Near-infrared Light Emission from Nanomolar-level Singlet Molecular Oxygen Generated with an Ultra low Concentration Mixture of Hypochlorite and Hydrogen Peroxide

Abstract— We report the detection of a weak near-infrared light emission originating from 8 nM singlet molecular oxygen (¹O₂) produced in a mixture of 1 m M hypochlorite (OC1^-) and 8 n M hydrogen peroxide (H₂O₂). The measurements were made with a highly sensitive detection system for ultraweak light emission in the 1.0-1.5 μm wavelength region. The emission intensity exhibited linear dependence for H₂O₂ concentrations in the range of 8-670 n M . The mixture containing a lower concentration (33 μ M ) of OCl^- pseudocontinuously emitted near-infrared light for 5 s. The rate constant for ¹O₂ production obtained from the kinetic analysis agrees with that previously reported. Our results demonstrate the possibility of measuring very low concentrations of ¹O₂ in a OCi-/H₂O² mixture as well as ¹O₂ production in intact living systems.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

EFFECT OF PHOSPHATE AND CHLORIDE ON THE PHOTODYNAMIC INACTIVATION OF YEAST CELLS

Abstract— Yeast cells are inactivated by treatment with hematoporphyrin and light. The inactivation, which is mediated by singlet oxygen (¹O₂), is enhanced by the presence of phosphate and chloride. Neither phosphate nor chloride has any influence on the yield of ¹O₂. Possible mechanisms for the enhancement are briefly discussed.     

NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN PRODUCED BY PHOTOSENSITIZATION WITH PROMAZINE and CHLORPROMAZINE

A sensitive near-infrared detection system has been used to study the steady-state emission of ¹O₂ at 1268 nra produced by promazine (PZ) and chlorpromazine (CPZ) during photo-illumination. Singlet molecular oxygen could be detected in a variety of ordinary and perdeuterated organic solvents, but was not detectable in water or deuterium oxide. The emission was enhanced in the perdeuterated organic solvents and could be eliminated by rigorous degassing or by addition of the singlet oxygen scavenger 2,3-dimethylfuran. Singlet oxygen could not be detected in any of the solvents during irradiation of the sulfoxides of PZ and CPZ. We conclude that in biological systems ¹O₂ production is not a major pathway to phototoxicity for the sulfoxides, while for the parent phenothiazines the formation of ¹O₂ is much more likely to be important in nonpolar environments such as cell membranes than in the aqueous parts of the cell.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.     

RELATION BETWEEN SOME PHOTOBIOLOGICAL PROPERTIES OF FUROCOUMARINS AND THEIR EXTENT OF SINGLET OXYGEN PRODUCTION

Abstract— The production of singlet oxygen (¹O₂) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8-methoxypsoralen (8-MOP) to generate ¹O₂.
The following compounds: 5-methoxypsoralen (5-MOP), psoralen, 4,5',8-trimethylpsoralen (TMP) and 5,8–dimethoxypsoralen (5,8–DMOP), are able to generate ¹O₂ when irradiated with long–wave ultraviolet light. With the photobiologically inactive angelicin no ¹O₂ production has been found. The relative extent of ¹O₂ formation has been determined for the various furocoumarins and has been compared with literature data for the skin photosensitizing effect. The observed relation between experimental data on the one side and the literature data on the other side is discussed.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

SINGLET OXYGEN FORMATION BY SENSITIZATION OF FUROCOUMARINS COMPLEXED WITH, OR BOUND COVALENTLY TO DNA

Abstract— The formation of singlet molecular oxygen (¹O₂) by sensitization of the furocoumarins 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen complexed with DNA was investigated. From the results it is concluded that 5-MOP complexed with native DNA is able to generate ¹O₂, even in a larger extent than 5-MOP free in solution. Also, with 8-MOP and especially with psoralen, ¹O₂ formation by the complexed compound could be observed. The ¹O₂ formation sensitized by covalently bound furocoumarin was demonstrated with psoralen as a model compound. 4',5'-Dihydropsoralen, a model compound for the UVA light absorbing 4',5'monoadducts of furocoumarins to DNA, is also able to generate ¹O₂.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (¹O₂) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of ¹O₂ from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in ¹O₂ generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0₂ quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by ¹O₂ which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of ¹O₂ was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of ¹O₂.     

Photochemical Reactions Involved in the Phototoxicity of the Anticonvulsant and Antidepressant Drug Lamotrigine (Lamictal®)

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal^®, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (¹O₂) with a quantum yield of 0.22 in CH₂Cl₂, 0.11 in MeCN and 0.01 in D₂O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak ¹O₂ quencher ( k _q = 3.2 × 10⁵  m ⁻¹ s⁻¹ in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched ¹O₂ very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated ¹O₂ twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of ¹O₂ prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.     

CHEMILUMINESCENCE FROM AUTOXIDIZING 6-HYDROXYDOPAMINE: THE INVOLVEMENT OF ACTIVATED FORMS OF OXYGEN

Abstract— The autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (20 μ M ) gave rise to a chemiluminescence which was greatly stimulated by FeSO₄ (20 μ M ) or by hydrogen peroxide addition (20 μ M to 2 m M ). The luminescence of both 6-hydroxydopamine alone or 6-hydroxydopamine plus hydrogen peroxide was strongly inhibited by catalase and by superoxide dismutase (both at 10 μg/m/); bovine serum albumin at 10 μg/m/ had no inhibitory effect. The luminescence was also strongly inhibited by several potent hydroxyl radical trapping agents and also by low concentrations of the ¹O₂ quencher DABCO (l,4-diazabicyclo-2.2.2.-octane). Chemiluminescence was greatly enhanced in D₂O, a solvent in which ¹O₂ has a prolonged lifetime. These data demonstrate the involvement of hydrogen peroxide, the superoxide radical and the hydroxyl radical in the chemiluminescence. The data are also consistent with some role for ¹O₂.     

TEMPORAL and SPECTRAL SEPARATION OF SINGLET OXYGEN LUMINESCENCE FROM NEAR INFRARED EMITTING PHOTOSENSITIZERS

Abstract— The luminescence emission of singlet molecular oxygen (¹O₂) generated by bacteriopheophytin a, a near-infrared-emitting photosensitizer, was measured using a new high-sensitivity spectrometer system for time- and spectral-resolved near-infrared detection. The instrument uses a low energy pulsed nitrogen laser (40 μJ per pulse) to excite the photosensitizer optically and is capable of a time resolution of 40 ns per data point and an instrument response function of 350 ns FWHM (full width at half maximum). The use of a low-energy (and relatively low cost) source provides sufficient system sensitivity to measure time-resolved spectra in the near infrared with high spectral and temporal resolution. The simultaneous detection, with high accuracy and repeatability, of both the temporal and spectral dependence of the photoprocesses of ¹O₂ generation, especially with near-infrared-emitting photosensitizers, may further stimulate the current intensive investigations concerning the activity of ¹O₂ to biomolecules.     

EFFECT OF SODIUM AZIDE ON PHOTODYNAMIC INDUCTION OF GENETIC CHANGES IN YEAST

Abstract— In view of the increasing attention to ¹O₂ (¹Δ_g) participation in the photodynamic action, different types of genetic changes in Saccharomyces cerevisiae by acridine orange sensitization were compared with respect to the response to N₃^-, a well known quencher of ¹O₂. The induction of mitotic crossing over with respect to ade 2 locus and mitotic gene conversion at trp 5 locus were suppressed by the addition of N₃^- suggesting the involvement of ¹O₂ as a major intermediate. However, the induction of reverse mutation at ilv 1 was only slightly suppressed. These results may indicate that there are two types of photodynamic DNA damage; one is produced via ¹O₂ and the other via non-¹O₂ reaction pathway which lead to mitotic gene conversion and mitotic crossing over, and to mutation, respectively.     

QUANTUM YIELD OF SINGLET OXYGEN PRODUCTION BY XANTHENE DERIVATIVES

The singlet oxygen quantum yield (φ₁o₂) of 11 purified fluorescein derivatives was determined by reaction with singlet oxygen acceptors in aqueous and ethanolic solutions; in both solvents φ₁o₂ was enhanced with increasing halogenation. Tryptophan and 2,2,6,6-tetramethylpiperidone were found to be unadapted for the determination of φ₁o₂, in our systems; however, the use of 9.10-dipropionic anthracene acid andp-nitrosodimethylaniline in conjunction with imidazole derivatives was suitable for ¹O₂ detection in water. Both methods lead to results in excellent agreement. As in ethanol. φ₁o₂, was equal to the triplet state quantum yield (φ_T), the comparison between the two solvents showed that φ_T in water was greater than in ethanol. The comparison between our values obtained with polychromatic light with published data obtained with monochromatic light suggests that the triplet quantum yield of fluorescein derivatives is wavelength independent.     

A NEW METHOD FOR THE DETECTION OF SINGLET OXYGEN IN AQUEOUS SOLUTIONS

Abstract— In experiments on the interception of reactive intermediates of strongly oxidizing character in dye (S) sensitized photooxidations using p -nitrosodimethylaniline (RNO) as a selective scavenger, it has been observed that some substrates (A) or ¹O₂ acceptors (like imidazole derivatives) induce the bleaching of RNO as followed spectrophotometrically at 440 nm. Since singlet oxygen (¹O₂) does not react chemically with RNO, this bleaching is a consequence of ¹O₂ capture by the imidazole ring which results in the formation of a trans-annular peroxide intermediate [¹O₂] capable of inducing the bleaching of RNO (-RNO). In the absence of RNO, [¹O₂] decomposes or rearranges into the final oxygenation product ¹O₂: ¹Δ_g Thus, the system imidazole plus RNO can be used as a sensitive and selective test for the presence of ¹O₂ in aqueous solutions. The method can also be applied in the presence of sensitizing dyes which, under visible irradiation, can partially bleach RNO even in the absence of imidazole derivatives. In such a case, the bleaching of RNO is strongly increased by the presence of imidazoles with a characteristic dependence on their concentration. The separation of the product of RNO bleaching by thin layer chromatography can serve as additional proof of the presence of ¹O₂ in the system. The imidazole plus RNO method has been applied to a number of sensitizing and non-sensitizing dyes.     

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (¹O₂) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub-mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe-S centers of nonheme iron proteins. Disruption of the Fe-S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of ¹O₂ generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe-S centers to the generation of ¹O₂ from SMP, we suggest that the Fe-S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of ¹O₂ is also discussed.     

RED EMISSION FROM CHLOROPLASTS ELICITED BY ENZYME-GENERATED TRIPLET ACETONE AND TRIPLET INDOLE-3-ALDEHYDE

—Enzyme-generated triplet acetone and triplet indole-3-aldehyde transfer energy very efficiently to chloroplasts, as indicated by the intensity of the sensitized red emission that is observed. The intermediacy of excited species of oxygen (¹O₂, O₂⁻, HO) has been excluded. Our results open the way for investigating energy transfer in architecturally organized systems in the absence of light.