo-bleaching of BR-DT was accelerated by the increasing concentration of UP and was inhibited, when UP was replaced by Cu2+UP. Formation of 2,2,6,6-tetramethylpiperidine N-oxyl through the irradiation of UP was diminished by sodium azide, a potent scavenger of singlet oxygen. The efficiency of singlet oxygen formation through visible light irradiation was in the order UP, coproporphyrin > Cu2+UP. Both bilirubin and BR-DT bound to human serum albumin (HSA) were photooxidized effectively in the presence of UP. The results indicate that irradiation of UP produces singlet oxygen with high efficiency which then rapidly oxidizes free and conjugated bilirubin.     

Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4-dihydroxyphenylalanine into eumelanins

The catechol oxidase-catalysed and autoxidative transformation of 3,4-dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λ_max = 480 nm) and dopalutin (λ_ex = 423 nm, λ_em = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10^?13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll-a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10^?9), the emission occurring in the 400-600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.     

A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation.

A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.     

Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

Oxygenation of anaerobically isolated brain and liver homogenates is associated with chemiluminescence and formation of lipid hydroperoxides, the latter determined by the thiobarbituric acid assay. Light emission and formation of malonaldehyde are 20-fold higher in the brain than in liver; chemiluminescence of both decays when accumulation of malonaldehyde ceases. Exogenous organic peroxides, such as t-butyl hydroperoxide, inhibit the light-emission response to oxygenation by brain homogenate, whereas they enhance that of liver homogenate. t-Butyl hydroperoxide-induced photoemission of liver homogenate shows a polyphasic kinetic pattern that is O2-dependent. The spectral analysis of chemiluminescence arising from brain and liver homogenates on oxygenation shows a spectrum with five emission bands at 420-450, 475-485, 510-540, 560-580 and 625-640 nm. These bands are subjected to intensity changes or shifts of the wavelength whenever t-butyl hydroperoxide is present, either inhibiting or stimulating light emission. The blue-band chemiluminescence, around 435 nm, is possibly due to the weak light emission arising from excited carbonyl compounds [Lloyd (1965) J. Chem. Soc. Faraday Trans. 61, 2182-2193; Vassil'ev (1965) Opt. Spectrosc. (USSR) 18, 131-135], whereas the presence of other bands suggests generation of singlet molecular oxygen either in the process triggered on oxygenation (lipid oxygenation) or after supplementation with organic hydroperoxides. We offer several explanations for the spectral analysis presented here.     

Studies on the properties of the singlet oxygen-like factor produced during lipid peroxidation

The singlet oxygen reaction product of various trapping agents is observed during enzymic and nonenzymic peroxidation of microsomes as well as during the peroxidation of pure lipids extracted from microsomes. We now wish to report that purified fatty acid hydroperoxide alone, as well as peroxidized microsomal lipid and cumene hydroperoxide also form the singlet oxygen reaction product with 2,5-diphenylfuran. The reaction product (cis-1,2-dibenzoylethylene) was observed to be formed in an anaerobic system, with or without EDTA. The data indicate that a reaction of hydroxyl radicals with 2,5-diphenylfuran cannot account for the formation of dibenzoylethylene in these systems. These results are consistent with a hypothesis that the singlet oxygen-like factor was formed from the lipid peroxides per se and, in addition, supports the possibility that either the peroxides can react directly with diphenylfuran to produce dibenzoylethylene or that the self-reaction of organic peroxides may form an intermediate product which can react directly with singlet oxygen-trapping agents to produce substances which are identical to a reaction of the trapping agents with singlets oxygen.     

Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.     

Chemiluminescence Emission during Reactions between Superoxide and Selected Aliphatic and Aromatic Halocarbons in Aprotic Media

The reactions between superoxide free radical anion (.O⁻₂) with the halocarbons CCl₄, CHCl₃, BrCH₂CH₂Br(EDB), decachloro-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O⁻₂ consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.     

Free radicals in primary photobiological processes.

Formation of a semiquinone free radical derived from chlorophyll in the reaction of photoreduction has been discovered by A. A. Krasnovsky, Sr. in 1953. This review consider the results obtained in the author's laboratory, concerning the participation of free radicals in photochemical reactions under UV-irradiation of aromatic amino acids, proteins, and lipids, as well as in the reactions of chemiluminescence (CL) in the protein and chlorophyll-containing systems. Free radicals are the very first products of photochemical reactions in all systems studied. The back reactions of radicals are accompanied with photon emission. From the point of view of the molecular energetics, the radiativeless electronic transition in molecules is the most probable event, the transition triplet level is less probable, and the transition to the singlet excited level is virtually impossible. This may explain the low quantum yield of CL, similarity of CL and phosphorescence (rather than fluorescence) spectrum of the reaction products, low quantum yield of CL, and its high temperature coefficient.     

Isolation and characterization of an apoprotein from the d less than 1.006 lipoproteins of human and canine lymph homologous with the rat A-IV apoprotein.

The singlet oxygen traps, 2,5-diphenylfurane and 1,3-diphenylisobenzofurane were oxidized to cis-benzoylethylene and o-dibenzoylbenzene during the decomposition of diisopropyl-N-nitrosamine catalyzed by peroxidase. Singlet oxygen quenchers inhibited this conversion and also the chemiluminescence accompaying the catalyzed reaction. The chemiluminescence is enhanced by 1,4-diazobicyclo (2.2.2) octane, fluorescein, eosin rhodamine B and rose bengal but little effect was detected in the presence of 9,10-dibromoanthracene-2-sulfonate, 9,10-diphenylanthracene-2-sulfonate and anthracene-2-sulfonate. An emission spectrum of the unsensitized reaction in 560 – 600 nm region was observed. It is concluded that singlet oxygen is formed during peroxidase catalyzed degradation of diisopropyl-N-nitrosamine.     

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

Chemiluminescence in the autoxidation of the pyruvic acid analogues of a thyroid hormone and related molecules

Since the hydroperoxide formed from 4-hydroxy-3,5-diiodopheyl-pyruvic acid and oxygen, a likely precursor of thyroxine, is also known to undergo cleavage to 4-hydroxy-3,5-diiodobenzaldehyde and oxalic acid, that is, to products expected from a dioxetane intermediate, we have investigated the chemiluminescence of aerated solutions of 4-hydroxy-3,5-diiodophenylpyruvic acid. Light was emitted in dimethyl-sulfoxide solution containing potassium t-butoxide. The emitting species is the excited singlet aldehyde. Other pyruvic acid analogues, including that of the hormone 3,5,3′-triiodothyronine, chemiluminesce in similar conditions. Extremely weak emission occurs in aqueous solution.     

Chemiluminescence from thermal oxidation of amino acids and proteins

Chemiluminescence (CL) with maximum emission in the range 550–650 nm is observed when proteins and certain amino acids are heated in air, and CL intensity is significantly reduced in nitrogen. Of the 20 common amino acids, lysine (Lys) has the highest thermal CL intensity by a factor of ~30 over arginine, threonine and asparagine. This finding differs from previous studies on amino acids and proteins oxidised using free radical initiators or singlet oxygen, where tryptophan was the dominant factor for CL emission. CL from heating solid Lys in air is accompanied by browning and the generation of fluorescent products which are characteristic of advanced glycosylation end products (AGEs) in thermally treated milk proteins. During thermal oxidation, Lys may react with its own carbonyl oxidation products to form fluorescent compounds similar to AGEs via the formation of Schiff bases. The mechanism of thermal oxidation of proteins may be similar to polyamide polymers, where reaction of free primary amino groups with carbonyls to form Schiff bases plays a key role.     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.