Weak chemiluminescence of bilirubin and its stimulation by aldehydes

Bilirubin in an alkaline solution exhibits a weak chemiluminescence (CL) under aerobic conditions. This spontaneous CL was markedly enhanced by the addition of various aldehydes. The fluorescent emission spectrum of bilirubin, excited by weak intensity light at 350 nm, coincided with its CL emission spectrum (peak at 670 nm). CL emission from bilirubin was not quenched by active oxygen scavengers. This suggests that triplet oxygen reacts with bilirubin, and forms an oxygenated intermediate (hydroperoxide) as a primary emitter (oxidative scission of tetrapyrrole bonds in bilirubin is not involved in this CL). The Ehrlich reaction (test for monopyrroles) and hydrolsulphite reaction (test for dipyroles) on the CL reaction mixture and unreacted bilirubin showed no differences. When the CL was initiated by singlet oxygen, rather than superoxide anion, monopyrrole, was detected in the reaction products by gel chromatography. The inhibitory effect of a scavenger of singlet oxygen on CL was eliminated in the presence of formaldehyde. Therefore, triplet carbonyl, formed by singlet oxygen through the dioxetane structure in bilirubin, is not an emitter. The reaction mechanism of bilirubin CL and the formation of a hydroperoxide intermediate is discussed in relation to the chemical structure of luciferin molecules from bioluminescent organisms.     

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.     

Are dioxetanes chemiluminescent intermediates in lipoperoxidation?

Ultraweak chemiluminescence arising from lipoperoxidation has been attributed by several authors to the radiative deactivation of singlet oxygen and triplet carbonyl products. The latter emitters have been suggested to come from annihilation of RO. and ROO. radicals as well as from the thermolysis of dioxetane intermediates formed by (2 + 2) cycloaddition of 1O2 to polyunsaturated fatty acids. This article questions possible dioxetane intermediacy in lipoperoxidation, as the literature clearly states that addition of 1O2 to alpha-hydrogen-containing alyphatic olefins yields only the corresponding allylic hydroperoxides. These compounds may undergo dark thermal or Lewis acid-assisted decomposition to the same product obtained from dioxetane cleavage. Here, reexamining the chemiluminescence properties of dioxygenated tetramethylethylene and linoleic acid and comparing them with those of tetraethyldioxetane, a hindered dioxetane, we corroborate the literature information that only steric hindrance leads to dioxetane formation upon singlet oxygen addition to electron-poor olefins, albeit in very low yields. Proton nuclear magnetic resonance (1H-NMR) analysis, quenching by dioxygen and energy transfer studies to 9,10-dibromoanthracene, as well as gas chromatography (GC) analysis of triphenylphosphine-treated and untreated photo- and chemically dioxygenated olefins support our final conclusion that dioxetane formation during lipoperoxidation can be safely excluded on the basis of the data presently available.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

What is beta-carotene doing in the photosystem II reaction centre?

During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.     

Singlet oxygen production in photosystem II and related protection mechanism

High-light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II (PSII) and causes photo-oxidative stress. In the PSII reaction centre, singlet oxygen is generated by the interaction of molecular oxygen with the excited triplet state of chlorophyll (Chl). The triplet Chl is formed via charge recombination of the light-induced charge pair. Changes in the midpoint potential of the primary electron donor P(680) of the primary acceptor pheophytin or of the quinone acceptor Q(A), modulate the pathway of charge recombination in PSII and influence the yield of singlet oxygen formation. The involvement of singlet oxygen in the process of photoinhibition is discussed. Singlet oxygen is efficiently quenched by beta-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress.     

UVC induced oxidation of chloropurines: excited singlet and triplet pathways for the photoreaction

The phototransformation of 2-chloro, 6-chloro and 2,6-dichloropurines under UVC excitation (254 nm) has been studied and the major photoproducts have been identified using absorption spectroscopy, HPLC and mass spectrometry. It was shown that hydroxypurines were formed as the main products for all three investigated compounds both in the presence and absence of oxygen. In the case of 6-chloro- and 2,6-dichloropurine, a photodimer is also formed as a minor photoproduct in the absence of oxygen but is efficiently quenched in the presence of oxygen. Nanosecond photolysis experiments also revealed significant intersystem crossing to the triplet state of the chloropurines which has been characterized (transient absorption spectra, triplet formation quantum yields and rate constants of quenching by oxygen, Mn(2+) ions and ground state). Experimental evidence allows to conclude that the triplet state is involved in photodimer formation whereas the hydroxypurine is formed from the reaction of the excited singlet state of chloropurines with the solvent (water addition) through heterolytic C-Cl bond rupture. Mass spectrometry and (1)H NMR results allowed to propose a chemical pathway for dimer formation in the case of 2,6-dichloropurine in a two-step process: first a homolytic rupture of C-Cl bond in the triplet state of the molecule with the formation of purinyl radicals, which subsequently react with an excess of ground state molecules and/or hydroxypurine primarily formed.     

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.     

Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation

Chinese hamster ovary cells (CHO-K1) photosensitized with 12-(1'-pyrene)dodecanoic acid (P12) are killed when exposed to long wavelength ultraviolet (UV) light (greater than 300 nm). Mutants deficient in plasmalogen biosynthesis are hypersensitive to this treatment. We now demonstrate that plasmenylethanolamine is rapidly and preferentially destroyed when CHO-K1 cells, photosensitized either with P12 or merocyanine 540, are irradiated with light of the appropriate wavelength. Using [2-14C]ethanolamine, [1-14C]hexadecanol, or [U-14C]hexadecanol to follow the turnover of plasmenylethanolamine, we show that 2-monoacylglycerophosphoethanolamine, formic acid, and pentadecanal are formed during P12/UV treatment of CHO-K1 cells, but not of mutant cells deficient in plasmalogen synthesis. The decomposition of plasmenylethanolamine is O2-dependent, is enhanced in D2O, and is reduced in the presence of sodium azide. The process may be explained, in part, by the cycloaddition of singlet oxygen to the vinyl ether linkage of plasmenylethanolamine, generating a dioxetane intermediate that would be expected to decompose under physiological conditions to the observed products. An additional possibility is the formation of an allylic hydroperoxide at the 1'-carbon of the alkyl moiety by an "ene" reaction of singlet oxygen, or by radical-mediated oxidation, followed by metabolism or chemical decomposition of the hydroperoxide. Given the P12/UV hypersensitivity of plasmalogen-deficient mutants, we suggest that plasmalogens might protect animal cell membranes from singlet oxygen and/or radical-initiated oxidation by functioning as scavengers and decomposing to products that can be reutilized.     

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by beta-carotene, alpha-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress.     

Chemienergized aromatic aldehydes from the peroxidase catalyzed oxidation of pyruvates: excited vanillin from vanylpyruvate.

Oxidation by molecular oxygen of vanylpyruvate in dimethylsulfoxide containing potassium t-butoxide results in formation of emissive, electronically excited (singlet) vanillin and of oxalate, that is, the products expected from the cleavage of a dioxetane intermediate. The reaction is a model for the peroxidase and laccase catalyzed processes that occur during lignin degradation by fungi. It is inferred that vanillin formed in the latter processes is generated in an electronically excited state, not necessarily emissive. This view is strengthened by (i) the emission, albeit very weak, observed from the enzyme system, and (ii) the alteration of the enzyme as a result of the reaction, the spectral changes being similar to those induced by uv irradiation of the enzyme alone. Also other peroxidase catalyzed oxidations of aromatic pyruvates should produce the electronically excited aldehyde.     

Production and chemiluminescent free radical reactions of glyoxal in lipid peroxidation of linoleic acid by the ligninolytic enzyme, manganese peroxidase.

Glyoxal is a key compound involved in glyoxal oxidase (GLOX)-dependent production of glyoxylate, oxalate and H2O2 by lignin-degrading basidiomycetes. In this paper, we report that glyoxal was produced from a metabolite of ligninolytic fungi, linoleic acid, by manganese peroxidase (MnP)-dependent lipid peroxidation. In the absence of the parent substrate of linoleic acid, the dialdehyde was oxidized by MnP and Mn(III) chelate to start free radical reactions with emission of chemiluminescence at 700-710 nm. The spectroscopic profile of the light emission is distinguishable from (a) singlet oxygen, (b) triplet carbonyls from dioxetane and alpha-hydroxyperoxyl radicals, and (c) biacyl triplet formed by the coupling of two acyl radicals. The photon emission of glyoxal by MnP was activated by co-oxidation of tartrate. The MnP-dependent oxidation of glyoxal in tartrate buffers continued for 10 days without addition of exogenous H2O2. The importance of these results is discussed in relation to the free radical chemistry of lignin biodegradation by wood rot fungi.     

The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.     

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.     

The cytotoxic activity of hematoheme: evidence for two different mechanisms

Hematoheme displays a potent cytolytic activity toward erythrocytes either in the presence of hydrogen peroxide and a halide ion (system I) or in the presence of oxygen and a reducing agent (system II). In system I it resembles the cytotoxic activity of various peroxidases, whereas in system II it resembles the destructive activity of bleomycin and a variety of metal complexes. Both types of reactions presumably involve the generation of active oxygen species, which are responsible for the damaging effects. In a first attempt to compare the chemical mechanisms of the two types of reactions we used various traps and scavengers of active oxygen species. Tryptophan as well as tyrosine and uric acid were found to be potent inhibitors of the hematoheme-H2O2-halide reaction but do not significantly inhibit the hematoheme-O2-ascorbate reaction. Pyridine, on the other hand, inhibits the oxygen-mediated reaction, but does not affect the peroxide-halide-mediated activity. The cytolytic activity of photoactivated hematoporphyrin, which involves the generation of singlet oxygen, is activated by pyridine and is strongly inhibited by diphenylisobenzofuran. The latter compound is a weak activator of both hematoheme reactions. We conclude that the two hematoheme reactions proceed by two different mechanisms and probably generate different toxic intermediates. The results further suggest that the toxic intermediate generated by photoactivated hematoporphyrin (singlet oxygen) does not play a dominant role in either of the two hematoheme reactions.     

Mechanisms involved in the chemical inhibition of the eosin-sensitized photooxidation of trypsin

A large series of compounds was screened for ability to protect trypsin from eosin-sensitized photodynamic inactivation. Eosin-sensitized photooxidation reactions of this type typically proceed via the triplet state of the dye and often involve singlet state oxygen as the oxidizing entity. In order to determine the mechanisms by which trypsin is protected from photoinactivation, a number of good protective agents (inhibitors) and some non-protective agents were selected for more detailed flash photolysis studies. Good inhibitors such as p-phenylenediamine, n-propyl gallate, serotonin creatinine sulfate and p-toluenediamine competed efficiently with oxygen and with trypsin for reaction with the triplet state of eosin. The inhibitors were shown to quench triplet eosin to the ground state and/or reduce triplet eosin to form the semireduced eosin radical and an oxidized form of the inhibitor. In the latter case, oxidized inhibitor could react by a reverse electron transfer reaction with the semi-reduced eosin radical to regenerate ground state eosin and the inhibitor. The good inhibitors also competed effectively with trypsin for oxidation by semioxidized eosin, thus giving another possible protective mechanism. Non-inhibitors such as halogen ions and the paramagnetic ions Co++, Cu++ and Mn++ reacted only slowly with triplet and with seimioxidized eosin. The primary pathway for the eosin-sensitized photooxidation of trypsin at pH 8.0 involved singlet oxygen, although semioxidized eosin may also participate.     

Substituent effects on the decomposition of chemiluminescent tricyclic aromatic dioxetanes

Three tricyclic 1,2‐dioxetane derivatives, 1a, 2a and 3a were synthesized from their corresponding 1,4‐dioxin acenaphthylene compounds, 1, 2 and 3, by reaction with singlet‐oxygen (¹O₂) in dichloromethane. Evidence for the formation of the dioxetanes 1a, 2a and 3a is provided by the chemiluminescence (CL) that corresponds to the emission from the electronically excited diesters 1b*, 2b* and 3b*, which are decomposed thermally from the dioxetanes 1a, 2a and 3a, respectively. The highly strained 1,2‐dioxetane ring decomposes from a twisted geometry by simultaneous cleavages of the O–O and C–C bonds, producing the electronically excited diester that emits CL. It was observed that the CL from compound 2a is red‐shifted relative to that of compounds 1a and 3a suggesting a higher degree of stabilization for the excited state by the electron‐donating methoxy group. Also, a study of the solvent effect on fluorescence shows a significant red‐shift in compound 2b, indicating a more polar excited state. The kinetics of the thermal decomposition of the 1,2‐dioxetanes clearly demonstrate that the CL characteristics of compound 2a are quite different from those of compounds, 1a and 3a. These results are consistent with the proposed intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism which is triggered by the electron‐donating group of compound 2a. Copyright © 2013 John Wiley & Sons, Ltd.     

Extra-weak chemiluminescence of drugs. VIII. Extra-weak chemiluminescence arising from the amino-carbonyl reaction

Extra-weak chemiluminescence (CL) from amino-carbonyl reactions of L-lysine with various sugars and aldehydes in aqueous solution was examined. Amongst the aldehydes and sugars tested, glycolaldehyde and D -arabinose produced the highest CL intensity. The CL of the amino-carbonyl reaction reached a maximum after about 60 minutes. The CL was pH dependent, and a linear relation between CL intensity and hydroge-ion concentration was demonstrated. Low oxygen levels inhibited CL and no CL was produced in nitrogen purged solutions. Addition of cupric or ferrous ion, decreased the CL. The involvement of free radical intermediates was demonstrated by ESR. Our findings suggest that the CL of the amino-carbonyl reaction arises from free radicals derived from melanoidines or their intermediates. CL should prove useful for evaluating the stability of crude drugs extracted from natural resources that contain various amino acid derivatives protein and sugar components.     

Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants

Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.