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PubMed1989

Singlet oxygen induced mutagenesis of benzo[a]pyrene derivatives.

Singlet oxygen activates the mutagenicity of several benzo[a]pyrene (BP) derivatives in the absence of mammalian metabolic action. This has been demonstrated using a separated-surface-sensitizer system for generating chemically pure singlet oxygen, eliminating most of the complications that arise with singlet oxygen generation by conventional photosensitization. Salmonella typhimurium bacteria were exposed to singlet oxygen in the presence of certain BP derivatives and the mutation frequency determined with an azaguanine forward mutation assay. The mutation frequency was increased by exposure to singlet oxygen compared to light-only controls for those BP derivatives that were saturated at either the 7,8 or 9,10 positions but not both. The increase in mutation frequency depends on both the concentration of BP derivative and on the dose of singlet oxygen. Mutation frequency was also significantly increased when bacteria were treated with a solution of trans-7,8-dihydrodiol-BP that had been separately exposed to singlet oxygen, unequivocally demonstrating that the mutagenicity is due to the formation of a product of BP derivative oxidation by singlet oxygen and that this product has a lifetime at least on the order of minutes in acetonitrile. The requirement for singlet oxygen rather than some other form of reactive oxygen was confirmed by determination of the gas phase lifetime of the intermediate responsible for activating mutagenicity. This was performed by measuring the dependence of the mutation frequency on the distance separating the sensitizer from the target. This gives a value of 88 +/- 35 ms, which is in excellent agreement with the mean value of 89 ms calculated from previous independent determinations of the gas phase lifetime of singlet oxygen reported in the literature.

Benzo(a)pyrene↗

PubMed1988

Singlet oxygen production by human eosinophils.

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.

Bromides↗

PubMed1987

Diffusion of singlet oxygen into human bronchial epithelial cells.

The respiratory epithelium undergoes morphological and functional changes following exposure to single oxygen. However, mechanisms by which singlet oxygen causes cellular injury are unclear. The present experiments were designed to investigate the possibility that singlet oxygen, a highly reactive species, diffuses into respiratory epithelial cells. Of the various methods for detection of singlet oxygen, the electron spin resonance (ESR) spectrometric technique was judged to be most compatible and sensitive for use with cell suspensions. ESR spectrometry was used to monitor the singlet oxygen-mediated conversion of 2-(9,10-dimethoxyanthracenyl)-tert-butylhydroxylamine, (I), to 2-(9,10-dimethoxyanthracenyl)-tert-butylnitroxide, (II), and its corresponding endoperoxide, (III), in human bronchial epithelial cells treated with extracellularly generated singlet oxygen. In a second series of experiments, bronchial epithelial cells labeled with (I) were treated with singlet oxygen in the presence of 1,4-diazabicyclo[2.2.2]octane, a singlet oxygen quenching agent. The addition of this quenching agent eliminated the ESR spectrum corresponding with (II) and (III). This result is consistent with the quenching of singlet oxygen by 1.4-diazabicyclo[2.2.2]octane. Collectively, our results indicate that extracellularly generated singlet oxygen diffuses into human bronchial epithelial cells and that this process is a potentially important step in the cytotoxic action of singlet oxygen to the respiratory epithelium.

Anthracenes↗

PubMed1979

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.

Bromides↗

PubMed1989

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen.

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.

Amino Acids↗

PubMed1991

Genotoxicity of singlet oxygen.

Singlet oxygen, 1O2 (1 delta g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.

Animals↗

PubMed1995

Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase.

Singlet oxygen has been postulated to be generated by Ultraviolet (UV) A irradiation of mammalian cells. We studied the role of singlet oxygen in the downstream signaling of the complex UV response leading to the induction of matrix-metalloproteinase-1 (interstitial collagenase/MMP-1). Exposure of cultured human fibroblasts to singlet oxygen, generated in a dark reaction by thermodissociation of the endoperoxide of the disodium salt of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) induced collagenase mRNA steady state levels in a dose dependent manner. The increase in collagenase expression after singlet-oxygen exposure generated with 3 mM NDPO2 was equivalent to that observed with UVA at a dose rate of 200-300 kJ/m2 and developed in a similar time course. In contrast, mRNA levels of TIMP-1, the specific tissue inhibitor of metalloproteinases, remained unchanged. Indirect evidence for the role of singlet oxygen in the UVA induction of collagenase comes from studies using singlet oxygen enhancer or quencher. Accordingly, incubation in deuterium oxide, an enhancer of singlet-oxygen lifetime, led to an additional increase in steady-state levels of collagenase mRNA after exposure to NDPO2 or to UVA irradiation. In contrast, sodium azide, a potent quencher of singlet oxygen, almost totally abrogated the induction of collagenase after exposure of fibroblasts to NDPO2 or to UVA irradiation. Similar results were obtained in studies of the proteins by radioimmunoprecipitation of MMP-1 and TIMP-1 using specific antibodies. Collectively, our data provide circumstantial evidence that singlet oxygen mediates the UVA induction of collagenase in vitro, whereas it does not exert any effect on TIMP-1 synthesis. The unbalanced synthesis of interstitial collagenase may contribute to the connective tissue damage in vivo related to photoaging and other photocutaneous disorders.

Azides↗

PubMed1997

Determination of singlet oxygen-specific versus radical-mediated lipid peroxidation in photosensitized oxidation of lipid bilayers: effect of beta-carotene and alpha-tocopherol.

Photosensitized oxidation reactions damage tissue by catalyzing the formation of oxyradicals and singlet oxygen. beta-Carotene is hypothesized to exert photoprotective effects by quenching singlet oxygen formed by Type II reactions and by scavenging free radicals formed by Type I reactions. beta-Carotene antioxidant mechanisms were studied in a phospholipid membrane model of photooxidation with a new isotope dilution gas chromatography-mass spectrometry (GC-MS) assay that quantitatively distinguishes singlet oxygen-mediated and radical-mediated lipid peroxidation. This assay measures 9- and 10-hydroxylinoleate methyl esters and was used to generate photooxidation profiles for the photosensitizers methylene blue, Rose Bengal, and tetraphenylporphine. These profiles indicate a shift from Type II to Type I photooxidation mechanisms in later stages of photooxidation. beta-Carotene (0.45 mol %) inhibited singlet oxygen-mediated lipid peroxidation at early stages of methylene blue-sensitized photooxidation. Production of radical-mediated products increased faster than singlet oxygen-mediated products at later stages. beta-Carotene-5,8-endoperoxide, a specific marker for singlet oxygen oxidation of beta-carotene in solution, was unstable under the incubation conditions and was not detected in this system. alpha-Tocopherol (0.45 mol %) was ineffective in inhibiting photosensitized lipid peroxidation, whereas 4.5 mol % alpha-tocopherol inhibited almost all radical-mediated lipid peroxidation as well as early-stage singlet oxygen-mediated lipid peroxidation. Cumene hydroperoxide stimulated radical-mediated lipid peroxidation, indicating that accumulation of hydroperoxides from Type II photooxidation may enhance Type I reactions. These data suggest that singlet oxygen quenching, rather than radical scavenging reactions, accounts for the photoprotective actions of beta-carotene.

Chromatography, High Pressure Liquid↗

PubMed1990

Quenching of singlet oxygen by human plasma.

Direct measurements of the decay of singlet oxygen phosphorescence at 1270 nm were made in human plasma diluted with various amounts of deuterium oxide. The Stern-Volmer plot of the singlet oxygen lifetimes was linear up to 15% plasma concentration (vol/vol). Extrapolation of these measurements to 100% plasma content gave a singlet oxygen lifetime of 1.04 +/- 0.03 microseconds in human plasma. Biological molecules accounted for 77% of the total singlet oxygen quenching while water accounted for 23% of the quenching. The contributions of various types of biological molecules to the total singlet oxygen quenching were calculated from their plasma concentrations and their quenching constants. Plasma proteins quenched most of the singlet oxygen. Uric acid also quenched a significant amount of singlet oxygen (12%). Tocopherols, carotenoids, ascorbic acid and bilirubin made only small contributions to the total singlet oxygen quenching (less than or equal to 4%).

Humans↗

PubMed1990

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.

Azides↗

PubMed1997

Cross-linking of collagen by singlet oxygen generated with UV-A.

Singlet oxygen (1O2), a highly reactive and toxic intermediate, may play a role in photo-induced aging. We examined singlet oxygen generation from hematoporphyrin (HP) with UV-A, by monitoring the emission at 1,268 nm corresponding to 1O2 --> 3O2. Singlet oxygen was formed HP-dose-dependently in this system. We then investigated the reaction of singlet oxygen generated by UV-A irradiation with collagen, which is related to skin elasticity and softness. Collagen from skin was rapidly and dose-dependently cross-linked by singlet oxygen. The reaction was inhibited by NaN3, a selective quencher of singlet oxygen. In contrast, SOD (superoxide dismutase) and mannitol had no effect. These results suggested that cross-linking of collagen was caused by UV-A-generated singlet oxygen, not by any other reactive oxygen species. Compared with another multisubunit protein, alcohol dehydrogenase, collagen was cross-linked much more efficiently. Further, the finding that semicarbazide inhibited cross-linking of collagen showed that cross-links were formed between photooxidized histidyl residues and amino groups. Singlet oxygen generated by UV-A irradiation may contribute to cross-linking of collagen in the process of skin photoaging.

Collagen↗

PubMed1991

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.

Animals↗

PubMed1995

UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen.

This study describes the damage that occurs to lipids and proteins that have been irradiated in vitro or in human skin fibroblasts with physiological doses of UVA radiation. Thiobarbituric acid-reactive species were formed from phosphatidylcholine after UVA radiation in vitro. By using iron chelators, this process was shown to involve iron. Ferric iron associated with potential physiological chelators was reduced by UVA radiation, but iron within ferritin was not. By enhancing the half life-time with deuterium oxide or by using scavengers, singlet oxygen was also shown to be involved in the UVA radiation-dependent peroxidation of phosphatidylcholine. UVA radiation-generated singlet oxygen reacted with phosphatidylcholine to form lipid hydroperoxides, and the breakdown of these hydroperoxides to thiobarbituric acid-reactive species was dependent on iron. We have shown that iron and singlet oxygen are also involved in the UVA radiation-dependent formation of thiobarbituric acid-reactive species in human skin fibroblasts, and we propose that a similar concerted effect of iron and singlet oxygen is involved in UVA radiation-dependent damage to fibroblast lipids. Sulphydryl groups of bovine serum albumin and human gamma-globulin were oxidised upon UVA irradiation in vitro. The use of scavengers and deuterium oxide showed that UVA radiation-dependent sulphydryl oxidation was dependent on singlet oxygen. By adding or chelating iron, UVA radiation-dependent oxidation of sulphydryl groups of bovine serum albumin and human gamma-globulin was shown to be iron-dependent. The use of catalase and hydroxyl radical scavengers demonstrated that hydrogen peroxide, but not the hydroxyl radical, was involved. The oxidation of sulphydryl groups of proteins in human skin fibroblasts that occurs as a result of UVA irradiation was also shown to involve iron, singlet oxygen, and hydrogen peroxide. We conclude that iron, singlet oxygen, and hydrogen peroxide are important redox active species involved in the deleterious effects of UVA radiation on lipids and proteins of human skin cells.

Dose-Response Relationship, Radiation↗

PubMed1995

Inactivation of viruses by chemically and photochemically generated singlet molecular oxygen.

Inactivation of viruses in blood plasma can be achieved by photodynamic procedures using methylene blue (MB) or other photoactive dyes. Singlet molecular oxygen (1O2) probably contributes to the virucidal effects of photosensitization. We report the inactivation of herpes simplex virus type 1 (HSV-1) and suid herpes virus type 1 (SHV-1) by chemically generated singlet oxygen, produced by thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate (NDPO2). We demonstrate that viruses can be inactivated by 1O2 generated by chemiexcitation in a reaction in the dark, even in the presence of human plasma. Virus inactivation in phosphate-buffered saline (PBS) was enhanced when water was replaced by deuterium oxide (D2O) and diminished when human plasma or quenchers (imidazole or histidine) were added. The singlet oxygen quenching activities of plasma, imidazole and histidine correlated with their inhibitory effects on virus inactivation. The production of 1O2 was assessed by an indicator reaction: the bleaching of p-nitrosodimethylaniline (RNO) with imidazole as 1O2 acceptor. Virus inactivation and singlet oxygen generation of NDPO2 were compared with those of MB/light-mediated photosensitization. Based on similar amounts of 1O2 generated by either procedure, virus inactivation by MB/light was more effective. Virus inactivation by MB/light was not affected by type I quenchers (e.g. mannitol), but was inhibited by human plasma or singlet oxygen quenchers. Furthermore, in D2O-based PBS, virus inactivation was more effective than that in H2O. These observations confirm that singlet oxygen is involved in virus inactivation by MB/light. Taken together, the results demonstrate that singlet oxygen produced by either procedure is virucidal. The enhanced effect of the photochemical procedure suggests that, in addition to type II, type I reactions and/or the binding affinity of the dye for the virus contribute to virus killing by MB/light.

Animals↗

PubMed1995

Singlet oxygen generation from the reaction of ozone with plant leaves.

Aqueous extracts of the intercellular fluid from Sedum album L. leaves generated singlet oxygen chemiluminescence at 1270 nm when exposed to a nitrogen gas stream containing ozone at 21 +/- 2 ppm. The concentration of ascorbic acid in the intercellular fluid extracts was 310 +/- 40 microM. The intensity of the singlet oxygen chemiluminescence from the intercellular fluid extracts was comparable with the chemiluminescence from a control solution containing 300 microM ascorbic acid. The intensity of the singlet oxygen emission from intercellular fluid treated with ascorbate oxidase was 0.19 +/- 0.07 of the intensity of the singlet oxygen chemiluminescence from untreated samples of intercellular fluid extract. The simplest explanation for the effect of ascorbate oxidase is that ascorbic acid is the major ozone target generating singlet oxygen. Much weaker singlet oxygen chemiluminescence was detected at 1270 nm when intact S. album L. plant tips were exposed to a nitrogen gas stream containing ozone at 22 +/- 5 ppm. Various explanations for the relatively low intensity of the singlet oxygen chemiluminescence from intact S. album L. plant tips are discussed.

Luminescent Measurements↗

PubMed1998

Oxidation of catalase by singlet oxygen.

Different bands of catalase activity in zymograms (Cat-1a-Cat-1e) appear during Neurospora crassa development and under stress conditions. Here we demonstrate that singlet oxygen modifies Cat-1a, giving rise to a sequential shift in electrophoretic mobility, similar to the one observed in vivo. Purified Cat-1a was modified with singlet oxygen generated from a photosensitization reaction; even when the reaction was separated from the enzyme by an air barrier, a condition in which only singlet oxygen can reach the enzyme by diffusion. Modification of Cat-1a was hindered when reducing agents or singlet oxygen scavengers were present in the photosensitization reaction. The sequential modification of the four monomers gave rise to five active catalase conformers with more acidic isoelectric points. The pI of purified Cat-1a-Cat-1e decreased progressively, and a similar shift in pI was observed as Cat-1a was modified by singlet oxygen. No further change was detected once Cat-1e was reached. Catalase modification was traced to a three-step reaction of the heme. The heme of Cat-1a gave rise to three additional heme peaks in a high performance liquid chromatography when modified to Cat-1c. Full oxidation to Cat-1e shifted all peaks into a single one. Absorbance spectra were consistent with an increase in asymmetry as heme was modified. Bacterial, fungal, plant, and animal catalases were all susceptible to modification by singlet oxygen, indicating that this is a general feature of the enzyme that could explain in part the variety of catalases seen in several organisms and the modifications observed in some catalases. Modification of catalases during development and under stress could indicate in vivo generation of singlet oxygen.

Catalase↗

PubMed1992

Quenching of singlet oxygen by biomolecules from L1210 leukemia cells.

Singlet oxygen lifetimes for detergent-dispersed L1210 leukemia cells in deuterium oxide buffer were measured by following the decay of 1270 nm phosphorescence. Four photosensitizers and two detergents were studied. Stern-Volmer plots were linear over the cell concentration range studied (0-10(7) cells/mL). The singlet-oxygen quenching constants obtained depended somewhat upon the specific combination of detergent and photosensitizer used. Extrapolation of the singlet-oxygen lifetime data to "100%" cell concentration (1.39 +/- 0.04 x 10(9) cells/mL) and correction for the contribution of the water solvent gave a singlet-oxygen lifetime between 0.17 and 0.32 microseconds for the L1210 leukemia cell. The theoretical contributions of various types of biological molecules within the L1210 cell to the total singlet-oxygen quenching were calculated from their concentrations and their quenching constants. These calculations suggest that proteins will quench most of the singlet-oxygen. Only about 7% of the singlet-oxygen is quenched by water.

Animals↗

PubMed1997

Singlet oxygen-mediated inactivation of acetylcholinesterase: a comparison of purified enzyme in solution and enzyme bound to K562 leukemia cells.

We have compared the singlet oxygen-mediated inactivation of acetylcholinesterase (ACE) in solution with the inactivation of ACE on the surface of K562 leukemia cells. In solution, the actions of the singlet-oxygen quenchers, methionine, azide, disodium [N,N'-ethylenebis (5-sulfosalicylideneimminato)]nickelate(II) (Ni-chelate 1) and disodium [(N,N'-2,3-propionic acid)bis(5-sulfosal-icylideneimminato)] nickelate(II) (Ni-chelate 2) could be explained quantitatively by assuming their only mechanism of action was to quench singlet oxygen. The singlet oxygen quenchers, azide, Ni-chelate 1 and Ni-chelate 2, caused smaller inhibitions in the rate of singlet oxygen-mediated inactivation of ACE on K562 cells than ACE in solution. The effects of these quenchers and of deuterium oxide were interpreted using a mathematical model of singlet-oxygen quenching and diffusion to estimate the lifetime of singlet oxygen near the cell surface. The azide quenching data and the deuterium-oxide data gave lifetimes of 0.9 +/- 0.2 microsecond and 0.45 +/- 0.15 microsecond, respectively. The increases in ACE inactivation lifetime caused by the nickel chelates were anomalously large. The unexpectedly large quenching due to the nickel chelates may have been due to a nonuniform distribution of the chelates in the cytoplasm with a large concentration of the chelate near the cell membrane.

Acetylcholinesterase↗