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.     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Effects of Singlet Oxygen Quenchers and pH on the Bacterially Induced Hypersensitive Reaction in Tobacco Suspension Cell Cultures

Suspension cell cultures of Nicotiana tabacum L. inoculated with the incompatible pathogen Pseudomonas syringae pv pisi undergo a hypersensitive reaction. Addition of the singlet oxygen quencher bixin to cell suspensions had no effect on hypersensitive cell death. Addition of the singlet oxygen quencher 1,4-diazabicyclo octane (DABCO) increased the medium pH and delayed the onset of cell death. This delay was eliminated when cell suspensions were buffered, and could also be induced by increasing medium pH with KOH. Bixin and DABCO also did not suppress the hypersensitive reaction in tobacco leaves. These data do not support a role for singlet oxygen in the hypersensitive reaction. Medium pH, however, appears to be a critical factor in cell suspension cultures.     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

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.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Direct observation of singlet oxygen phosphorescence at 1270 nm from L1210 leukemia cells exposed to polyporphyrin and light.

Near-infrared emission (1170-1475 nm) was studied from L1210 leukemia cells incubated with polyporphyrin (fractionated hematoporphyrin derivative), suspended in deuterium oxide buffer, and then exposed to light. Following pulsed laser excitation, the near-infrared emission decayed in two phases. The first phase of the emission (0-2 microseconds) was principally due to polyporphyrin fluorescence. The second phase of the emission (20-90 microseconds) was due mainly to singlet oxygen. Evidence supporting the assignment of the second phase emission to singlet oxygen included a spectral analysis showing a peak near 1270 nm and reductions in the second phase emission caused by the singlet oxygen quenchers, histidine, carnosine, and water. The second phase emission decayed in a biexponential manner with lifetimes of 4.5 +/- 0.5 and 49 +/- 4 microseconds. Most of the singlet oxygen in the second phase emission was likely due to singlet oxygen that was generated near the surface of the L1210 leukemia cells and then diffused into the deuterium oxide buffer. Direct measurements of singlet oxygen phosphorescence at 1270 nm may prove to be a useful analytical technique for studying photochemical generation of singlet oxygen in cultured cells.     

Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate

Maize malic enzyme was rapidly inactivated by micromolar concentrations of cupric nitrate in the presence of ascorbate at pH, 5.0. Ascorbate or Cu2+ alone had no effect on enzyme activity. The substrate L-malate or NADP individually provided almost total protection against Cu2+-ascorbate inactivation. The loss of enzyme activity was accompanied by cleavage of the enzyme. The cleaved peptides showed molecular mass of 55 kDa, 48 kDa, 38 kDa, and 14 kDa. Addition of EDTA, histidine and imidazole provided protection. The results of protection experiments with sodium azide, DABCO and catalase suggested that reactive oxygen species were generated resulting in loss of enzyme activity. This was further supported by experiments showing that the rate of enzyme inactivation was higher in D2O than in water. It is suggested that maize malic enzyme is modified by reactive oxygen species like singlet oxygen and H2O2 generated by Cu2+-ascorbate system and the modified amino acid residue(s) may be located at or near the substrate-binding site of the enzyme.     

Cellular toxicity of elsinochrome phytotoxins produced by the pathogenic fungus, Elsino? fawcettii causing citrus scab.

Elsinochromes are the red/orange pigments produced by many Elsino? fungal species and are structurally similar to the phytotoxin, cercosporin. Here, pigments were extracted from cultures of a citrus pathogen, Elsino? fawcettii and tested for cellular toxicity. On irradiation with light, elsinochromes rapidly killed suspension cultured citrus and tobacco cells. The toxicity was decreased by adding the singlet oxygen ((1)O(2)) quenchers (bixin (carotenoid carboxylic acid), DABCO (1, 4-diazabicyco octane), ascorbate or reduced glutathione). Application of elsinochromes onto rough lemon leaves resulted in necrotic lesions, whereas lesion development was inhibited by the addition of bixin, DABCO or ascorbate, but not a-tocopherol. Incubation of rough lemon leaf discs with elsinochromes in the light induced a steady increase of electrolyte leakage. Compared with two photosensitizing compounds, hematoporphyrin and cercosporin, the accumulation of (1)O(2) induced by elsinochromes after irradiation was indicated by successful detection of the cholesterol oxidation product, 5a-hydroperoxide. Addition of a potent quencher, beta-carotene prevented 5alpha-hydroperoxide production. Elsinochromes generated superoxide ions (O(2)(*-)), whereas accumulation of O(2)(*-)was blocked by addition of the superoxide dismutase, a scavenger of O(2)(*-), but not the (1)O(2)-quencher, DABCO. Our study indicated that elsinochromes are functioning as photosensitizing compounds that produce (1)O(2)and O(2)(*-), and exert toxicity to plant cells.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.     

Spatial energy transfer with observation of bimolecular singlet oxygen emission using quantum dots as donors and zinc‐phthalocyanine as acceptors

This study reports the influence of CdSe–ZnS core–shell quantum dots (QDs) for formation of singlet oxygen using zinc‐phthalocyanine (ZnPc) dyes in colloidal solutions. Using a microluminescence surface scan technique it was possible to measure accurately the photon diffusion length, or photon mean free path, inside the medium. Analyses were performed for a range of QD concentrations. Photon diffusion length was assigned to the bimolecular singlet oxygen emission at 707 nm. Related singlet oxygen emission was predicted by observing quenching of the photon diffusion length measured at the specific oxygen emission as a function of QD concentration, being a nontrivial phenomenon related to the QD donors. Diffusion length measured at 707 nm increased with QD concentration; in the absence of QDs, as in pure ZnPc samples, the emission peak at 707 nm was not observed.     

Mechanistic investigations of the novel non-heme vanadium bromoperoxidases. Evidence for singlet oxygen production

Three newly discovered non-heme bromoperoxidases isolated from marine algae were found to catalyze the production of singlet oxygen in reactions composed of the bromoperoxidase, hydrogen peroxide, and bromide. The bromoperoxidases studied were vanadium bromoperoxidase (V-BrPO) from Ascophyllum nodosum, native non-heme bromoperoxidase from Corallina vancouveriensis (which contains vanadium and iron), and the vanadium-reconstituted bromoperoxidase derivative from C. vancouveriensis. These enzyme systems generated near infrared emission, characteristic of singlet oxygen. The emission had a peak intensity near 1268 nm, was greatly increased in 2H2O-containing buffers, and was greatly decreased by the singlet oxygen quenchers, histidine and azide. The yield of singlet oxygen was approximately 80% of the theoretical yield. A unique feature of the non-heme bromoperoxidases distinct from the iron heme haloperoxidases, was the remarkable stability of the non-heme enzymes in the presence of singlet oxygen and oxidized bromine species. V-BrPO turned over multiple aliquots of 2 mM hydrogen peroxide without losing efficiency. In contrast, iron heme lactoperoxidase was completely inactivated after turnover of the first aliquot of 2 mM hydrogen peroxide, and iron heme chloroperoxidase was 50% deactivated. The profile of singlet oxygen formation by V-BrPO and the near stoichiometric yield of singlet oxygen suggest that the mechanism of singlet oxygen formation is the same as the mechanism of dioxygen formation determined by oxygen probe measurements.     

Chemically shifted singlet oxygen spectrum.

The estimated light emission spectrum was determined for a singlet oxygen (1O2)-producing system, NaOCl + H2O2, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of 1O2 and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of 1O2.     

Vanillin as an antioxidant in rat liver mitochondria: Inhibition of protein oxidation and lipid peroxidation induced by photosensitization

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (¹O₂), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.     

L-ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: generalized antioxidant property of vitamin C

L-ascorbic acid quenches singlet (1 delta g) molecular oxygen in aqueous media (pH 6.8 for [1H]H2O and pD 7.2 for [2H]D2O) as measured directly by monitoring (0,0) 1 delta g leads to 3 sigma-g emission at 1.28 micron. Singlet oxygen was generated at room temperature in the solutions via photosensitization of sodium chrysene sulfonate; this sulfonated polycyclic hydrocarbon was synthesized to provide a water soluble chromophore inert to usual dye-ascorbate photobleaching. A marked isotope effect is found; kHQ2O is 3.3 times faster than kDQ2O, suggesting ascorbic acid is chemically quenching singlet oxygen.     

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.     

The role of EDS1 (enhanced disease susceptibility) during singlet oxygen-mediated stress responses of Arabidopsis

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.     

Ultraviolet radiation drives methane emissions from terrestrial plant pectins

Recent studies demonstrating an in situ formation of methane (CH(4)) within foliage and separate observations that soil-derived CH(4) can be released from the stems of trees have continued the debate about the role of vegetation in CH(4) emissions to the atmosphere. Here, a study of the role of ultraviolet (UV) radiation in the formation of CH(4) and other trace gases from plant pectins in vitro and from leaves of tobacco (Nicotiana tabacum) in planta is reported. Plant pectins were investigated for CH(4 )production under UV irradiation before and after de-methylesterification and with and without the singlet oxygen scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO). Leaves of tobacco were also investigated under UV irradiation and following leaf infiltration with the singlet oxygen generator rose bengal or the bacterial pathogen Pseudomonas syringae. Results demonstrated production of CH(4), ethane and ethylene from pectins and from tobacco leaves following all treatments, that methyl-ester groups of pectin are a source of CH(4), and that reactive oxygen species (ROS) arising from environmental stresses have a potential role in mechanisms of CH(4) formation. Rates of CH(4 )production were lower than those previously reported for intact plants in sunlight but the results clearly show that foliage can emit CH(4) under aerobic conditions.