A STUDY OF LIGHT-INDUCED ELECTRON PARAMAGNETIC RESONANCE SIGNALS IN WALLS OF MOSS SPORES AND POLLEN GRAINS

Abstract —Light-induced EPR signals can be observed in moss spores and pollen grains. Their characteristics are studied in this paper. The modification of these signals during spore maturation or various physical and chemical treatments shows that they probably originate in the spore or pollen grain wall. This hypothesis is confirmed by the similarity between the spore (or isolated spore wall) absorption spectrum and the light-induced signal action spectrum.     

AN IDENTIFICATION OF THE RADICAL GIVING RISE TO THE LIGHT-INDUCED ELECTRON SPIN RESONANCE SIGNAL IN PHOTOSYNTHETIC BACTERIA

Abstract— A reaction-center fraction isolated from Rhodopseudomonas spheroides chromatophores exhibits light-induced changes in its optical and electron spin-resonance (ESR) spectra. In particular, a bleaching at 870nm (P870) has been found to be closely correlated with the appearance of an ESR signal with a g factor of 2.0025 and a peak-to-peak line width of 10 G. The ESR signal is indistinguishable from light-induced signals found in chromatophores or whole cells.
A careful measurement of the spin concentration showed that the ratio of the light-induced spins to bleached P870 molecules is 1.1 ± 0.1. In addition the formation and decay kinetics are identical within experimental error under a variety of experimental conditions.
Previous work has shown that P870 is a bacteriochlorophyll molecule in a specialized environment and that the bleaching signifies oxidation. The present work provides strong evidence that the photo-bleaching of P870 produces the radical cation of bacteriochlorophyll, P870⁺, and that this radical is the source of the ESR signal in whole cells.
The quantum yield for the bleaching of P870 in reaction centers has been measured, using actinic light of wavelengths 880, 800, 760 and 680 nm. For light absorbed at 880 or 800 nm the efficiency is close to 100 per cent. In a coupled reaction, the oxidation of mammalian cytochrome c by P870⁺ proceeds with nearly the same efficiency.
The above results place definite limits on the possibilities for the identity of the primary acceptor. These possibilities are discussed.     

PHOTOSENSITIZATION OF MELANIN: AN ELECTRON SPIN RESONANCE STUDY OF SENSITIZED RADICAL PRODUCTION AND OXYGEN CONSUMPTION

Abstract Rose Bengal is shown to photosensitize free-radical production and oxygen consumption in solutions of melanin from autooxidation of 3,4-dihydroxyphenylalanine (DOPA). In anaerobic solutions the sensitizer enhances rates of free-radical production by up to a factor of 20. In aerobic solutions, rates of oxygen consumption can be increased by a factor of several hundred. The reactions appear to involve the triplet state of the sensitizer. The effect of the sensitizer in increasing oxygen consumption is quenched by low concentrations of azide and enhanced by D₂O, suggesting that a singlet oxygen mechanism is involved.     

ELECTRON PARAMAGNETIC RESONANCE OF SOME TRIPHENYLMETHANE DYES IN THE TRIPLET STATE

Abstract— Electron paramagnetic resonance spectra of the triplet states of several triphenylmethane dyes in glassy solutions at 90 K have been measured and their zero-field splitting parameters estimated. Crystal violet and para rosaniline do not possess trigonal symmetry in their triplet states, and the unusually broad absorptions in the Δ M ₈= 1 region of the spectra have been attributed to the presence of different rotational isomers of the dye cations. A number of malachite green derivatives were investigated, but absorption of the triplet states of these dyes was only observed in the low field Δ M ₈= 2 region of the spectrum.     

ELECTRON SPIN RESONANCE STUDY OF PHOSPHORANYL RADICALS I. INFLUENCE OF STERIC AND ELECTRONIC EFFECTS ON RADICAL FORMATION IN SOLUTION

Abstract

An electron spin resonance study of phosphoranyl radicals, generated by u.v. irradiation of solutions containing a trialkylphosphite and a dialkylperoxide, enabled us to examine the influence of steric and electronic factors on radical formation. It was demonstrated that the influence of steric factors, such as α- or β-branching of the alkyl groups, on the magnitude of radical formation was present in both trialkyl- and methyldialkylphosphites, but not in dimethylalkylphosphites. Furthermore, the study of additional hetero atoms in the alkyl ligand of the dimethylalkylphosphites clearly revealed the influence of the gauche-effect. It was demonstrated that the presence of the gauche-effect, which causes a conformational preference in which a larger steric hindrance is produced, resulted in a decrease of radical formation.     

LIGHT-INDUCED ELECTRON TRANSFER REACTIONS BETWEEN CHLOROPHYLL AND QUINONE IN LIPOSOMES: RADICAL FORMATION AND DECAY IN NEGATIVELY AND POSITIVELY CHARGED VESICLES*

Abstract— The incorporation of relatively small amounts (≤ 20 mol%) of a negatively charged surfactant into otherwise electrically neutral phosphatidylcholine vesicles containing chlorophyll in the presence of benzoquinone has been shown to produce large effects on radical formation and decay as measured by laser flash photolysis. When salt ions are present in the aqueous phase, increasing the level of negative surfactant leads to a small increase in radical yield, followed by a larger decrease in radical yield. When the salt concentration is low, increasing the negative surfactant concentration leads to a suppression of the fast radical recombination process, an increase in slow radical decay and, at the highest surfactant concentration, an approximately 35% increase in total radical yield. An analysis of these effects is given in terms of the influence of a negative electrostatic field on radical pair stabilization and recombination, radical pair separation and expulsion of the acceptor radical anion from the vesicle. The incorporation of relatively small amounts (≤ 20 mol%) of a positively charged surfactant into egg phosphatidylcholine (EPC) vesicles containing chlorophyll and benzoquinone also produces large effects on radical formation and decay. When the electrolyte concentration in the suspending aqueous medium is high, radical yields are decreased as the surfactant concentration is increased, without any appreciable effect on decay kinetics. When deionized water is used, the slow recombination component of the decay is specifically suppressed by the presence of the positive surfactant, whereas the fast decay component decreased and then increased in amount as the surfactant concentration is increased. In all cases, however, the total radical yield is less than in pure EPC vesicles. These results can be understood in terms of the influence of a positive electrostatic field on radical pair separation and acceptor radical anion mobility. When equimolar amounts of both positively and negatively charged surfactants are incorporated into EPC vesicles, the radical yields and decay kinetics are relatively unaffected, but a large effect is observed on the radical difference spectrum. This may be a consequence of clustering of oppositely charged molecules within the bilayer surface.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

AN ELECTRON SPIN RESONANCE STUDY OF MEROCYANINE 540-MEDIATED TYPE I REACTIONS IN LIPOSOMES

The merocyanine 540 (MC540)-mediated reduction of nitroxide spin labels in a liposomal system was examined using electron spin resonance (ESR) spectroscopy. Spin label reduction was light driven, and occurred in liposomes composed of both fully-saturated (dimyristoyl) and mono-unsaturated (1-palmitoyl-2-oleoyl) phosphatidylcholine. Loss of the nitroxide ESR signal was enhanced by the physiological electron donors glutathione, cysteine, and NADPH; and was strongly inhibited by the presence of molecular oxygen. Nitroxides reduced in the presence of MC540 alone could be regenerated either by purging the sample with air or by the addition of ferricyanide, indicating that the ESR signal loss was due to reduction to the corresponding hydroxylamines. Only partial regeneration was attained for nitroxides reduced in the presence of glutathione, cysteine, or NADPH. Reduction rates for the lipophilic spin labels, 5-, 12-, and 16-doxyl stearic acid, were not influenced by the position of the nitroxide moiety along the alkyl chain, however reduction of spin labels occupying primarily the aqueous phase was much slower. These studies demonstrate that MC540 can initiate oxidation/reduction (Type I) reactions. Such Type I processes may augment the effects of singlet oxygen in MC540-mediated photodynamic therapy.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE: A KINETIC STUDY OF ENDOGENOUS LIGHT-INDUCED FREE RADICALS IN REACTION CENTER PREPARATIONS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have detected, by means of a kinetic analysis, a rapidly decaying signal in reaction center preparations from the R26 blue-green mutant of Rhodopseudomonas spheroides. This signal, which we designate Signal B3, is essentially the same as that seen previously in iron-free preparations. Signal B3 decays at 20°C with a 1/ e time of ˜ 3 ms and exhibits an activation energy of 5 ± 1 kcal mol^-1 over the temperature range 0–30°C. Extraction with isooctane completely eliminates Signal B3, whereas readdition of exogenous ubiquinone-30 completely restores the signal. o -Phenanthroline has no effect on Signal B3. We discuss these results in terms of a model in which the primary acceptor is an iron-ubiquinone complex with excess ubiquinone serving as a secondary electron acceptor pool.     

VARIATION IN ELECTRON PARAMAGNETIC RESONANCE SIGNALS OF PHOTOSYNTHETIC SYSTEMS WTTH THE REDOX LEVEL OF THEIR ENVIRONMENT

Abstract— The redox dependence of the light-induced electron paramagnetic resonance signal at g=2 in R. rubrum, R. spheroides and Chromatium chromatophore particles and quantasonie particles from spinach chloroplasts has been determined qualitatively over the range —0.3 to +0.6 V and quantatively over the range +0.3 to ±0.6 V. A light-induced EPR signal has been titrated and demonstrated to have a midpoint potential of +0.44 v at pH 7 and 20°C. Concentration, ionic strength and pH dependence for this transition in R. rubrum chromatophores is reported. In addition to the dark signal which replaces the light signal, in chromatophore material another dark signal, occurring in the seine location as the light signal, has been demonstrated to occur at high potential.
Selective chemical oxidation with K₂lrCl₆ of chromatophore particles from the three bacteria resulted in the removal of some 95 per cent of the absorbance in the near infrared and left the photoactive pigments.
Two light-induced EPR signals were found in quantasome particles by their dependence upon the redox level. Of particular interest is a signal observed at quite high potential (e.g. + 0.60 V). It was demonstrated that oxygen evolution by these quantasonie particles in the presence of K³Fe(cN)₀ occurred at the same rate at +0.55 V as at +0.40 V.     

STUDIES OF ELECTRON SPIN RESONANCE ON BILIRUBIN FREE RADICALS

The nature of ESR signals derived from bilirubin-Ⅸα has been studied by focusing onthe samples treated with free radical generating and inhibiting systems, i.e. X-XOD, Fe/EDTA, SOD, mannitol/ascorbate, CO, KCN, etc. In all the cases, the stable signals comprisethose originated from semiquinone radical (g = 2.0012) and superoxide free radical (g_∥=2.041, g_⊥= 2.0040). The superoxide is shown binding with certain metal ions chelated bybilirubin. The free radical scavengers are able to destroy these radicals. The kinetic curveof the regeneration of bilirubin radicals has been determined and the reaction follows azero order mechanism. It is likely that both the physiological and toxic actions of bilirubinare related to the characters of its free radicals. Bilirubin is discussed as "active oxygensink" in mammalians.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS-XIII. pH DEPENDENCE OF THE PHOTOCHEMISTRY OF PHOTO ALLERGENS BITHIONOL AND FENTICHLOR: AN ELECTRON spIN RESONANCE STUDY OF THE FREE RADICAL PHOTOPRODUCTS

Abstract The photoallergens bithionol (BT) and fentichlor (FT) generated free radical photoproducts upon UV photolysis which were observable by direct electron spin resonance (ESR). Both the yield and the type of free radical photoproducts were affected by pH, and to some extent, concentration of oxygen and concentrations of the photosensitizers. At pH 8.5, bithionol (0.9 mM) generated a semiquinone type free radical (BI) via a mechanism which probably involves substitution of the 4-chlorine by hydroxyl to form the corresponding hydroquinone followed by oxidation. The photolysis of 4-chlorophenol, 4-chlorocatechol and 2,2'-methylene-bis(4-chlorophenol) also generated the corresponding semiquinone radicals, suggesting that this mechanism is shared by other 4-chlorophenols. At pH 8.5, only photoproduct BI was observed during the irradiation of BT; FT related photoproducts were not observed at this pH. However, at higher pH values (pH 10.7 or pH 12), FT photoproducts were also observed in addition to BI upon prolonged irradiation. Moreover, the yield of BI increased drastically at higher pH. Oxygen did not play any role at pH 10.7, although it enhanced the yield of BI at pH 8.5. At pH 8.5, irradiated fentichlor generated, in roughly equal amounts, a semiquinone radical (Fla) and an unidentified species which contained two inequivalent protons (FII). At higher pH values (pH 10.7 and pH 12), at least four species were observed. All of the species are believed to be semiquinone radicals and two have been unambiguously identified. The yield of FI increased by a factor of 50 as the pH was increased from 8.5 to 12. Oxygen played only a minor role at pH 10.7 and above. However, at pH 8.5, it also enhanced the yield of FI.     

ELECTRON PARAMAGNETIC RESONANCE SIGNAL II IN SPINACH CHLOROPLASTS—I. HIGH RESOLUTION SPECTRA AND MORPHOLOGICAL LOCATION

Abstract —The electron paramagnetic resonance Signal II of spinach chloroplasts was recorded under high-resolution conditions. The signal is asymmetric, has five lines of unequal intensity and splitting, a derivative linewidth of 20.0 G, and a g -value of 2.0051. The latter value varies between 2.0045 and 2.0052 since other signals contribute to the center portion of the spectrum. Signal II is present in dark-adapted leaves and chloroplasts at a concentration of 50 per cent of the maximum light signal. It is not exhibited by stroma lamellae but is seen in grana thylakoid fractions. The maximum concentration of the material providing Signal II is found in a fraction (40 K) which has a very low Photosystem II activity. The unknown substance giving rise to Signal II may not be required for either Photosystem I or Photosystem II activity. Evidence presented here indicates that Signal II probably is not due to a free, mobile plastosemiquinone or a free, mobile plastochromanoxyl radical.     

LIGHT-INDUCED FREE RADICALS IN ORIENTED DNA-PROFLAVINE COMPLEXES

Abstract— Oriented wet DNA-proflavine complexes were illuminated with visible light, Λ > 395 nm, at 77 K, in the presence or absence of oxygen. The electron paramagnetic resonance spectra at 77 K and low microwave power (3 μ W) indicated formation of anionic free radicals in thymine and cationic free radicals, probably in guanine, identical to those induced by y rays at 77 K in similar samples of pure DNA.
The free-radical formation rate showed a quadratic dependence on light intensity, indicating a biphotonic mechanism. The proflavine triplet spectrum was observed during illumination. If the exciting light includes wavelengths below 390 nm, significant amounts of hydrogen addition radicals in thymine are found.     

ELECTRON PARAMAGNETIC RESONANCE SIGNAL II IN SPINACH CHLOROPLASTS—II. INFLUENCE OF PHOSPHORYLATION AND ELECTRON-TRANSPORT INHIBITORS

Abstract —The influence of several groups of inhibitors on the electron paramagnetic resonance Signal II of spinach chloroplasts was investigated. It was found that phosphorylation uncouplers that do not inhibit electron transport (gramicidin, valinomycin) have no effect. Likewise, inhibitors that block electron transport between the two photosystems are ineffective. However, reagents that can inhibit electron transport and also uncouple phosphorylation, such as phenylhydrazones (FCCP) or anilino-thiophenes (ANT 2p), will completely abolish Signal II in the dark. The signal is regenerated by red light and also by far-red light, but in the latter case only if cyclic electron transport is possible. These agents, in addition to discharging the water-splitting enzyme [ADRY effect, G. Renger, Biochim. Biophys. Acta (1972) 256, 428] were also found to inhibit cyclic electron flow. Light activation of Signal II in ANT 2p- or FCCP-treated chloroplasts by either red or far-red light can be suppressed by simazine or dichlorophenyl dimethylurea (DCMU) but not by other inhibitors that also inhibit linear electron flow. Evidence is provided which indicates that one functioning electron-transport system, either linear or cyclic, is necessary and sufficient for the enhancement of Signal II. The requirement for Signal II activation seems to be the reduction of plastoquinone. In ANT 2p- and FCCP-treated chloroplasts, a link probably exists between the water-splitting enzyme and the plastoquinone pool, bypassing Photosystem II; the unknown substance giving rise to Signal II may be located on such a sidepath.     

PHOTOIONIZATION OF TRYPTOPHAN: AN ELECTRON SPIN RESONANCE INVESTIGATION

The photoionization of tryptophan has been studied by ESR spectroscopy using the free radical scavenger 5, 5 dimethyl-1-pyrroline-l-oxide as a spin trap for hydrated electrons. A special set-up has been devised to irradiate more than one aliquot of the bubbled aqueous solutions in the cavity without removing the ESR cell out of the cavity.
Quantum yields of electrons production have been estimated as a function of irradiation wavelength. No photoionization threshold has been observed in these experiments.     

ELECTRON SPIN RESONANCE STUDIES OF ULTRAVIOLET IRRADIATED KERATIN AND RELATED PROTEINS

Abstract— The formation of free radicals in keratin and related proteins by exposure to u.v. light has been examined by electron spin resonance spectroscopy. From a comparison of the spectra obtained and the effects of different wavelengths and the stabilities of the radicals produced, it has been shown that several species of free radicals are produced by the u.v. irradiation of keratin. However it has been possible to identify only those free radicals associated with the cystine residues. The free radicals produced in keratin by exposure to shorter wavelength irradiation (below 3250 Å) were found to be quite different to those produced at longer wavelengths.     

LIGHT-INDUCED ELECTRON TRANSFER REACTIONS BETWEEN CHLOROPHYLL AND QUINONE IN ELECTRICALLY-CHARGED VESICLES: EFFECTS OF COUNTERIONS LOCATED IN EITHER THE INNER OR OUTER AQUEOUS PHASES ON RADICAL FORMATION AND DECAY*

Abstract— The presence of relatively low concentrations of counterions (millimolar in the case of univalent ions and micromolar in the case of polyvalent ions) in either the inner or outer aqueous compartments of electrically charged lipid bilayer vesicles containing chlorophyll in the presence of benzoquinone has been shown to produce large effects on radical formation and decay as measured by laser flash photolysis. With negatively charged vesicles having no added salt inside, salt ions added to the external phase caused radical yields to markedly decrease, with no change in decay kinetics. When salt was present only in the interior phase, radical decay became biphasic and the ability of externally added salt to affect radical yields was greatly diminished. With positively charged vesicles having no added salt inside, salt addition to the external medium caused both the radical decay rate and yield to decrease. The presence of interior salt, however, caused radical decay to become faster and, as was the case with negative vesicles, greatly reduced the effects of salt added externally. These results have been interpreted in terms of electrostatic and structural effects of charge neutralization by counterions on the dynamics of radical-ion separation and recombination.     

FREE RADICAL PRODUCTION BY CHLORPROMAZINE SULFOXIDE, AN ESR SPIN-TRAPPING AND FLASH PHOTOLYSIS STUDY

Abstract— Using the spin-trapping technique we have investigated the photolysis of chlorpromazine sulfoxide and promazine sulfoxide. Photolysis of these sulfoxides in aqueous solution resulted in a species which is capable of oxidizing ascorbate, cysteine, glutathione, NADH, and azide by one electron, in addition to extracting hydrogen atoms from ethyl alcohol and dimethyl sulfoxide. These oxidations were not dependent on the presence of dissolved oxygen. The oxidizing species is proposed to be the hydroxyl free radical arising from the homolytic cleavage of the S-O bond of the sulfoxide. Flash photolysis of the chlorpromazine and promazine sulfoxides demonstrated the formation of cation radicals consistent with the loss of the hydroxyl radical from the sulfoxides. In addition we present a simple direct method for the quantitative synthesis of promazine and chlorpromazine sulfoxides from the parent promazine derivatives.     

ESR DETECTION OF ENDOGENOUS ASCORBATE FREE RADICAL IN MOUSE SKIN: ENHANCEMENT OF RADICAL PRODUCTION DURING UV IRRADIATION FOLLOWING TOPICAL APPLICATION OF CHLORPROMAZINE

Using electron spin resonance spectroscopy, we observed that UV radiation (330 nm) increased the endogenous ascorbate free radical concentration in hairless mouse (HRS/J) skin. When the skin was topically treated with a chlorpromazine solution prior to illumination, UV irradiation caused the ascorbate free radical concentration to increase even more. This observation suggests that there is an increased UV-induced oxidative stress in the presence of chlorpromazine, probably caused by the production of free radicals from chlorpromazine.