HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (¹O₂) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D₂O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that ¹O₂ was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D₂O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Ų for the inactivation of subtilisin BPN' by ¹O₂ and 20 Ų for the oxidation of tryptophan in BSA.     

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.     

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.     

MECHANISMS OF THE PHOTOSENSITIZED OXIDATION OF TYRAMINE

Abstract— …According to the criteria of enhancement in D₂O and inhibition by sodium azide, the oxidation of tyramine photosensitized by methylene blue is largely a singlet oxygen or Type II process. Its quantum yield approximates 0.3 in D₂O at pH 10. There is a less efficient reaction not quenched by azide, which is assigned to a dye-substrate or Type I process. It gives rise to products with distinct bands at 320 and 285nm. Products of the Type I reaction are further oxidized by singlet oxygen and thereby compete with tyramine for this reagent. Kinetic parameters were estimated by computer simulation of the dependence of quantum yield on extent of reaction. The rate constant for reaction of O₂ (¹Δ_g) with tyramine was estimated to be 2.8 × 10⁸ M ^-1 s ^-1± 20% at pH 10. The reaction was also sensitized by hypericin in what appears to be a Type II process.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

THE LIPID SOLUBILITY OF PORPHYRINS MODULATES THEIR PHOTOTOXICITY IN MEMBRANE MODELS

The photosensitizing activities of hematoporphyrin, Cu-hematoporphyrin, protoporphyrin. Zn-protoporphyrin, deuteroporphyrin and uroporphyrin are studied. The relative yields of ¹O₂ are measured in buffer (pH 8.0) and compared to the yields of Type II photodamages induced on cholesterol embedded in egg lecithin liposomes. Results show that the solubilization of the sensitizer in the lipid bilayer is a prerequisite for its photosensitizing activity at the membrane level. In this context, the partition coefficient represents an important parameter.     

SINGLET OXYGEN: A MAJOR REACTIVE SPECIES IN THE FUROCOUMARIN PHOTOSENSITIZED INACTIVATION OF E. COLI RIBOSOMES

Abstract— The skin photosensitizing furocoumarins, 8-methoxypsoralen (MOP) and 4,5',8-trimethylpsoralen (TMP), inactivate E. coli ribosomes in vitro , on UV irradiation at 313 nm. Purging the solutions with N₂ protects the ribosomes considerably against photoinactivation (75% with MOP and 80% with TMP). In air, the ribosome photoinactivation is mainly due to singlet oxygen (¹O₂), since the presence of NaN₃ and other ¹O₂ quenchers protects the system and the inactivation is enhanced in D₂O. Although ¹O₂ dominates as the inactivating species, the possibility of additional (∼15%) minor mechanisms involving free radicals exists. However, O^-₂ does not appear to be the damaging species, since superoxide dismutase does not provide any protection.
Photosensitization of the partially purified enzyme, phe-tRNA-synthetase with MOP or TMP shows inactivation and protection curves similar to those seen with the ribosomes. On the other hand, unfrac-tionated tRNA^phc is not photosensitized under similar conditions, although it shows self-photosensitization. It is likely that in the furocoumarin-sensitized ribosomes, the primary events of photoinactivation are associated with the proteins.     

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.     

EFFECTS OF QUENCHING AGENTS ON THE PHOTODYNAMICALLY-INDUCED CHEMOTACTIC RESPONSE OF THE COLORLESS FLAGELLATE Polytomella magna

Abstract In the presence of the photosensitizer riboflavin at high fluence rates a photoproduct, most probably H₂O₂, is formed which causes negative phototaxis in the colorless flagellate Polytomella magna . The aim of this study was to find out whether H₂O₂ is produced in a type I or II reaction. As has been shown, ¹O₂ quenchers either do not influence the photodynamic action of riboflavin (furfuryl ethanol, DPBF, l -histidine, crocetin) or show slight quenching effects only at very high concentrations ≧ 10⁻² M (DABCO, DMF, imidazole). D₂O is toxic to P. magna even in 1:1 and 1:2 mixtures with H₂O. On the other hand, the quenching effect of 1,4-benzoquinone, highly indicative for the type I pathway, is more than two orders of magnitude stronger than the one of the above mentioned ¹O₂ quenchers. The results suggest that H₂O₂ is produced in a type I reaction. Superoxide does not seem to be involved since superoxide dismutase does not diminish the photodynamic effect of riboflavin.     

SELECTIVE TYPE I PHOTOOXIDATIONS IN MIXTURES OF PORPHYRINS AND ELECTROPHILIC NITROIMIDAZOLES

Abstract— Electrophilic compounds metronidazole (METRO) and misonidazole (MISO), considerably enhance the photooxidation quantum yield of Type I photodynamic substrates such as Trp, Tyr and Cys. For the latter, the quantum yield of photooxidation which can be much greater than one, suggests radical chain reactions. On the other hand, METRO and MISO inhibit ¹O₂ formation because they react at diffusion controlled rate (∼10⁹ M⁻¹ S⁻¹) with porphyrin triplets forming, porphyrin radical cations. As a result, the porphyrins are still able to photosensitize the destruction of Type I substrates even in the absence of O₂. These results are discussed with respect to the possibility of increasing the sensitivity of hypoxic cells to porphyrin photosensitization.     

KINETIC STUDIES OF SELF-SENSITIZED PHOTOOXYGENATION IN H2O AND D2O OF A WATER-SOLUBLE RUBRENE DERIVATIVE

Abstract A continuous argon laser has been used to study the self-sensitized photooxidation of potassium rubrene-2,3,8,9-tetracarboxylate in oxygen-saturated H₂O and D₂O. An analysis of the data obtained in concentrated solutions leads to an unexpected high value of the ratio of ¹O₂ lifetimes in D₂O and H₂O, ^T d ₂o/^T h ₂o =17 ± 1. Results obtained in diluted aqueous solutions are interpreted in terms of a re-encounter of ¹O₂ and ground state substrate molecules generated in the same triplet—triplet annihilation act.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

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.     

A PRELIMINARY INVESTIGATION OF THE USE OF LIMONENE FOR DETECTING SINGLET OXYGEN AS A COMPONENT OF POLLUTED AIR

Abstract— Exploratory experiments using simulated conditions indicate that the terpene, (+)-limonene can serve to detect O₂(¹Δ_g) in polluted atmospheres. ¹O₂ attack on limonene results in the formation of specific oxidation products in a specific distribution; quantitative identification of these products is a highly specific test for ¹O₂.     

INACTIVATION OF NORMAL AND MUTANT NEUROSPORA CRASSA CONIDIA BY VISIBLE LIGHT AND NEAR-UV: ROLE OF 1O2, CAROTENOID COMPOSITION AND SENSITIZER LOCATION

Abstract— Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-UV light has been investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV is virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, ¹O₂ is not involved in any substantial way in the formation of lethal lesions. The finding that carotenoid deficient strains are similar to wild-type strains in sensitivity to near-UV inactivation is consistent with ¹O₂ not being involved.
Photodynamic inactivation of conidia by visible light occurs in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains are more sensitive to such inactivation only when MB and TB are used. These results support the contention that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids.
A newly isolated, lemon–yellow mutant, mapping to the al -1 locus, exhibits sensitivities to photodynamic inactivation similar to other pure-white mutants at the same locus. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation (seven to nine double bonds) of the two colored carotenoids, zeta–carotene and neurosporene, produced by the mutant.     

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.     

USE OF TRIS (2,2'-BIPYRIDINE) RUTHENIUM(II) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

Abstract— A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H₂O and D₂O. The technique uses tris (2,2'-bipyridine) ruthenium(II) dication (Ru(bipy)₃₂₊) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of Ru(bipy)₃2+* can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (±0.3) x 10⁶ mol^-1 dm³ s^-1 has been determined for the reaction of ¹O₂ with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (±0.4) x 10⁶ and 3.1 (±2.0) x 10⁵ mol^-1 dm³ s^-1; for swine skin gelatin this value is 3.9 (±2.4) × 10⁵ mol^-1 dm³ s^-1. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and ¹O₂.     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

PHOTOOXYGENATION OF MESO-TETRAPHENYLPORPHYRIN METAL COMPLEXES

Abstract Cadmium(II), zinc(II), magnesium(II), and bis-[thallium(I)] complexes of meso -tetraphenyl-porphyrin undergo unsensitized photooxygenation to yield a bilitriene derivative as the final product. The reaction has previously been shown to involve singlet oxygen (Matsuura et al. , 1980); on the basis of mass spectrometry of products from ¹⁶O₂ and ¹⁸O₂ experiments, and characterization of the initial photoproduct, the reaction is shown to proceed by a mechanism in which one oxygen molecule adds to the metalloporphyrin to give a metallobilitriene. In the subsequent work-up, demetalation and addition of two hydrogen atoms gives the final product.     

THE PHOTOOXIDATION OF DIETHYLHYDROXYLAMINE BY ROSE BENGAL IN MICELLAR AND NONMICELLAR AQUEOUS SOLUTIONS

The photooxidation of N,N -diethylhydroxylamine (DEHA) by Rose Bengal (RB) has been investigated in micellar and nonmicellar aqueous solutions. We measured the quantum yield of oxygen consumption forming H₂O₂ and monitored two intermediates, the superoxide and diethylnitroxide radicals. When the pH was vaned, the quantum yield of oxidation remained constant for 6 < pH < 10.5, decreased in acidic pH, and increased considerably in NaOH solution; these changes could be attributed to the protonation and dissociation processes of the >N-OH moiety of DEHA. The formation of diethylnitroxide radical was enhanced by superoxide dismutase or strong alkaline solution. Around neutral pH, the oxidation proceeded mainly via electron transfer from DEHA to the RB triplet ( k _q = 10⁷ M ^-1 s^-1) with little ¹O₂ participation ( k_q < 10⁵ M ^-1 s^-1). However, when RB was incorporated into micelles in alkaline solution, the contribution of the singlet oxygen pathway increased at the expense of electron transfer, which was inhibited by the less polar micellar environment. Dark autoxidation of DEHA was accelerated by heavy metal impurities and increased very strongly in NaOH solution.