LONG-LIVED AFTERGLOW OF PHOTOSYNTHETIC PIGMENTS IN SOLUTION: PHOTOCHEMILUMINESCENCE

Abstract —Our recent research on photochemiluminescence (PCL) of pigments in solutions is reviewed. PCL was observed in the course of photooxidation by oxygen of chlorophyll a , bacteriochlorophyll, protochlorophyll, their analogs, synthetic dyes and aromatic hydrocarbons. The PCL of chlorophyll was studied in detail. It depends on oxygen concentration, intensity of exciting light, pH, nature of pigments, solvents etc. The thermochemiluminescence was observed after illumination of liquid and solid pigment solutions at low temperature (down to - 170C). The excitation spectra of PCL coincide with the pigment absorption spectra. The PCL emission spectra in most cases differ from those of pigment fluorescence. Electron acceptors, electron donors, radical inhibitors and β-carotene quench PCL. The quenching efficiency of electron acceptors is similar to their action on the chlorophyll triplet state. The quenching effect of radical inhibitors and β-carotene correlates with their activity in reaction with singlet oxygen. The effect of quenchers on the chlorophyll fluorescence, photobleaching and pigment sensitized oxygenation was studied. Analysis of experimental data allowed the assumption that chemiluminescence accompanies the decomposition of labile pigment peroxides. The accumulation of peroxides is probably due to the reaction in the complex of pigment and singlet oxygen, formed as a result of energy transfer from photoexcited (triplet) pigment molecules to oxygen. The terminal chemiluminescence emission proceeds from the singlet excited states of molecules of pigments and products of their oxidation.     

The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: properties of the triplet state and singlet oxygen photogeneration and quenching

Measurements of pigment triplet-triplet absorption, pigment phosphorescence and photosensitized singlet oxygen luminescence were carried out on solutions containing monomeric bacteriochlorophylls (Bchl) c and d, isolated from green photosynthetic bacteria, and their magnesium-free and farnesyl-free analogs. The energies of the pigment triplet states fell in the range 1.29-1.34 eV. The triplet lifetimes in aerobic solutions were 200-250 ns; they increased to 280 +/- 70 microseconds after nitrogen purging in liquid solutions and to 0.7-2.1 ms in a solid matrix at ambient or liquid nitrogen temperatures. Rate constants for quenching of the pigment triplet state by oxygen were (2.0-2.5) x 10(9) M-1 s-1, which is close to 1/9 of the rate constant for diffusion-controlled reactions. This quenching was accompanied by singlet oxygen formation. The quantum yields for the triplet state formation and singlet oxygen production were 55-75% in air-saturated solutions. Singlet oxygen quenching by ground-state pigment molecules was observed. Quenching was the most efficient for magnesium-containing pigments, kq = (0.31-1.2) x 10(9) M-1 s-1. It is caused mainly by a physical process of singlet oxygen (1O2) deactivation. Thus, Bchl c and d and their derivatives, as well as chlorophyll and Bchl a, combine a high efficiency of singlet oxygen production with the ability to protect photochemical and photobiological systems against damage by singlet oxygen.     

单线态分子氧的产生和体外叶绿素α的结构状态

叶绿素α(Chl α)是光合作用光量子的接受器和光能最初传递体。早期研究表明,在光合器官中,Chlα长波主吸收带位于红区。1969年Krasnovsky指出,Chlα受光照射时有可过氧化还原反应。     

Rational design of boron dipyrromethene (BODIPY)-based photobleaching-resistant fluorophores applicable to a protein dynamics study

We studied the photobleaching of a library of boron dipyrromethene (BODIPY) derivatives with a range of electron densities, and found that the photobleaching rate is influenced by the electron-withdrawing capacity of the substituents. Electron-deficient BODIPYs generated less singlet oxygen, were less reactive to singlet oxygen, and were highly resistant to photobleaching. We confirmed the utility of one of these fluorophores, 2,6-diCO(2)R-BDP, for visualizing EGF receptor dynamics in cells expressing an SNAP-tagged EGF receptor.     

Photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen

The photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen (AcPso), proposed for the photochemotherapy of some skin diseases, were investigated. The photoreaction of AcPso with DNA is weaker in the presence of air than in a nitrogen atmosphere, in terms of total photobinding and DNA cross-linking; when UVA irradiation is performed in air, AcPso behaves as a monofunctional reagent. The quenching effect of oxygen is related to the high capacity of AcPso to produce singlet oxygen. Furthermore, it is demonstrated that AcPso photoadducts are better producers of singlet oxygen than free AcPso in solution. Using DNA sequencing methodology, two modes of DNA photosensitization by AcPso are shown, these lead to the formation of photoadducts mainly at T residues (and at C to a lesser extent) and to photo-oxidized G residues probably via singlet oxygen. Chemical or enzymatic cleavage were used as probes in these experiments. A rapid assay for the detection of the photodynamic effect of a photosensitizer on DNA, involving oxygen, is also described. Finally, the cytotoxicity and genotoxicity of AcPso on E. coli WP2 cells appear to be related to its ability to form photoadducts, in particular cross-links, rather than to its capacity to produce singlet oxygen.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

Single cell responses to spatially controlled photosensitized production of extracellular singlet oxygen

The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed postirradiation "waiting period" before necrosis became apparent depended on: (1) the distance between the cell membrane and the domain irradiated, (2) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced and (3) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach of using extracellularly generated singlet oxygen to induce cell death can provide a solution to a problem that often limits mechanistic studies of intracellularly photosensitized cell death: it can be difficult to quantify the effective light dose, and hence singlet oxygen concentration, when using an intracellular photosensitizer.     

A Tandem Mass Spectrometric Method for Singlet Oxygen Measurement

Singlet oxygen, a harmful reactive oxygen species, can be quantified with the substance 2,2,6,6‐tetramethylpiperidine (TEMP) that reacts with singlet oxygen, forming a stable nitroxyl radical (TEMPO). TEMPO has earlier been quantified with electron paramagnetic resonance (EPR) spectroscopy. In this study, we designed an ultra–high‐performance liquid chromatographic—tandem mass spectrometric (UHPLC‐ESI‐MS/MS) quantification method for TEMPO and showed that the method based on multiple reaction monitoring (MRM) can be used for the measurements of singlet oxygen from both nonbiological and biological samples. Results obtained with both UHPLC‐ESI‐MS/MS and EPR methods suggest that plant thylakoid membranes produce 3.7 × 10^?7 molecules of singlet oxygen per chlorophyll molecule in a second when illuminated with the photosynthetic photon flux density of 2000 μmol m^?2 s^?1.     

THE 75°C THERMOLUMINESCENCE BAND OF GREEN TISSUES: CHEMILUMINESCENCE FROM MEMBRANE-CHLOROPHYLL INTERACTION

Abstract— Exposure of thylakoid membranes of green plants to high temperature promotes the appearance of free radicals resulting in a thermoluminesccnce (TL) band peaking around 75°C. The occurrence of this band with the same intensity in prcilluminated and in dark-adapted samples demonstrates that, contrary to several other TL bands, it is not a result of charge recombination. The high temperature TL band is oxygen dependent. Parallel to TL emission singlet oxygen is formed, as demonstrated by spin trapping EPR measurements and by the decrease of TL intensity in the presencc of sodium-azide, a singlet oxygen scavenger.
We suggest that the 75°C TL band is a result of a temperature-enhanced interaction between molecular oxygen and the photosynthetic membrane, possibly involving lipid peroxidation. The spectral maximum of the emission (around 720 nm) implics that light emission occurs upon energy transfer from an excited product to chlorophyll molecules destablized from pigment-protein complexes.     

Ratiometric singlet oxygen nano-optodes and their use for monitoring photodynamic therapy nanoplatforms

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel-based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen-sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen-insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen-free dye probe, anthracene-9,10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by "dynamic nanoplatforms" that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.     

Spatial and temporal electrochemical control of singlet oxygen production and decay in photosensitized experiments

Active spatial and temporal modulation of domains of singlet oxygen activity is demonstrated using electrochemical tools. Using singlet oxygen microscopy in photosensitized experiments, it is shown that singlet oxygen concentrations around an ultramicroelectrode can be controlled by applying a bias voltage to the electrode. Two phenomena that can be exploited separately or collectively are examined: (1) the singlet oxygen concentration can be altered by local oxidation or reduction of the photosensitizer, which is the precursor to singlet oxygen, and (2) the reduction of oxygen to produce the superoxide anion which, among other things, is an effective singlet oxygen quencher, results in a local decrease in the concentration of singlet oxygen around the electrode. Both of these phenomena depend significantly on the diffusion of molecules along concentration gradients established by the biased electrode. The results reported herein demonstrate that one can indeed exert local electrochemical control and readily manipulate the population of singlet oxygen produced in a photosensitized process.     

Optical detection of singlet oxygen from single cells

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen, O(2)(a (1)Delta(g)), is a reactive species involved in many chemical and biological processes. To better understand the roles played by singlet oxygen in biological systems, particularly at the sub-cellular level, optical tools have been developed to create and directly detect this transient state in time- and spatially-resolved experiments from single cells. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extracellular environment. On one hand, these results demonstrate that the behavior of singlet oxygen in an intact cell can be significantly different from that inferred from model bulk studies. More generally, these results provide a new perspective for mechanistic studies of intra- and inter-cellular signaling and events that ultimately lead to photo-induced cell death.     

Singlet Oxygen-Mediated Inactivation of Acetylcholinesterase: A Comparison of Purified Enzyme in Solution and Enzyme Bound to K562 Leukemia Cells

Abstract— 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 '-ethylene-bis(5-sulfosalicylideneinuninato)]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 oxygenmediated 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 μs and 0.45 ± 0.15 μs, 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.     

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

Photodynamic therapy (PDT) is a field with many applications including chemotherapy. Graphene quantum dots (GQDs) exhibit a variety of unique properties and can be used in PDT to generate singlet oxygen that destroys pathogenic bacteria and cancer cells. The PDT agent, methylene blue (MB), like GQDs, has been successfully exploited to destroy bacteria and cancer cells by increasing reactive oxygen species generation. Recently, combinations of GQDs and MB have been shown to destroy pathogenic bacteria via increased singlet oxygen generation. Here, we performed a spectrophotometric assay to detect and measure the uptake of GQDs, MB and several GQD‐MB combinations in MCF‐7 breast cancer cells. Then, we used a cell counting method to evaluate the cytotoxicity of GQDs, MB and a 1:1 GQD:MB preparation. Singlet oxygen generation in cells was then detected and measured using singlet oxygen sensor green. The dye, H₂DCFDA, was used to measure reactive oxygen species production. We found that GQD and MB uptake into MCF‐7 cells occurred, but that MB, followed by 1:1 GQD:MB, caused superior cytotoxicity and singlet oxygen and reactive oxygen species generation. Our results suggest that methylene blue's effect against MCF‐7 cells is not potentiated by GQDs, either in light or dark conditions.     

Functional in situ evaluation of photosynthesis-protecting carotenoids in mutants of the cyanobacterium Synechocystis PCC6803

Cyanobacteria possess different carotenoids as scavengers of reactive oxygen species. In Synechocystis PCC6803, zeaxanthin, echinenone, beta-carotene and myxoxanthophyll are synthesized. By disruption of the ketolase and hydroxylase genes, it was possible to obtain mutants devoid of either zeaxanthin, echinenone, or a combination of both carotenoids. With these mutants, their function in protecting photosynthetic electron transport under high light stress as well as chlorophyll and carotenoid degradation after initiation of singlet oxygen or radical formation was analyzed. Wild type Synechocystis is very well protected against high light-mediated photooxidation. Absence of echinenone affects photosynthetic electron transport to only a small extent. However, complete depletion of zeaxanthin together with a modification of myxoxanthophyll resulted in strong photoinhibition of overall photosynthetic electron transport as well as the photosystem II reaction. In the double mutant lacking both carotenoids the effects were additive. The light saturation curves of photosynthetic electron transport of the high light-treated mutants exhibited not only a lower saturation value but also smaller slopes. Using methylviologen or methylene blue as a radical or singlet oxygen generators, respectively, massive degradation of chlorophyll and carotenoids, indicative of photooxidative destruction of the photosynthetic apparatus, was observed, especially in the mutants devoid of zeaxanthin.     

Photoinduced DNA Cleavage and Photocytotoxic of Phenanthroline-Based Ligand Ruthenium Compounds

The photophysical and biological properties of two new phenanthroline-based ligand ruthenium complexes were investigated in detail. Their DNA interaction modes were determined to be the intercalation mode using spectra titration and viscosity measurements. Under irradiation, obvious photo-reduced DNA cleavages were observed in the two complexes via singlet oxygen generation. Furthermore, complex 2 showed higher DNA affinity, photocleavage activity, and singlet oxygen quantum yields than complex 1. The two complexes showed no toxicity towards tumor cells (HeLa, A549, and A375) in the dark. However, obvious photocytotoxicities were observed in the two complexes. Complex 2 exhibited large PIs (phototherapeutic indices) (ca. 400) towards HeLa cells. The study suggests that these complexes may act as DNA intercalators, DNA photocleavers, and photocytotoxic agents.     

MEMBRANE IONIC CURRENT PHOTOMODIFICATION BY ROSE BENGAL and MENADIONE: ROLE OF SINGLET OXYGEN

Abstract –Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.     

Oxidation of human catalase by singlet oxygen in myeloid leukemia cells

Catalases are oxidized by singlet oxygen giving rise to more acidic conformers detected in zymograms after electrophoresis in polyacrylamide gels. This shift in catalase mobility can be indicative of singlet oxygen production in vivo. Catalase from human cells, as from many organisms, is susceptible to in vitro modification by singlet oxygen. Human myeloid leukemia (U937) cells were treated under different stress conditions and catalase activity and its electrophoretic mobility was monitored. The U937 cells were found to have high levels of catalase activity, as compared to cultured fibroblasts, and to be very resistant to oxidative stress. Hydrogen peroxide did not modify the electrophoretic mobility of catalase, even at doses that produced cell damage. Conditions that primarily generate superoxide, such as treatment with paraquat or heat shock, also failed to modify the enzyme. In contrast, photosensitization reactions using rose Bengal gave rise to a more acidic conformer of catalase. Singlet oxygen quenchers prevented catalase modification by rose Bengal and light. The growth medium had a photosensitizing activity. Catalase was not modified in cells illuminated in phosphate buffer but was modified in cells illuminated in phosphate buffer containing riboflavin. Intense light per se also generated a slight shift in the electrophoretic mobility of catalase. Ultraviolet light (350 or 366 nm) did cause a change in catalase, but to a less acidic catalase conformer, indicating other modifications of the enzyme. The main effect of photosensitization with methylene blue was crosslinking of the enzyme, although some shift to acidic conformers was observed at a low concentration of the photoactive compound. Results indicate that catalase can be modified by singlet oxygen generated intracellularly, even though the enzyme is predominantly inside peroxisomes. Under some photosensitization conditions, catalase modification can be used as a marker to detect intracellular singlet oxygen.     

IRON-SULFUR CENTERS AS ENDOGENOUS BLUE LIGHT SENSITIZERS IN CELLS: A STUDY WITH AN ARTIFICIAL NON-HEME IRON PROTEIN

The possible involvement of Fe-S clusters in photodynamic reactions as endogenous sensitizing chromophores in cells has been investigated, by using an artificial non-heme iron protein (ANHIP) derived from bovine serum albumin and ferredoxins isolated from spinach and a red marine algae. Ferredoxins and ANHIP, when exposed to visible light, generate singlet oxygen, as measured by the imidazole plus RNO method. Irradiation with intense blue light of the ANHIP-entrapped liposomes caused severe membrane-damage such as liposomal lysis and lipid peroxidation. In the presence of ANHIP, isocitrate dehydrogenase and fructose-1,6-diphosphatase were photoinactivated by blue light. However, all of these photosensitized reactions were significantly suppressed by a singlet oxygen (1O2) quencher, azide, but enhanced by a medium containing deuterium oxide. Further, the Fe-S proteins with the prosthetic groups destroyed did not initiate the blue light-induced reactions. In addition, the action spectrum for 1O2 generation from ANHIP was very similar to the visible absorption spectrum of Fe-S centers. The results obtained in this investigation appear consistent with the suggestion that Fe-S centers are involved in photosensitization in cells via a singlet oxygen mechanism.     

MEMBRANE IONIC CURRENT PHOTOMODIFICATION BY ROSE BENGAL AND MENADIONE: ROLE OF SINGLET OXYGEN

Abstract: Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.