Showing papers in "Photochemistry and Photobiology in 1978"

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: 1Δg 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.

Reevaluation of the spectral and kinetic properties of ho2 and o2‐ free radicals

Abstract: — The spectra and molar absorbances of the HO2 and O2- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by 60Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are 225= 1400 ± 80 M-1 cm-1 and 225= 2350 ± 120 M-1 cm-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO2 and O2-: KHo2+HO2= (8.60 ± 0.62) × 105M-1 s-1; KHo2+O2-= (1.02 ± 0.49) × 108M-1 s-1; KHo2+O2- < 0.35 M-1 s-1. For the equilibrium HO2 O2-+ H+ the dissociation constant is KHo2= (2.05 ± 0.39) × 10-5M or pKHO2= 4.69 ± 0.08. G(O2-) has been evaluated as a function of formate concentration.

Cellular and subcellular mechanisms of photodynamic action: the 1o2 hypothesis as a driving force in recent research

Abstract: — Since 1972 the protective (quenching) effect of N3- and the enhancing effect of D2O have been used in combination to demonstrate 1O2 (1Δg) as the major reactive intermediate in the dye-sensitized photooxygenation of biomolecules in in vitro systems. Extended application to in vivo systems has recently begun, producing some results which generally support the involvement of 1O2 pathways in the photosensitization processes. The use of other 1O2 quenchers and acceptors has also been increasing. In the application of these techniques as diagnostic tests for 1O2 participation in in vivo systems, careful examination of the experimental conditions should be made with respect to the quenching capability and accessibility of D2O in the specified cellular environment. Furthermore, the diffusive nature of 1O2, generation sites, generation efficiencies, the location and type of sensitizers, and the reactivity of 1O2 with presumed target structures in the cell should also be taken into account in the interpretation. Recent studies illustrating the importance of these factors are discussed. Finally, a tentative picture of the mode of in vivo photodynamic activity of common dye sensitizers is outlined.

The effect of iron on the distribution of superoxide and hydroxyl radicals as seen by spin trapping and on the superoxide dismutase assay

Abstract: — Using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide we have demonstrated the presence of OH in the xanthine-xanthine oxidase system when iron and/or iron-EDTA (ethylenediamine-tetraacetic acid) is present. With increasing iron (or iron-EDTA) concentration the intensity of the O2- spin adduct decreased while that of OH increased. However, use of diethylenetriaminepentaacetic acid (DETAPAC) as a metal chelator in the reaction mixture suppressed the OH spin adduct signal while maintaining the intensity of the signal from the O2- spin adduct. Use of EDTA to eliminate the interfering effects of metal ions in the superoxide dismutase assay employing xanthine oxidase and nitroblue tetrazolium introduces an artifact from the iron present. The interference in the assay from metal ions, including iron, can be eliminated with use of DETAPAC as a metal chelator. Thus, it is possible to make comparisons of measured superoxide dismutase activities even when there are variations in the amount of iron present in the samples.

The haber-weiss cycle

Abstract: — The Haber-Weiss cycle: was investigated at low pH by radiolysis of oxygen or nitrogen saturated solutions of hydrogen peroxide. It was found that reaction 2 has a low rate constant: k2= 3.0 ± 0.6 M-1 s-1 (pH 2.3, 22°C). The rate determining step of reaction 2 is most probably the transfer of an electron from a π8* orbital of HO2 to the empty su* orbital of H2O2. Overlap between these two orbitals is hindered by the filled π8* orbitals of H2O2. Fe(HI)EDTA catalyses reaction 2.

The formation of free radicals and the consequences of their reactions in vivo.

Abstract: — Mechanisms for the initiation of free radical reactions in vivo are reviewed, including ozone and 1O2 reactions, radiation, one-electron transfer processes, and enzymatic reactions. The roles that radical reactions might play in aging processes and in carcinogenesis are discussed.

CHEMISTRY OF SINGLET OXYGEN—XXVI. PHOTOOXYGENATION OF PHENOLSy†

Abstract: — The aerobic dye-sensitized photooxygenation of monohydric phenols proceeds by way of singlet oxygen under the conditions studied. Various phenols give different proportions of reaction with and quenching of singlet oxygen. Para-substituted 2,6-di-t-butylphenols show a linear correlation between the log of the total rate of singlet oxygen removal and their halfwave oxidation potentials; the same correlation is given for certain phenol methyl ethers. A Hammett plot using s+ gives ρ - 1.72 ± 0.12, consistent with development of some charge in the quenching step. Reaction of photo-chemically generated singlet oxygen with 2,4,6-triphenylphenol gives 2,4,6-triphenylphenoxy radical as an intermediate in singlet oxygen quenching, although no overall reaction occurs. Kinetic analysis indicates that the radical is derived exclusively from the interaction of 2,4,6-triphenylphenol with singlet oxygen. A charge-transfer mechanism for quenching of singlet oxygen by phenols is proposed.

Comparative study of oxidation of nucleic acid components by hydroxyl radicals, singlet oxygen and superoxide anion radicals.

Abstract: — Superoxide radicals, singlet oxygen and hydroxyl radicals are individually or in combination involved in radiation or photochemical processes and in various enzymatic reactions. The reactivity and the mechanism of reaction of these oxygen species with some biologically significant DNA components were investigated through the characterization of the final oxidation products. Superoxide radicals appear to be unreactive with purine and pyrimidine 2'-deoxyribonucleosides. However, the autoxidation reaction of 6-hydroxydopamine leads to extensive degradation of thymine through the intermediary of hydroxyl radicals. Chemically and microwave-discharge generated singlet oxygen oxidation is specific to 2'-deoxyguanosine. The main oxidized products of these reactions were also characterized as well as an as yet unidentified nucleoside in the methylene-blue photooxydation of 2-deoxyguanosine. These results, in addition to specific deuterium effect experiments, lend support to the involvment of singlet oxygen (type II mechanism) in the methylene-blue photosentization. No singlet oxygen effect was observed in aqueous irradiated system.

The role of superoxide and singlet oxygen in lipid peroxidation.

Abstract: — An investigation into the mechanism of lipid peroxidation catalyzed by xanthine oxidase showed a dependence upon superoxide, singlet oxygen and adenosine 5'-diphosphate chelated iron (ADP-Fe3+). In the absence of ADP-Fe3+ or in the presence of superoxide dismutase there is complete inhibition of enzymatic peroxidation. Initiation of peroxidation likely occurs through an ADP-perferryl ion complex formed by ADP-Fe3+ and superoxide. Use of the singlet oxygen trapping agent 2,5-diphenylfuran showed that singlet oxygen does not participate in the initiation of peroxidation but rather in the propagation of peroxidation. The mechanisms of NADPH-cytochrome P450 reductase-catalyzed and ADP-Fe2+ catalyzed lipid peroxidation parallel that of xanthine oxidase in that initiation occurs through a superoxide dismutase-sensitive reaction and that singlet oxygen is present during propagation of lipid peroxidation.

Laser intensity and the comparative method for determination of triplet quantum yields

Abstract: — In the comparative method of determining the triplet quantum yield oT by laser flash absorption spectroscopy, general equations are established (1) for describing the dependence of oT with laser intensity and (2) for absolute actionometry of a laser pulse. Applications to specific examples are discussed.

The energetics of electron-transfer reactions of chlorophyll and other compounds*

Abstract: — Quite often the primary photochemical reaction of an excited state molecule is transfer of an electron to or from another molecule in its ground state. Rates of such reactions are closely dependent on differences between ground and excited state redox potentials of the reagents. The solvent also plays an important role in stabilizing ion pairs formed by the electron transfer. This Review discusses experimental data relating rates to electrochemical energy parameters in the context of a scheme which portrays the energy and electron transactions in a unified manner. Three consequences of reaction of a singlet excited state are distinguished: (S1) quenching without detectable products, (S2) exciplex fluorescence, (S3) transient radical ion production, and energetically necessary conditions are derived for each. Similarly, four kinds of reactions involving the triplet state are distinguished, which depend on the relation between the energy of the triplet state and that of the ion pair states: (TI) rapid quenching, (T2) slow quenching, (T3) accelerated intersystem crossing and (T4) generation by reaction between radical ions of like spin. The last may be followed by electrochemiluminescence. Classes of compounds for which data are available include chlorophylls, porphyrins and a few other molecules of biological interest, aromatic hydrocarbons and their derivatives, heterocyclic systems, carbonyl compounds, dyes, and complexes of Ru and U. A Table compiling median or selected values of ground and excited state electrochemical potentials of chlorophylls, some porphyrins, and a few other compounds is presented.

Superoxide radicals, superoxide dismutases and the aerobic lifestyle

Abstract: — The superoxide radical (O-2) is a commonplace product of the biological reduction of molecular oxygen and plays an important role in oxygen toxicity. Superoxide dismutases (SOD) catalytically scavenge this radical and are the primary defense against its cytotoxicity. The data which support these statements have been briefly reviewed. Oxygen enhances the lethality of certain antibiotics, such as streptonigrin, and of ionizing radiation as well. The role of O-2 in the enhancements is presented and the basis of the radioprotective effects of SOD discussed. Several more recent developments are presented in detail including: (a) The induction of the MnSOD of E. coli upon exposure to very low levels of oxygen, (b) The changes in SOD in E. coli as a function of nutritional state, during culture in a chemostat. (c) A convenient new assay and activity stain for SOD.

Photodynamic inactivation of E. coli by rose bengal immobilized on polystyrene beads.

Abstract: . The photodynamic inactivation of E. coli by visible light and O2 was found to occur in the presence of the sensitizer rose bengal, immobilized by covalent bonding to polystyrene beads. The demonstrated absence of significant amounts of dissolved rose bengal indicated that an inactivation mechanism based on penetration of sensitizer molecules into the cell's interior could not be operating. Survival curves typically exhibited induction periods followed by rapid exponential death, with 99.99% kill requiring 1–2 h depending on conditions. A mechanism involving the participation of photo-generated singlet excited oxygen O2(1δ) in inactivation of E. coli is proposed. The photodynamic inactivation rate increased significantly in H2O compared with H2O, which is evidence supporting singlet oxygen as an active intermediate, since O2(1δ) has a much longer lifetime in H2O than in H2O. H2O did not act as a short term poison in the absence of sensitizer.

Molecular properties of phytochrome

Abstract: The physiological experiments which led to the discovery of the photoreversible, morphogenically active plant chromoprotein phytochrome and to predictions concerning its molecular properties represent a classic example of a successful photobiological investigation (Hendricks, 1964). Biochemical and biophysical studies which followed verified many of these predictions. However, hypotheses concerning the molecular mode of action of phytochrome (Mohr, 1966; Borthwick et al., 1969; Mancinelli and Rabino, 1975; Schafer, 1975a) have not yet been definitively tested. A principal purpose of this review will be to summarize our present understanding of the molecular properties of phytochrome in order to provide a framework within which these latter hypotheses may 1976; Kendrick and Spruit, 1977; Marmk, 19771, monographs (Mohr, 1972; Smith, 1975) and symposium volumes (Mitrakos and Shropshire, 1972; Smith, 1976) concerning phytochrome have appeared in recent years. Because most of these reviews deal more with phytochrome-mediated events within intact tissues or whole organisms than with the molecular properties of the pigment itself, I shall emphasize those studies which involve direct in vitro biochemical or biophysical assay of phytochrome. In addition, since available reviews of the molecular properties of phytochrome generally cover literature up to about 1973, I shall further emphasize information which has appeared since that year.

Triplet excited state of furocoumarins: reaction with nucleic acid bases and amino acids

Abstract: . The triplet-triplet absorption spectra of psoralen, xanthotoxin, angelicin and bergapten have been determined using the technique of pulse radiolysis and laser flash photolysis in benzene and water. The extinction coefficients of the triplet transitions have been measured and used to determine the singlet → triplet intersystem crossing quantum yields for 353 nm excitation. These yields vary considerably in passing from benzene to water as solvent. The highest yields were obtained for psoralen and angelicin in water, being 0.45 and 0.33, respectively. Reaction rate constants of the psoralen and angelicin triplets with various pyrimidine and purine nucleic acid bases and amino acids have been measured. The high reactivities support the suggestion that the photosensitising properties of furocou-marins are mediated by the corresponding furocoumarin triplet states. The similarly high S → T quantum yields, and triplet reactivities with pyrimidines of psoralen and angelicin, also support the suggestion that the observed differences in photosensitising properties of these two psoralens may be due to their differing geometries, which allow psoralen itself to intercalate DNA more easily than angelicin. The efficient reactions also found for psoralen and angelicin triplets with amino acids suggest that such triplets in vivo may also react with these components. Significant damage may thus be caused to protein as well as nucleic acid components of the cell.

HO2 ELIMINATION FROM α‐HYDROXYALKYLPEROXYL RADICALS IN AQUEOUS SOLUTION

Abstract: — In aqueous solutions α-hydroxyalkylperoxyl radicals undergo a spontaneous and a base catalysed HO2 elimination From kinetic deuterium isotope effects, temperature dependence, and the influence of solvent polarity it was concluded that the spontaneous reaction occurs via an HO2 elimination followed by the dissociation of the latter into H+ and O2- The rate constant of the spontaneous HO2 elimination increases with increasing methyl substitution in α-position (k(CH2(OH)O2) 7000s-1) Because of the HO2 eliminations the peroxyl radicals derived from d-glucose do not undergo a chain reaction in contrast to peroxyl radicals not containing an α-OH group In competition with the first order elimination reactions the α-hydroxylalkylperoxyl radicals undergo a bimolecular decay These reactions are briefly discussed

Photophysical properties of phycobiliproteins from phycobilisomes: fluorescence lifetimes, quantum yields, and polarization spectra

Abstract: — Absorption and fluorescence polarization spectra, as well as absolute fluorescence quantum yields, and lifetimes of phycobiliproteins separated from intact phycobilisomes of Porphyridium cruentum, Nostoc sp. and Fremyella diplosiphon were measured. Two different types of phycoerythrin, in addition to phycocyanin and allophycocyanin, were separated from both Porphyridium cruentum and Nostoc sp. phycobilisomes. They were distinguishable by the shape of their absorption spectra, values of fluorescence quantum yields and their limiting polarization. Phycobilisomes of Fremyella diplosiphon had a type of phycoerythrin that was different from the above kinds. By the use of fluorescence quantum yields and lifetime data, the values of natural lifetimes, the decadic molar extinction coefficients, as well as Forster's critical distances R0 for excitation energy transfer, between phycobiliproteins in phycobilisomes, were estimated. The values obtained of Forster's critical distances indicate that for most efficient energy transfer from phycoerythrin to allophycocyanin, the outer layers of Porphyridium cruentum and Nostoc sp. phycobilisomes should be composed of bangiophycean, phycoerythrin and cyanophytan phycoerythrin-II respectively.

The reaction center of photosynthetic bacteria

Abstract: . Investigations on the bacterial photosynthetic reaction center have recently made several important steps forward. Progress has been made in measuring the time course of the light-driven reactions, and in understanding the thermodynamics of these processes, in determining the chemical-structural properties of the protein and its constituents, and in elucidating the functional relationship of the reaction center with the chromatophore membrane. Although the well-characterized Rhodopseudomonas sphaeroides reaction center has been the main exploratory vehicle in many of these studies, we now have an ever increasing body of information from bacteria of other species and genera. This work is providing information from which we can underline features that are common to bacterial reaction centers, but it also reveals differences which may reflect different selection pressures on the separate species. In this report we shall describe the early photochemical events of the reaction center, summarize the comparative biology of the reaction center, and discuss some of the current physical-chemical problems pertaining to the redox components of the reaction center.

Reactions of O-.2, H2O2 and other oxidants with sulfhydryl enzymes.

Abstract: — The inactivation of a number of sulfhydryl enzymes by H2O2. OH and O-2 is discussed. and the efficiencies of inactivation for the free radicals are compared with those of halide radicals. The enzymes with essential acyl-binding SH groups (papain and GAPDH) exhibit a higher sensitivity to inactivation by O-2 and H2O2 than those with “unactivated” SH groups (LDH and ADH). The sulfenic acid derivatives from the reactions of papain and GAPDH with H2O2 are repairable by RSH molecules, the rates of repair depending on the nature of the R group. Comparisons of rate constants show that competitive scavenging by RSH must be a very important mechanism for the protection of sensitive SH-containing proteins against OH, and for papain in oxygen-free solutions the efficiency of this protection increased in the order GSH < PenSH < CysSH < dithiothreitol. On the other hand, catalase and superoxide dismutase are more effective than RSH compounds in rendering protection against H2O2 and O-2. In fact in oxygen-containing systems the effects of RSH are highly complex, because the OH- and/or O-2-initiated chain oxidation of these molecules produces reactive species, which may inactivate essential SH groups. Experiments with papain showed that the yields and nature of the RSH-derived species depended on the structure of the R group. Also, very large yields of papain Cys25SSR were produced from the reactions of the radicals and H2O2. In effect, it is apparent that under certain conditions the oxidation of “activated” SH groups of important target molecules could become coupled to the OH or O-2 -initiated oxidation of RSH molecules. This should be taken into account in assessing the overall effectiveness of RSH compounds as cellular protectors.

On the generation of the hydroxylation agent from superoxide radical. can the haber–weiss reaction be the source of oh radicals?

Abstract: — In many biological systems, the role of O2- in hydroxylation and toxic processes was assumed to be due to the formation of OH radicals. The Haber-Weiss reaction (Haber and Weiss, 1934)—(H2O2+ O2-→ OH + OH-+ O2) was suggested as the origin of this activity. In this study it is shown that this reaction pathway is too slow, and that OH is probably formed from the reaction of complexed superoxide with H2O2 or/and from the reduction of Fe(III), bound to biological compounds, by O2-; the reduced Fe(II) can then react with H2O2 as a Fenton reagent, to yield OH. It is also shown that singlet oxygen cannot be formed in these biological systems neither from the dismutation of OJ nor from the reaction of O2- with OH. Singlet oxygen may be formed from the reduction of metal complexes by O2-.

Impact of enhanced simulated solar ultraviolet radiation upon a marine community

Abstract: —There is evidence to indicate that an increased exposure to solar radiation in the UV-B region (specifically, 290–320 nm) may occur as a result of anthropogenic degradation of stratospheric ozone. The fact that present levels of solar UV radiation can detrimentally affect marine organisms led to experiments to quantify the impact of increased UV radiation upon a marine community. Two 720–l seawater chambers (continuous flow-through design) were exposed to simulated solar UV radiation. Fluorescent sunlamps filtered by a 290 nm cutoff filter (a 0.13 mm thickness of cellulose triacetate film) were used as the radiation source. Utilization of three different weighting factors for the spectral irradiances at the surface of the chambers yielded differences of 18%, 35% and 40% in biologically effective fluence rate between the two chambers. Analysis of attached forms of algae at various depths demonstrated that a surface exposure of 1.4W/m2 in the 290–315nm waveband as contrasted with the chamber receiving a surface exposure of 1.0W/m2 resulted in depressed Chl a concentrations, reduced biomass, increased autotrophic indices, and decreased community diversity. These results indicate a potential for adverse effects of increased solar UV-8 radiation: decreased community diversity, community structure shifts, and decreased productivity.

The mode of interaction between blue (uv) light photoreceptor and phytochrome in anthocyanin formation of the sorghum seedling

Abstract: Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (Pfr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without Pfr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.

Differential causes of mutation and killing in Escherichia coli after psoralen plus light treatment: monoadducts and cross-links.

Abstract: — On treatment with 8-methoxypsoralen plus near UV light, an excisionless (uvrB-) strain of Escherichia coli showed about 3– and 10 times higher sensitivities to killing and mutation, respectively, than its parental strain. On re-irradiation with near UV in the absence of unbound psoralen, the uvrB- strain pretreated with psoralen plus near UV showed a decrease in both survival and mutation. After treatment with psoralen plus near UV, re-irradiation of T7 DNA in the absence of unbound psoralen caused an increase in the cross-linked fraction with an equivalent decrease in the non-cross-linked fraction. From these and previous results, we conclude that monoadducts produced by treatment with psoralen plus near UV are converted to cross-links by further irradiation and that, in E. coli, monoadducts are responsible for the mutation induced by psoralen-plus-light whereas cross-links are the major cause of its lethal action.

Genetic and physiological studies of the effect of light on the development of the moss, physcomitrella patens

Abstract: The germination of Physcomitrella patens spores only occurs when wet spores are exposed to light. Depending on their ripeness, spores require from 44 to 64 h illumination to bring about maximum germination. There is a lag period of about 15 h between the reception of sufficient light to elicit germination before germination can be observed. Wavelengths in the range 640-680 nm are much more effective in inducing germination than longer or shorter wavelengths, but far-red reversal of red light induction of germination has not been demonstrated. Light also has very marked effects on protonemal and gametophore development. In darkness, only caulonemata are produced, and these grow negatively geotropically. No new gametophores develop but existing gametophores grow negatively geotropically, etiolate and bear only scale leaves. In light, chloronemata, as well as caulonemata are produced, the former grow positively phototropically, while the latter grow at right angles to the direction of light, and neither cell type is sensitive to gravity. In the light, gametophores grow positively phototropically, are indifferent to gravity, produce large leaves and do not etiolate. All these responses to light by protonemata and gametophores are shown by cultures growing in a 23 h dark/1 h red light cycle, but if this red light treatment is followed by 15 min far-red light, the effect of the red light is reversed, indicating an involvement of phytochrome in the mediation of these responses. Mutants showing abnormal growth in the dark have been isolated, as well as mutants having abnormal phototropic responses. The latter type has lost the phototropic response of both the protonemal cell types, as well as of gametophores, indicating that these different responses may share a common component.

Cytotoxic reactions of free radical species of oxygen.

Abstract: — We report on autooxidation reactions related to selective cell killing in vivo and present oxygen uptake and EPR data on the molecular nature of these changes, with a note on a reaction used to test for singlet oxygen. Selective obliteration by 6-hydroxydopamine, 6-aminodopamine and their congeners of beta-adrenergic cell receptors and of adrenergic and dopaminergic neurons in vivo and in vitro have been reported recently. These effects devolve from autoxidation of the compounds in situ after appropriate biological concentration, but it is unclear whether cytotoxicity is mediated by the reactive oxygen species themselves or by nucleophilic reactions of the product quinones. Because of the possibility of harnessing these reactions for a generalized “chemical surgery” if the former mechanism is operative, and because many other oxidative damage reactions involving unsaturated lipids of biomembranes seem to share similar autoxidative initiating and chain-carrying steps, we have studied the autoxidation of ascorbate and other reductants catalyzed by polyhydroxy quinols related to 6-hydroxydopamine. We find that superoxide anion mediation may or may not be important, depending on redox potentials and reaction kinetics of particular compounds. To the extent that the cellular toxicity of these compounds is oxidative in nature, it is facilitated by—and may depend upon—hydroxyl radical production due to Fenton-type reactions. Likely Fenton reactants in vivo are hydrogen peroxide produced from the quinol-catalyzed autoxidations and non-heme iron reduced cyclically by superoxide anions from the same autoxidations. Studies using flow systems adapted for EPR at 35 GHz have provided support for some of the reaction mechanisms proposed. In nonaqueous solvents eerie oxidation produced transient free radicals from hydrophobic diphenyl and isobenzo furans, which are often cited as “specific” probes for singlet oxygen detection in biochemistry.

The involvement of the hydroxyl radical in the destructive photooxidation of chlorophylls in vivo and in vitro

Abstract: The photooxidation of chlorophylls in vim and in aqueous micellar solutions has been inves- tigated. Through spin trapping experiments, it was found that hydroxyl radicals are photo-produced in these systems if oxygen is present. Optical studies indicate that the rate of photo-destruction of chlorophyll is spin trap dependent. This suggests that the hydroxyl radical may play a key role in the destruction of chlorophyll. INTRODUCHON The destructive photooxidation of chlorophyll$ (Chl) within the thylakoid membrane of chloroplasts in green plants and algae is thought, at least in part, to be prevented by the presence of carotenoids in the membrane (Griffith et al., 1955). The same is true for Bchl in the chromatophore membranes of photosyn- thetic bacteria (Clayton, 1965). In fact, blue-green mutant bacteria, which lack any carotenoids, die if exposed to both oxygen and light. The mode of pre- ventive action by carotenoids and the mechanism of photooxidation of Chl are interrelated. If one assumes that the carotenoid functions only by acting as an energy trap for triplet Chl (see e.g. Kung and De Vault, 1976), then three mechanisms emerge as most probable for the photooxidation of Chl:

Studies of chlorophyll‐chlorophyll and chlorophyll‐ligand interactions by visible absorption and infrared spectroscopy at low temperatures

Abstract: — A comparison of the visible absorption and infrared spectra of various chlorophyll-chlorophyll (Chl) and Chi-nucleophile aggregates at room temperature and at low temperatures has been made The IR data provide structural information indispensable for the interpretation of the visible spectra As a necessary preliminary, it is shown that Chl a solutions in nonpolar solvents can be prepared by appropriate drying techniques that contain at a conservative estimate ≤ 3 mol % of water (ie Chl a/H2O > 30:1) Very dry solutions of Chl a or Pyrochl a(≥ 10 mM) in toluene or methylcyclohexane-isopentane solution show only slight changes in visible spectra on cooling to 77 K From IR, additional Chl-Chl aggregation occurs on cooling in methylcyclohexane-isopentane but not to a significant extent in toluene Dilute (10 μM) solutions of Chl a or Pyrochl a in nonpolar solvents form a new absorption peak near 700 nm at low temperatures, which we attribute to traces of water in the solvent or other residual nucleophiles not removed during the Chl purification Addition of stoichiometric amounts of water increases the size of the ˜700 nm peak even in dilute Chl solutions Chlorophyll a, Pyrochl a, but not pheophytin a are shown to interact with nucleophiles of the general type RXH (where R= H or alkyl, and X = O, N, or S) Such nucleophiles can coordinate to the Mg atom of one Chl molecule by lone pairs on O, N, or S, and hydrogen bond to oxygen donor functions in another Chl molecule A ˜01 M solution of Chl a or Pyrochl a in toluene containing 15 equivalents of ethanol is converted almost entirely to a species absorbing at ˜700 nm at 77 K Infrared spectroscopy shows conclusively that it is the keto C=O function that is involved in the cross-linking by hydrogen bonding, a conclusion supported by the observation that Pyrochl a forms a very similar red-shifted species at low temperatures, despite the absence of a carbomethoxy C=O function n-Butylamine and ethanethiol interact in much the same way as does ethanol to form species red shifted to ˜700 nm A variety of possible structures for the low temperature forms is discussed, and the use of these red shifted species as paradigms for photoreaction center Chl is described

Regulation of chloroplast development by red and blue light

Abstract: There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.

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 tRNAphc 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.

The generation of hydroxyl radicals in biologic systems: toxicological aspects.

Abstract: — The formation of hydroxyl radicals in vitro was studied through their reaction with 2-keto-4-thiomethylbutyric acid to form ethylene gas. The autoxidation reaction of 6-aminodopamine served as a model source of hydroxyl radicals. Ethylene production was suppressed by catalase and by superoxide dismutase, indicating that both hydrogen peroxide and superoxide were involved in the reaction. Hydroxyl radical scavengers (thiourea > benzoate > ethanol) suppressed ethylene production in good agreement with their respective rate constants for reaction with hydroxyl radicals. Urea served as a negative control. Several substituted thiourea derivatives also suppressed ethylene production to a similar degree as thiourea itself. Biologic studies centered on several cytotoxic agents whose mechanisms of action are thought to involve hydroxyl radicals. These agents included alloxan, which destroys the beta cells of the pancreas, and 6-hydroxy- and 6-aminodopamine, which destroy sympathetic nerves. Damage to tissues in vivo was blocked to varying degrees by pretreatment of animals with hydroxyl radical scavengers such as ethanol or the thiourea derivatives. In addition, hydroxyl radical scavengers blocked the action of 5,7-dihydroxytryptamine, a neurotoxin whose effects on noradrenaline neurons were previously shown to be blocked by inhibitors of monoamine oxidase. The data indicate that these cell toxins produce their damaging actions on specific target cells through the intracellular generation of hydroxyl radicals.