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.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: FLASH PHOTOLYSIS STUDIES OF THE CHLOROPHYLL-QUINONE REACTION IN ALCOHOL SOLVENTS*

Abstract— Flash photolysis of chlorophyll a alone in CBE (cyclohexanol-t-butanol-ethanol) yields a difference spectrum similar to those obtained upon steady illumination of chlorophyll a-quinone mixtures in this solvent. Decay kinetics in CBE and dimethylsulfoxide are faster at the Soret band than at 460–580 nm and red band regions. This difference is not obtained in other solvents (CHCI₃, CCI₄, t-butanol, ethanol), implying that two or more species are obtained in CBE and DMSO. β-Carotene in CBE increases the rate of decay of the flash-induced chlorophyll transients at 430 and 660 nm but only decreases the magnitude of the signal at 470 nm. This implies that the 470 nm absorbance is due to a product formed from the triplet state. This effect is not observed in ethanol. Adding quinone to chlorophyll solutions results in slowly decaying species being generated by flash excitation in CBE. Three components can be distinguished: the first (t_1/2? 0.2 msec) corresponds to the triplet state; the second (t_1/2= 5–10 msec) is quinone concentration and species independent; the third (t_1/2= several seconds) is dependent upon quinone concentration and species (rate is faster for higher concentrations and lower potential quinones). The ESR signal decay rate is approximately equal to the third component flash decay rate when the chlorophyll and quinone concentrations are equal. With excess quinone, the flash decay rate becomes faster, and the ESR decay rate decreases slightly. These slowly-decaying species are not produced when quinone is added to chlorophyll a in ethanol or t-butanol, or to pheophytin in CBE. One observes merely a decrease in signal height with no accompanying increase in decay rate. Mechanisms to account for all of these phenomena are presented which involve an initial chlorophyll triplet-solvent reaction with the subsequent formation of several species of chloro-phyll-quinone radical complexes.     

SINGLET AND TRIPLET MECHANISMS IN PHOTOCHEMISTRY

A perturbation method is proposed for determining the photochemical quantum yields of the singlet and triplet mechanisms for an organic compound in solution. Some common fallacies about singlet and triplet mechanisms in the photochemical literature are discussed.     

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.     

EFFECT OF CHLORIDE ON CHLOROPHYLL PHOTOCHEMISTRY IN SOLUTION: ENHANCEMENT OF CATION RADICAL AND SEMIQUINONE YIELDS

Abstract—Illumination with red light at low temperatures of a degassed ethanol solution of chlorophyll and ferric chloride reversibly produces an EPR signal due to the chlorophyll cation radical (Chl+). The magnitude of this signal is about ten times larger than is obtained with chlorophyll alone. When benzoquinone is also present, an EPR signal due to Chl+ and the neutral semiquinone radical (QH.) is photoproduced. The semiquinone signal is about five times larger than in the absence of ferric chloride. Both of these systems show first order radical decay when the light is turned off, suggesting that radical complexes are being formed. These results indicate that iron is capable of serving as an electron transfer bridge between chlorophyll and quinone.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

CHLOROPHYLL PHOTOCHEMISTRY IN CONDENSED MEDIA—II. TRIPLET STATE QUENCHING AND ELECTRON TRANSFER TO QUINONE IN LIPOSOMES*

The fate of excitation energy and electron transfer to quinones within Chl-a-containing phosphatidyl choline liposomes has been investigated. The bilayer membrane of the liposome stabilizes the Chl triplet state, as evidenced by a three-fold increase in the lifetime over that observed in ethanol solution. The relative triplet yield follows the relative fluorescence yield, indicative of quenching at the singlet level. Triplet state lifetimes are markedly shortened as the Chl concentration is increased, demonstrating that quenching occurs at the triplet level as well. This process is shown to be due to a collisional de-excitation. In the presence of quinones, the Chl triplet reduces the quinone resulting in production of long-lived electron transfer products. The percent conversion of Chl triplet to cation radical when benzoquinone is employed as acceptor is approximately 60 ± 10%, which is slightly less than in ethanol solution (70 ± 10%). The lifetime of the radical, however, can be as much as 1900 times longer. With respect to potentially useful photochemical energy conversion, the magnitude of this increased lifetime is far more significant than is the decreased radical yield.     

CHARACTERIZATION OF RADICAL ADDUCTS FORMED DURING PHOTOCHEMICAL SPIN TRAPPING IN LIPOSOMES

There exists an urgent need to monitor radical reactions in biological membranes. With the exception of a few studies in model and natural membranes, most biological spin trapping reactions have been confined to homogeneous media. In the present work we have devised a methodology by which spin trapping reactions can be investigated in liposomes composed of a fully-saturated phospholipid, dimyristoylphosphatidylcholine. Using photochemical spin trapping, we have detected and characterized the formation and partitioning of spin adducts in liposomes. Benzophenone (a lipid-soluble photosensitizer) and benzoyl peroxide (a lipid-soluble radical initiator) have been used to generate free radicals from hydrogen donors during photolysis in liposomes. Both water-soluble and lipid-soluble spin traps have been used to trap the radicals.     

RELATIONS BETWEEN PHOTOCHEMISTRY AND FLUORESCENCE IN CELLS AND EXTRACTS OF PHOTOSYNTHETIC BACTERIA*

Abstract— Light-induced changes in the yield of bacteriochlorophyll fluorescence have been measured in cells and chromatophores of photosynthetic bacteria, and coordinated with light-induced absorbancy changes. Comparisons were drawn during transitions between dark and light steady states and also between steady states established at different light intensities. Aerobic cell suspensions of Rhodospirillum rubrum, Rhodopseudomonas spheroides, Chromatium and Rhodopseudomonas sp. NHTC 133 showed a strict correspondence between changes in the fluorescence yield and the bleaching of P870 (P985 in Rps. sp. NHTC 133), as reported by Vredenberg and Duysens for R. rubrum cells. The relationship shows that singlet excitation energy in bacteriochlorophyll is quenched by P870 at a rate proportional to the concentration of unbleached P870. This implies that the photosynthetic units are not independent with respect to energy transfer. In anaerobic cell suspensions the change in fluorescence did not follow the bleaching of P870 in the manner described by Vredenberg and Duysens. Here a change in fluorescence may have resulted from the reduction of a primary photochemical electron acceptor as well as from the oxidation (bleaching) of P870. In chromatophore preparations there were further deviations from the Vredenberg and Duysens relationship which could be attributed to changes in the rate constants for quenching of singlet excitation energy. Finally there was a light-induced increase in the fluorescence yield which was related to a band shift of bacteriochlorophyll and not to the bleaching of P870. Aerobic cell suspensions presented a limiting case in which these complications were absent. No change in the fluorescence was associated uniquely with the oxidation of cytochrome or band shifts of carotenoid pigments. These results, when coordinated with earlier findings about the fluorescence of bacteriochlorophyll and P870, indicate that the singlet excitation quantum is the only energy carrier linking the absorption of light with the initiation of photochemistry in bacterial photosynthesis.     

TYPE I AND TYPE II MECHANISMS IN THE PHOTOSENSITIZED LYSIS OF PHOSPHATIDYLCHOLINE LIPOSOMES BY HEMATOPORPHYRIN

Abstract The lysis of phosphatidylcholine (PC) liposomes was sensitized to visible light (>500nm) by hematoporphyrin (HP) incorporated in the liposomes (0.09-1.5%, wt/wt) or in the external buffer (1-15 μM). The lytic mechanism changed from the Type II pathway mediated by singlet oxygen (¹O₂) at low HP concentrations to the anoxic, Type I pathway at high HP concentrations. Spectral measurements of HP in aqueous and organic solvents indicate that the HP was not aggregated (monomers and/or dimers) for Type II sensitization and aggregated for Type I conditions. High concentrations of azide (>0.1 M) or DABCO (>0.5 M) were protective with high HP concentration under oxic and anoxic conditions, which cannot involve the scavenging of ¹O₂. Feasible protective mechanisms are quenching of the HP triplet state by high azide and repair of the damaged membrane by DABCO via an electron transfer process. There was significant protection against lysis under Type I conditions by low concentrations of ferricyanide (>1 mM), indicative of an electron transfer mechanism. The incorporation of 22 mol % cholesterol in PC liposomes with 1% HP had no effect on the lytic efficiency for oxic and anoxic conditions. Dipalmitoylphosphatidylcholine liposomes incorporating 1% HP showed negligible photosensitized lysis at 50°C compared with PC liposomes with 1% HP at 25°C. The promotion of photosensitized lysis by hydrodynamic agitation observed in prior work with methylene blue (Grossweiner and Grossweiner, 1982) was significant with HP sensitization for both Type I and Type II conditions. Actinometry with PC liposomes incorporating 1% HP indicated that photosensitized lysis was very inefficient, requiring many absorbed quanta per lysed liposome. Preliminary experiments with crude hematoporphyrin derivative (Hpd) showed similar concentration effects on lytic efficiency, where PC liposomes incorporating 0.1% (wt/wt) Hpd were strongly sensitized by oxygen, whereas sensitization by oxygen was insignificant with 3.1% Hpd. The results with HP and crude Hpd indicate that lytic damage in a biomembrane does not necessarily require oxygenation.     

SIMULTANEOUS MEASUREMENT OF FREE RADICAL DECAY IN POLYMERIZATION OF MMA INITIATED BY AIBN USING ESR AND ITS KINETIC MODEL*

 The kinetics of free radical decay in the polymerization of MMA initiated by AIBN was studied by means of ESR spectroscopy. It was found that the curves of radical decay are strongly associated with the reaction temperature, the initiator concentration and the solvent. In the case of the radical polymerization carried out at high temperature or in solution, the radical concentration first reached a maximum, then declined monotonously with reaction time. It was also found that the greater the amount of initiator or the higher the temperature, the more rapidly the radicals decay. When the bulk polymerization was implemented at a relatively low temperature, the curves of radical decay became more complicated, i.e.,the radical concentration rapidly rose to a maximum, then dropped to a minimum, finally increased again with reaction time.Taking into account the diffusion effect, a semi-empirical equation is suggested to describe the kinetics of propagating radical decay.     

TRANSIENT SPECIES IN THE PHOTOCHEMISTRY OF EOSIN*

Abstract— Photochemical reactions of eosin in aqueous solution were studied using the flash photolysis technique. In deaerated solution the dye was converted quantitatively to the triplet state during flashing. The triplet dye decayed by first and second order reactions which partly regenerated the dye in the ground state and partly produced semioxidized and semireduced eosin. These radical species were formed in an electron dismutation reaction between two triplet molecules and also in a reaction between one triplet and one unexcited molecule. The radicals recombine rapidly to give the dye in the ground state.
An efficient reversible photooxidation reaction was observed in eosin solutions containing potassium ferricyanide. Semioxidized eosin was formed in high yield by reaction between the triplet dye and the oxidant. The dye was regenerated rapidly in a reverse reaction between the products of the oxidation reaction.
An analogous type of reaction was found to occur in eosin solutions containing p -pheny-lene diamine. This reagent reduced the triplet dye to semireduced eosin; the dye was regenerated in the ground state in a very efficient reverse reaction. The protolytic behaviour of semireduced eosin was studied by varying the pH.
Absorption spectra of the transient products were determined and rate constants for the observed reactions were measured. The results are compared with results from previous studies of fluorescein.     

HYDRODYNAMIC EFFECTS IN THE PHOTOSENSITIZED LYSIS OF LIPOSOMES*

Abstract— The photosensitized lysis of phosphatidylcholine liposomes incorporating methylene blue in the membrane or in the presence of external methylene blue is promoted by hydrodynamic agitation concurrent with or subsequent to irradiation with red light. The results implicate the attack of singlet oxygen on an unsaturated lipid component as the key photochemical step, which is followed by additional membrane damage induced by hydrodynamic action.     

WAVELENGTH DEPENDENCE AND KINETICS OF UV-INDUCED FREE RADICAL FORMATION IN THE HUMAN CORNEA AND LENS

Abstract— The wavelength dependence of free radical formation in the human lens and cornea was obtained in the ultraviolet-visible (UV-VIS) range with electron paramagnetic resonance. The spectral feature of having their maxima close to 290 nm, negligible intensity above 320 nm, and appreciable intensity toward 260 nm is common to both of these tissues.
The kinetics of free radical formation in the lens and cornea was interpreted in terms of a two component exponential function with characteristic rate constants k _l and k ₂, the latter of which has the same value for both the lens and cornea.     

ESR STUDIES OF CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: KINETICS AND MECHANISM OF REVERSIBLE HYDROQUINONE OXIDATION IN SOLUTION*

Abstract— ESR studies have been made of the kinetics of semiquinone radical formation and disappearance resulting from the reversible photosensitization by chlorophyll of hydroquinone oxidation in a pyridine-water solvent. The rate of radical decay was found to be second order with respect to the radical concentration, with a rate constant of 6.7 × 10⁵ l./mole sec at -30°C and an activation energy of 6900 cal/mole. The rate of radical formation was recombination-limited and, through the use of β-carotene as a quencher, the rate constant was determined to be 8.81 × 10⁵ l./mole sec at -30°C. The effect of light intensity and hydroquinone concentration on the rate of semiquinone radical formation and on the steady state radical concentration was also investigated and possible mechanisms to explain the results are discussed.     

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL*

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-a-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.     

ALKOXYL AND METHYL RADICAL FORMATION DURING CLEAVAGE OF TERT-BUTVL HYDROPEROXIDE IN THE PRESENCE OF WLYSAGCHARIBES

Abstract

The cleavage of fed' -butyI hydrspersxlde by ferrous ions at pH 4 8 yrerdea two distinctive radlcals, alkoxyl and methyl radicals, detected by the electron spin resonance (ESW) technieue with the sF;n trap 5 5-dimethyl-? pyrrslsne-N-oxide (DMPOO) Free radicail formation was found to be dependent span the ferrous ran concentration Usrng drfferent BMPB concentrations, it was proved that aBkoxy l radtcais were formed intinally polysaccharzdes (PS), such a s giucomannan, lscust bean gum, xanthan, and carrageenan, retarded free radical formation, but no similar effect was obsermd far chztosan It is csnciuded that the suppressing effect is caused by the tron blndlng abrlnty of each PS.     

聚甲基丙烯酸甲酯的燃烧特性以及CO和CO2的生成机理

采用质谱，色谱和锥形量热仪研究了自由基聚合的聚甲基丙烯酸甲酯（PMMA）燃烧过程中CO和CO2的生成机理及恒温条件下的燃烧情况，结果表明，在有氧条件下，自由基聚合的PMMA的解聚方式有三种，除了链末端的双键断裂和主链上的无规则C－C键的断裂外，还存在侧链酯基的断裂，其主要产物为CO2和CH3OH；在燃烧过程中，单体MMA的氧化主要的耗氧反应，其产物为CO2和少量的CO，并且CO2和CO的产率与反应温度以及样品的厚度基本无关，通过耗氧量估算得出，PMMA在空气中燃烧反应的热效应大约为22．9MJ/kg。     

PHOTOCHEMISTRY OF 1,2,3-INDANETRIONE*

The photochemistry of 1,2,3-indanetrione (1) has been examined in solution at room temperature by steady state and laser flash photolysis. The triplet state of 1 (T = 6.5 μs, δ_mux, = 360 and 570 nm, in dry acetonitrile) reacts preferentially via an a-cleavage process followed by a considerably slower loss of carbon monoxide. Triplet 1 shows a remarkably fast hydrogen abstraction rate constant when in the presence of 1,4-cyclohexadiene (k_r= 1.4 times 10⁶M^?1s^?1) in spite of its low excitation energy (E_T= 42 kcal/mol). This behavior can be explained by assuming that the vicinal carbonyls coplanar to the ketyl radical play an important role in its stabilization.     

PAIRWISE RECOMBINATION IN THE DECAY OF PHOTOCHEMICAL HYDRATED ELECTRONS*

Abstract— The decays of hydrated electrons generated by 265 nm laser flash photolysis of aqueous I^-, tyrosine, tryptophan, lysozyrne. and ribonuclease have been analyzed by numerical computing. Homogeneous reactions do not explain the data without e^- _aq—radical back reactions significantly faster than diffusion limited. The results are explained by postulating pairwise recombination persists at times where conventional diffusion theory predicts randomization, particularly at low intensities and in the absence of effective e^- _aq scavengers. The physical mechanisms responsible for these effects are considered.