COOPERATION OF QUERCETIN WITH ASCORBATE IN THE PROTECTION OF PHOTOSENSITIZED LYSIS OF HUMAN ERYTHROCYTES IN THE PRESENCE OF HEMATOPORPHYRIN

Abstract— Quercetin(20–100 μ M ) suppressed photohemolysis sensitized by hematoporphyrin, while ascorbate(10–100 μ M ) stimulated it. However, in the presence of 40 μ M quercetin, ascorbate promoted the suppression. The suppression by quercetin was due to scavenging of both singlet oxygen generated by a photosensitized reaction and radicals generated by decomposition of lipid peroxides formed by a singlet oxygen-dependent reaction. In scavenging, quercetin was oxidized and the oxidation was suppressed by ascorbate. Ascorbate was oxidized by illumination in the presence of quercetin. It is suggested that the cooperation of quercetin with ascorbate in photohemolysis is due to reduction of oxidized quercetin by ascorbate regenerating the flavonol.     

THE ROLE OF SINGLET OXYGEN IN THE PHOTOHEMOLYSIS OF RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINE SULFONATES

Abstract The post-illumination photohemolysis of human red blood cells was used as a model system for studying membrane damage sensitized by various phthalocyanine sulfonates. With a constant fluence and photolysis time at 670 ± 10 nm and with a fixed optical density of the sensitizer, the percentage of hemolysis was measured as a function of time after illumination; and from the usual sigmoidal shaped curves, the times for 50% hemolysis were determined. The most effective central cation was Al, followed by Zn; the metal-free compound and those containing the paramagnetic ions Cu, Fe and Co were inactive. The sensitizer bound to the red blood cells was found to be effective for photohemolysis. Studies of the Al compound in 98.0% deuterated media (as measured by NMR) showed a definite deuterium isotope effect, suggesting the participation of singlet oxygen. This result was supported by the large protective effects of 3.3 raM and 13.3 mM tryptophan present during photolysis. Tryptophan reacts rapidly with singlet oxygen and with OH radicals. The much smaller effects observed with the same concentrations of mannitol and glycerol, OH radical scavengers which do not react with singlet oxygen, are consistent with the predominant role of singlet oxygen in photohemolysis sensitized by Al phthalocyanine sulfonate.     

MAGNETIC FIELD EFFECTS ON THE PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES BY KETOPROFEN AND PROTOPORPHYRIN IX

Abstract— Application of a static external magnetic field (3350 G) during UV-irradiation (>300 nm) reduced the time for 50% photohemolysis of human erythrocytes by the phototoxic drug ketoprofen (3-benzoyl-α-methylbenzoacetic acid) from 96 min to 78 min. This observation can be attributed to a magnetic field induced decrease in the rate of intersystem crossing (k_ISC) of the geminate triplet radical pair generated by the reduction of ketoprofen in its triplet excited state by erythrocyte membrane constituents, probably lipids. The decrease in k_JSC results in an increase in the concentration and/or lifetime of free radicals that escape from the triplet radical pair. Thus the critical radical concentration needed to cause membrane damage and cell lysis is reached sooner in the presence of the magnetic field. In contrast, the photohemolysis induced by the photodynamic agent protoporphyrin IX was not affected by the magnetic field. Protoporphyrin IX photohemolysis, which is initiated by singlet oxygen, does not involve the initial generation of a triplet radical pair and so is not influenced by the magnetic field. The enhancement of ketoprofen-induced photohemolysis by an externally applied magnetic field is the first example of a magnetic field effect on a toxicological process involving free radicals.     

SINGLET OXYGEN INVOLVEMENT IN PHOTOHEMOLYSIS SENSITIZED BY MEROCYANINE-540 and ROSE BENGAL

Merocyanine-540 is currently in use in experimental protocols for the treatment of leukemia and neuroblastomas. The mechanism of cellular phototoxicity of this membrane binding sensitizer is unexplored. We have used sodium azide and deuterium oxide to examine the role of singlet oxygen in photomodification of cell membranes in a photohemolysis assay. The effects of these agents on the photomodification process were separated from the effects on the ion fluxes leading to lysis (lytic phase). Azide significantly inhibited photohemolysis sensitized by merocyanine-540 or rose bengal. The inhibition was equal for both sensitizers. Azide had no effect on the lytic phase. Deuterium oxide significantly potentiated lysis with both sensitizers to approximately the same degree. There was little effect of deuterium oxide when added after illumination. The results indicate that singlet oxygen plays a significant role in membrane photomodification sensitized by both rose bengal and merocyanine-540.     

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

PHOTOMODIFICATION OF HUMAN ERYTHROCYTES: EXTERNAL HEAVY ATOM EFFECT, SELECTIVE PERMEABILITY AND PROPERTIES OF THE ANION PERMEATION PATHWAY

Abstract— Anion permeability was studied in normal and photomodified erythrocytes and the role of anion species in photomodification and singlet oxygen generation was evaluated. Relative permeability to halides and nitrate was assessed using lysis rates in valinomycin-treated cells to make anion permeation rate-limiting. In non-photomodified cells the normal high temperature dependence (E:, = 67 kJ/mol; 16 kcalhol), selectivity sequence (NO, > I > Br > F > Cl) and sensitivity to block by the stilbene derivative HzDIDS were confirmed. In cells photomodified by illumination in the presence of phloxine B the anion permeability was severely perturbed. The temperature dependence was strongly reduced, the permeability to ions other than fluoride increased, leading to a reversal of the F/CI selectivity, and the capacity to be blocked by HIDIDS was lost. Pretreatment with NEM and posttreatment with DTE did not affect rates of photohemolysis. The amount of photomodification varied with species of anion present in the reaction medium, being greatest for F and smallest for I. Rates of singlet oxygen generation in aqueous solution measured by the RNO bleaching method followed the same anion sequence, suggesting a strong external heavy atom effect.     

HYPERICIN PHOTOSENSITIZATION IN AQUEOUS MODEL SYSTEMS

Abstract Photosensitization of lysozyme, liposomes, ghosts and intact red blood cells (RBC) was investigated for aqueous hypericin. The effects of azide ion, 1,4-diazabicyclo(2.2.2)octane, and superoxide dismutase on photosensitized inactivation of lysozyme in 0.5% Triton X-100 indicate that singlet oxygen is the major inactivating intermediate with a contribution from superoxide. The singlet oxygen quantum yield (Φ_Δ) scaled to methylene blue is 0.49 ± 0.06 at monochromatic wavelengths from 514 to 600 nm. Relative values of Φ_Δ based on lysozyme inactivation for different vehicles are: 0.5% Triton X-100 (1.13), human serum albumin (0.65), Cremophor-EL(0.76), Cremophor-RH40 (0.98), egg phosphatidylcholine (EPC) liposomes (0.04), hydrogenated soy phosphatidylcholine (HSPC) liposomes (<0.01). Hypericin photosensitized lipid peroxidation of EPC liposomes and RBC ghosts. Extensive cross-linking of ghost membrane proteins occurred during the initial stages of lipid peroxidation. Prompt photohemolysis was used as the assay of RBC membrane damage. The photohemolysis curves are modeled with multihit target theory based on the "hit number" (n) and the target cross section (v). The values of v and the conventional "1/t₅₀" parameter are equivalent determinants of the photohemolysis rate. The photohemolysis curves are in good agreement with n = 15 for incubation in phosphate-buffered saline at different hypericin concentrations and with additives. The measurements for other vehicles led to n = 19 for Cremophor-EL and n = 3 for EPC and HSPC liposomes, indicating that the kinetics of photohemolysis depend on the conditions of incubation.     

PRIMARY MECHANISMS OF ERYTHROCYTE PHOTOLYSIS INDUCED BY BIOLOGICAL SENSITIZERS AND PHOTOTOXIC DRUGS

Abstract— The elucidation of the molecular mechanism of photosensitized hemolysis of red blood cells may give important clues to the primary events underlying the phototoxic reactions observed in pathological conditions such as porphyria and induced by photosensitizing drugs. Sensitizers effective in photo-hemolysis are porphyrins, the tryptophan metabolite kynurenic acid, and phototoxic drugs such as chlor-promazine and demethylchlortetracycline. Utilizing the singlet oxygen quenchers. jS-carotene and histi-dine and the large deuterium effect on the lifetime of singlet oxygen previously described by us, good evidence of the participation of this excited molecular species in the photohemolysis in the presence of kynurenic acid was obtained. Chlorpromazine and demethylchlortetracycline clearly act by a non-singlet oxygen pathway. The situation observed with haematoporphyrin is less clear and may represent a mixed Type I-Type II mechanism.     

PHOTODEGRADATION AND in vitro PHOTOTOXICITY OF FENOFIBRATE, A PHOTOSENSITIZING ANTI-HYPEIUIPOPROTEINEMIC DRUG

The phototoxic anti-hyperlipoproteinemic drug fenofibrate was found to be photolabile under aerobic and anaerobic conditions. Irradiation under argon of a methanol solution of this drug produced the photoproducts isopropyl 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate, 1,2- bis (4-chlorophenyl)-1,2- bis (4-[isopro-poxycarbonylisopropoxy]phenyl)ethane-1,2-diol and 4-(4-chlorobenzoyl)phenol, while under oxygen the photoproducts were 4-chloroperbenzoic acid, methyl 4-chlorobenzoate, 4-chlorobenzoic acid and singlet oxygen, as evidenced by trapping with 2,5-dimethylfuran. These results can be rationalized through hydrogen abstraction by excited fenofibrate, to afford a free radical as key intermediate. Biologically active antioxidants such as glutathione and cysteine efficiently reduced 4-chloroperbenzoic acid to 4-chlorobenzoic acid. The involvement of an electron transfer mechanism is suggested by detection (UV-vis spectrophotometry) of the radical cation TMP⁺ during the oxidation of tetramethylphenylenediamine (TMP) with 4-chloroperbenzoic acid. Fenofibrate was phototoxic in vitro when examined by the photohemolysis test, both under oxygen and argon atmosphere, although the photohemolysis rate was markedly lower under anaerobic conditions. The photoproducts 4-(1-[4-chlorophenyl]-1,2-dihy-droxy)ethylphenoxyisobutyrate and 4-chloroperbenzoic acid induced hemolysis in the dark however, this effect was quantitatively less important than photohemolysis by fenofibrate. On the other hand, fenofibrate photosensitized peroxidation of linoleic acid, monitored by the UV detection of dienic hydroperoxides. Based on the inhibition of this process upon addition of butylated hydroxyanisole, a radical chain (type I) mechanism appears to operate. In summary, fenofibrate is phototoxic in vitro . This behavior can be explained through the involvement of free radicals, singlet oxygen and stable photoproducts.     

PHOTOOXIDATION OF CELL MEMBRANES USING EOSIN DERIVATIVES THAT LOCATE IN LIPID OR PROTEIN TO STUDY THE ROLE OF DIFFUSIBLE INTERMEDIATES

Eosin derivatives that bind primarily to lipid or protein sites in erythrocyte membranes were studied in solution and as sensitizers of erythrocyte membranes. In 50% ethanol-water mixtures eosin maleimide (EYMA) and 5-N-hexadecanoyl amino eosin (E16) had nearly identical absorption spectra. Higher ethanol concentrations did not change peak absorbances. In the presence of neutral detergent both sensitizers had equivalent absorbance at all ethanol concentrations. In water, EYMA was more effective than E16 at bleaching RNO, probably because of E16 aggregation into micelles, while in ethanol-water mixtures E16 was slightly more effective at bleaching DPBF, indicating equivalent singlet oxygen generation when the sensitizers are in monomeric form. In water with neutral detergent, azide in the 20 microM range inhibited the majority of RNO bleaching with both sensitizers; in 50% ethanol-water mixtures azide at 1 mM showed a 50% inhibition of DPBF bleaching with both sensitizers. Iodide in the 30 mM range reduced DPBF bleaching by 50% in 50% ethanol-water mixtures. When matched for amount loaded in erythrocyte membranes these sensitizers were about equally effective at sensitizing induction of cation permeability, assayed as rate of delayed photohemolysis, while E16 was slightly more effective at sensitizing loss of cholinesterase (AchE) activity. The relation of lysis rate to load was somewhat steeper for E16 than EYMA. For both sensitizers lysis rate increased at about the 1.5 power of light dose. Deoxygenation of the reaction media with argon totally blocked detectable photomodification. Ghost membranes made from sensitizer-treated cells were effective generators of singlet oxygen, assayed by RNO bleaching. However, when mixtures of EYMA-treated and untreated cells were illuminated together, only the EYMA-treated cells showed evidence of photomodification. Azide at 5 mM slowed the initial rate of AchE loss by about 75% with E16 and EYMA. Azide partially slowed photohemolysis. Azide decreased RNO bleaching by sensitizer-treated ghosts as it did in water with detergent micelles. A deuterium oxide solvent increased photohemolysis rate with E16 by 41%, but did not increase photohemolysis rate with EYMA. Deuterium oxide had a positive, but statistically insignificant effect on loss of AchE with both sensitizers. Deuterium oxide following illumination slowed lysis sensitized by both sensitizers more than 50%. Iodide exerted a modest inhibition of photohemolysis and loss of AchE sensitized by E16, but had virtually no influence on sensitization by EYMA. The results in solution indicate that EYMA and E16 have nearly identical photochemical properties when in monome     

PHOTOSENSITIZATION BY 2-CHLORO-3, 11-TRIDECADIENE-5, 7, 9-TRIYN-1-OL: DAMAGE TO ERYTHROCYTE MEMBRANES, Escherichia coli, AND DNA

The natural product 2-chloro-3,11-tridecadiene-5,7,9-triyn-1-ol (1) photosensitized the inactivation of Escherichia coli in the presence of near-ultraviolet light (320-400 nm; NUV) under both aerobic and anaerobic conditions. A series of E. coli strains differing in DNA repair capabilities and catalase proficiency exhibited indistinguishable inactivation kinetics following treatment with the chemical plus NUV. The presence of carotenoids did afford some protection to E. coli against inactivation under aerobic conditions, consistent with the involvement of singlet oxygen. The photosensitized hemolysis of human erythrocytes occurred more rapidly in the absence than in the presence of oxygen. Aerobically, the onset of hemolysis was partially inhibited by NaN3 and by 2,6-di-t-butyl-4-methylphenol (BHT) but not by superoxide dismutase (SOD). The aerobic lipid peroxidation observed in the membranes of erythrocyte ghosts was completely inhibited by BHT, and partially by NaN3, but not by SOD. These results suggest that either lipid peroxidation of the membrane is not the main cause of photohemolysis or that BHT has insufficient access to intact erythrocyte lipids to protect them. Aerobically, crosslinking of membrane proteins was also observed; it was not affected by SOD, but was partially inhibited by BHT and NaN3. The anaerobic photosensitized hemolysis of erythrocytes was more rapid; a radical mechanism was suggested since BHT inhibited the hemolysis to a greater extent than under aerobic conditions. Neither lipid peroxidation nor protein crosslinking was observed under conditions believed to be anaerobic. A light-dependent electron transfer to cytochrome c was obtained under argon but not under oxygen. Although induced mutations were not observed in the experiments with E. coli, 1 was capable of damaging both supercoiled pBR322 and Haemophilus influenzae transforming DNA in a manner that seemed to be equivalent under aerobic and anaerobic conditions. In conclusion, 1 can behave as typical photodynamic molecule under aerobic conditions but, in contrast to most photodynamic molecules, it is also phototoxic under anaerobic conditions. The extent to which the radical reactions detected under anaerobic reactions compete with the photodynamic processes when oxygen is present is not known.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

THE PHOTOCHEMICAL PROPERTIES OF FLUOROALUMINUM PHTHALOCYANINE

Abstract Fluoride is known to inhibit the photodynamic activity of aluminum phthalocyanine in a variety of biological systems. In order to gain insight into this phenomenon, the effect of fluoride on the photophysical properties of free and albumin-bound chloroaluminum phthalocyanine sulfonate (AlPcS_n.) were studied. The association constant of NaF with AlPcS_n, in aqueous solution was measured as 500 ± 20 M^?1. This binding affects the photophysical properties of the dye: the absorption bands in the visible range are blue-shifted by 6–8 nm, and this effect is mirrored in the fluorescence emission spectrum. Human serum albumin significantly quenched the dye fluorescence independent of the presence of fluoride ion. The transient absorption spectrum of the excited dye triplet is unchanged by NaF, but the quantum yield for its generation is increased by 50%, with no decrease in its lifetime. Formation of fluoroaluminum phthalocyanine complexes was also observed in tetrabutylammonium fluoride-assisted solutions in wet acetonitrile. The fluoro-AlPcS_n, complex is a better photosensitizer for generation of singlet oxygen than the original dye-hydroxyl ion complex, as confirmed using the imidazole-N,N-dimethyl-4-nitrosoaniline method. On the other band, the fluoro-AlPcS_n. complex exhibits an intense inhibitory effect on photohemolysis of red blood cells (RBC) even after the cells are washed to remove free dye and fluoride prior to irradiation, indicating that once the dye is attached to the cellular site, the fluoride ligand is no longer prone to displacement (by hydroxyl ion, for example). Nonetheless, it is clear from the spectroscopic data that the new fluoro complex is an efficient sensitizer for photo-oxidation. Therefore, the reduced photodynamic action of the fluoro-AlPcS_n. complex on RBC (Ben-Hur et al., Photochem. Photobiol. 58 , 351–355, 1993) may result from a lowering of the efficiency of interaction of the fluorodye complex with sensitive cell target moieties.     

Molecular mechanism of naproxen photosensitization in red blood cells

Red blood cell lysis photosensitized by naproxen was investigated. The photohemolysis rate was enhanced by deuterium oxide and inhibited by butylated hydroxyanisole, reduced glutathione, sodium azide and superoxide dismutase. Photohemolysis was also observed under anaerobic conditions. In the absence of red cells the irradiation of deaerated solutions underwent a decarboxylation process via intermediate radicals, while under aerobic conditions photo-oxidation leading to the photoproduct 6-methoxy-2-acetonaphthone occurred. A molecular mechanism involving free radicals and singlet oxygen as important intermediates and consistent with the overall results is proposed.     

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (³H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium-neon (HeNe) laser and TBO was investigated by using deuterium oxide (D₂O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L-tryptophan. There were 9.0 log₁₀ and 2 log₁₀ reductions in the presence of D₂O and H₂O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm²), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm²) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre-gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.     

Photodegradation pathways and the in vitro phototoxicity of pyrazinamide, a phototoxic antitubercular drug

The phototoxic antitubercular drug pyrazinamide (1) is photolabile under irradiation with UV-A light as well as with a N2 laser (at 337 nm) in aerobic conditions. Irradiation in methanolic and in aqueous solutions of 1 produces four and three photoproducts, respectively. Their formation involves primary alpha-cleavage between the excited carbonyl of the amido group and the aromatic ring followed by hydrogen abstraction and dimerization. Pyrazinamide was able to cause photohemolysis in human erythrocytes and peroxidation of linoleic acid. Inhibition of both processes on addition of reduced glutathione (GSH) or ascorbic acid suggests the involvement of radicals. The absence of inhibition of the photohemolysis and lipid peroxidation processes in the presence of sodium azide (NaN3), or irradiation under argon, and the absence of singlet oxygen during the photolysis confirmed with 2,5-dimethylfuran rules out the possibility of participation of 1O2 in this process. Glutathione depletion was also observed. A radical intermediate was evidenced by thiobarbituric acid that was used as a radical probe, as well as by the dimerization of cysteine. No photohemolysis was detected in presence of the isolated photoproduct. We have also determined the relative efficiencies for the formation of single strand breaks after the irradiation of pBR322 DNA and pyrazinamide, which was also reduced in the presence of GSH.     

QUENCHING OF SINGLET OXYGEN BY HUMAN RED CELL GHOSTS

Time resolved measurements of singlet oxygen phosphorescence at 1270 nm were made from unsealed red cell ghosts, labeled with 5-(N-hexadecanoyl)aminoeosin and suspended in deuterium oxide buffer. The singlet oxygen emission lifetime was long, 23 +/- 1 microseconds. The lifetime of the singlet oxygen phosphorescence from intact unsealed ghosts was not a measure of the singlet oxygen lifetime within the red cell ghost membrane, however. The prolonged singlet oxygen emission was due to singlet oxygen escaping from the thin membrane into the buffer, since the emission lifetime was significantly shortened by adding azide ion or water to the deuterium oxide buffer. The lifetime of singlet oxygen within the red cell ghosts membrane was estimated by dispersing the ghosts with detergent and then measuring the singlet oxygen lifetime in deuterium oxide buffers containing various dilutions of the dispersed ghosts. Apparent singlet-oxygen quenching constants were measured using four different photosensitizing dyes and two different detergents. The apparent quenching constant was independent of the dye used, but varied significantly with different detergents. Extrapolation of this data to "100%" ghost concentration gave a singlet oxygen lifetime from 24 and 130 ns. A ghost concentration of "100%" was defined as that concentration of red cell ghost molecules which would be contained within a red cell ghost membrane pellet containing no buffer solutions. Most of the singlet oxygen quenching was due to proteins. Lipids extracted from red cell ghosts accounted for only 2-7% of the total singlet oxygen quenching.     

Photoinactivation of Virus Infectivity by Hypocrellin A

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.     

SELECTIVE PHOTOOXIDATION OF THIOLS SENSITIZED BY AQUEOUS SUSPENSIONS OF CADMIUM SULFIDE

-Finely powdered cadmium sulfide in aqueous, air-saturated, phosphate buffered suspension sensitizes the photooxidation of cysteine to cystine with good efficiency; several additional thiols and inorganic sulfides are also photooxidized. The other amino acids (histidine, methionine, tryptophan, tyrosine) known to be rapidly photooxidized with typical organic photosensitizers are photooxidized only very slowly. The quantum yield of oxygen uptake during cysteine photooxidation is pH dependent with a maximum (0.021) at pH 9.5; the yield is not increased in D₂O and is not decreased appreciably by sodium azide, suggesting that singlet oxygen is not involved in the photooxidation process. The slow rate of photooxidation of histidine, which is known to react efficiently with singlet oxygen, also suggests that little if any singlet oxygen is produced by illuminated cadmium sulfide. Superoxide dismutase inhibits the yield of cysteine photooxidation to a maximum of approximately 50%, suggesting the partial involvement of superoxide in the reaction mechanism. The quantum yields of the photooxidation of cysteine, ethylenediaminetetraacetate and inorganic sulfides decrease as the wavelength of the exciting light is increased. Yeast alcohol dehydrogenase, a sulfhydryl enzyme, is inactivated by photodynamic treatment with cadmium sulfide; lysozyme, which has no free sulfhydryl groups, is not.     

INHIBITORY EFFECTS OF ETHANOL ON THE PHOTODYNAMIC CELL INACTIVATION AND PETITE INDUCTION BY EUFLAVINE IN YEAST, Saccharomyces cerevisiae

Abstract— Effects of active oxygen scavengers on cell inactivation and petite induction of yeast by the photodynamic action of euflavine were examined. Histidine, sodium azide, 1,3-diphenyl-isobenzofuran and p-carotene, which are singlet oxygen scavengers, inhibited photodynamic cell killing. Histidine and sodium azide inhibited petite induction, too. These results suggested that photobiological effects of euflavine are induced via singlet oxygen-mediated Type II reaction process. In this work, however, we found that ethanol, which is reported to be a hydroxyl radical scavenger, notably inhibited photodynamic cell inactivation and petite induction by euflavine. Inhibition of petite induction was increased with increasing concentration of ethanol. Decrease of absorbance of euflavine by irradiation was also inhibited by the addition of ethanol.
These results suggested that ethanol possibly acts as a singlet oxygen scavenger.