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.     

PARTICIPATION OF THE TWO PHOTOSYSTEMS IN LIGHT DEPENDENT HYDROGEN EVOLUTION IN Scenedesmus obliquus

Abstract— The H₂-photoproduction in the presence of dithionite measured in wild type and mutant cells of Scenedesmus obliquus demonstrates two sequential phases. In mutants showing only PS I activity phase 1 of H₂-photoproduction is visible with its core activity. When PS II is developed during greening, considerable activity is added to the core of phase I and phase II activity appears. Addition of DCMU reduces H₂-photoproduction by about 90%. The residual activity is completely attributed to the core of phase I. It was concluded that the core of phase I is dependent upon PS I only and can use sources different from water as electron donors. Phase II is dependent upon the capacity of PS II, a functioning photosynthetic apparatus and water as electron donor. The results are supported by studies of wavelength dependent activity of the two separate phases of H₂-photoproduction.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

Amplified Degradation of Photosystem II D1 and D2 Proteins under a Mixture of Photosynthetically Active Radiation and UVB Radiation: Dependence on Redox Status of Photosystem II

Abstract— Plants exposed to a mixture of photosynthetically active radiation (PAR) and UVB radiation exhibit a marked boost in degradation of the D1 and D2 photosysteni II (PS II) reaction center proteins beyond that predicted by the sum of rates in PAR and UVB alone (amplified degradation). Becausee degradation driven by visible or UVB radiation alone is uncoupled from PS II redox status, it was therefore assumed that the mixed-light-amplified component of degradation would behave similarly. Surprisingly, amplified degradation proved to be coupled tightly to the redox status of PS II. We show that inactivation of the PS II water oxidation by heat shock or oxidation of the plastosemiquinone (Q_A-) by silicomolybdate nullifies only the amplified component of degradation but not the basic rates of degradation under PAR or UVB alone. The data are interpreted to indicate that formation of plastosemiquinone or an active water-oxidizing Mn₄ cluster, is the UVB chromophore involved in amplified degradation of the D1 and D2 proteins. Furthermore, accumulation of Q_A-by 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2-bromo-3-methyl-6-isopropyl-4-nitrophenol stimulated the mixed-light-amplified degradation component. Thus, amplified degradation of the D1 and D2 proteins in mixed radiance of PAR plus UVB (which simulates naturally occurring radiance) proceeds by a mechanism clearly distinct from that involved in degradation under PAR or UVB alone.     

DCMU-INDUCED STIMULATION OF PHOTOSYSTEM I ELECTRON TRANSPORT. INVOLVEMENT OF MEMBRANE STRUCTURAL CHANGES

DCMU-induced stimulation of the rate of photosystem I (PS I) electron transport in DCIPH₂→ MV photoreaction occurs through the action of DCMU on the rate-limiting step which contains the site of electron donation of DCIPH₂ (Ramanujam et al. , 1981). The magnitude of stimulation of the rate by 50 μ M DCMU decreased with increasing concentration of chlorophyll (Chl), implying that DCMU is stoichiometrically related to Chl with respect to the stimulation of the PS I rate.
DCMU-induced stimulation was sensitive to the ionic condition of the thylakoids, the effect being reduced at low cation concentration. Cation-induced scattering changes in thylakoid suspension were partially reversed by DCMU, and the percent Chl in the 10 K fraction of the thylakoid decreased upon addition of DCMU, indicating that grana structure is disrupted by DCMU. Hydroquinone-mediated reduction of cytochrome f in thylakoids in the dark was accelerated in the presence of DCMU. The DCMU effect was not observed in isolated PS I particles.
It is concluded that DCMU binds to the thylakoid membranes and brings about structural changes leading to unstacking of the thylakoids accompanied by an altered interaction of the electron transfer chain components with the added electron donor. This binding of DCMU must have an affinity lower than the well-known binding of DCMU to photosystem II (PS II), because the concentration required is markedly higher.     

KINETIC STUDIES ON THE STABILIZATION OF THE PRIMARY RADICAL PAIR P680+ Pheo- IN DIFFERENT PHOTOSYSTEM II PREPARATIONS FROM HIGHER PLANTS*

Abstract— The stabilization of the primary radical pair P680+ pheophytin (Pheo)^- through rapid electron transfer from Pheo to the special plastoquinone of photosystem II (PS II), Q_A, was analyzed on the basis of time-resolved (40 ps) UV-absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of¹Chi* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of ¹Chi* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of QA formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q^-_A formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q^-_A formation was found to be (300 ± 100 ps)^-1 in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.     

INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (¹O₂), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal-sensitized inhibition of PS II was similar to that of high light-induced damage to PS II: (1) the secondary quinone (Q_B)-dependent electron transfer through PS II is inactivated much faster than the Q_B-independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the Q_B domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe-S centers resulted in a marked reduction of high light-induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by ¹O₂ and that Fe-S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.     

PICOSECOND FLUORESCENCE AND ELECTRON TRANSFER IN PRIMARY PHOTOSYNTHETIC PROCESSES

Abstract. Under conditions that drive the reaction centers (RC's) into the "closed" state, the lifetime ( T ) of the fluorescence emitted by antenna molecules increases from 80 to 200 ps in PS I, from 300 to 600 ps in PS II, and from 200 to 500 ps in bacterial chromatophores. In Rhodopseudomonas sphaeroides strain 1760-1, the decay curve for fluorescence from the RC's has a component with T ₂= 15 ps due to the bacteriochlorophyll of the RC, and a second component with T ₂= 250 ps due to bacteriopheophytin.
Data on electron transfer at low temperatures and under different redox conditions are analyzed. along with the ps fluorescence kinetics. The hypothesis is discussed that electron transfer in RC's is coupled to conformation changes in the interacting molecules.     

Effect of Supplementary UVB Radiation on Chlorophyll Synthesis and Accumulation of Photosystems during Chloroplast Development in Spirodela oligorrhiza

Abstract— Although the effects of ultraviolet B (UVB, 290–320 nm) radiation have been studied in plants extensively, little is known about the potential impacts on maturation of chloroplasts. To address this problem, the effects of supplementary UVB on chloroplast development were examined in the aquatic higher plant Spirodela oligorrhiza. Dark-grown Spirodela-containing proplastids were exposed to photosynthetically active radiation (PAR) and ultraviolet A (UVA, 320–400 nm), plus supplementary UVB equivalent to 1% of PAR on a photon basis. The biosynthesis and assembly of chlorophyll (Chi) into reaction centers was followed for 4 days in situ by low temperature (77 K) Chi fluorescence. Impacts on chloroplast development were detected after only 1 h incubation in light with supplementary UVB. Fluorescence emission signals from Chi associated with the photosystem (PS) II antenna, PSII reaction centers and PSI reaction centers were detected at the same time with or without UVB, but the magnitude of PS fluorescence was diminished up to 60% in plants incubated in UVB. The Chi content was also lower in UVB-treated plants, but to a lesser degree than anticipated by low temperature fluorescence, suggesting lack of organization and/or association of Chi with PS. Electron transport, measured with room temperature fluorescence induction, was not consistently different in plants exposed to UVB. These results suggest that with UVB, fewer and/or smaller PS form during chloroplast development, but there is not a large inhibition of Chi synthesis or PSII activity.     

Ultraviolet Radiation Effects on a Microscopic Green Alga and the Protective Effects of Natural Dissolved Organic Matter

Abstract— The population and photosynthetic responses of a microscopic green alga ( Selenastrum capricornutum ) to realistic levels of UV radiation (UVA and UVB) were assessed in natural lake waters of different dissolved organic carbon (DOC) concentration. Specific growth rates and photosynthetic competence (as reflected by F_v/F_m [measure of maximal quantum efficiency of photosystem II] and t_1/2 [estimate of electrons transported to the plastoquinone pool] measured by in vivo variable chlorophyll a fluorescence) were compared between two exposure levels of UVR and two concentrations of DOC (2.5 mg C L⁻¹ 7.7 mg C L⁻¹). Exposure periods of 6–9 days (five to nine generations) were used. Exposure to UVA primarily affected the efficiency of photosystem II, as evidenced by significant decreases of F_v/F_m but not growth rates or t_1/2 Exposure to UVB, in the presence of UVA, did not cause significant additional decreases of F_v/F_m but did diminish growth rates. In the low DOC water, t_1/2 was also diminished, suggesting different proximate sites of action from those for UVA. The high DOC water decreased the effective exposure to both UVA and UVB and diminished the negative impact of UV radiation on the cells, but the apparent protection was not explicable solely by the shading action of the DOC. Control values for F_v/F_m, growth rates and t_1/2 were all lower in the high DOC water, suggesting a negative side effect to the apparent protective action of the DOC against UVB.     

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme-linked immunosorbent assay, has been modified to detect UV-induced damage in cellular DNA of monolayer-grown human melanocytes. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar-simulated light or γ-rays. Following physiological doses of UVA, UVB or solar-simulated light, a dose-related DNA unwinding comprising a considerable number of single-strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/10¹⁰ Da/kJ/m² were detected and after UVB irradiation 1.9 ssb/10¹⁰ Da/kJ/m² were seen. One minimal erythema dose of solar-simulated light induced 2.25 ssb/10¹⁰ Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

SPECTROSCOPY AND PHOTOCHEMISTRY OF 8-SUBSTITUTED 5-DEAZAFLAVINS

Abstract— Absorption, fluorescence and phosphorescence spectra as well as fluorescence and phosphorescence quantum yields of 8-X-5-deazaflavins (X = C1, NO₂, p -NO₂-C₆H₄, N(CH₃)₂, NH₂, p -NH₂-C₆H₄, p -N(CH₃)₂-C₆H₄-N=N) were determined. It was found that all these data are highly influenced by the substituent at position 8 of the 5-deazaisoalloxazine skeleton. Also the photoreduction of 8-X-5-deazaflavins in the presence of electron donors was studied. It was established that the photoreduction leads to the formation of a 5,5'-dimer and/or a 6,7-dihydro compound. Reduction of the C(6)-C(7) bond is promoted by strong electron-donating substituents and bulky electron donors. 5-Deazaftavins with a reducible substituent at position 8 exhibit reduction of the substituent prior to the reduction of the 5-deazaisoalloxazine skeleton.     

ABSENCE OF OXYGEN-EVOLVING CAPACITY IN DARK-GROWN CHLORELLA: THE PHOTO ACTIVATION OF OXYGEN-EVOLVING CENTERS*

Abstract— Chlorella cells were cultured in darkness on glucose and examined for pigment composition, capacity to evolve oxygen, and capacity to photo-oxidize artificial electron donors to Photosystems II and I. Evidence was obtained which indicated that such cells lack oxygen-evolving centers and the Mn pool associated with such centers but possess fully active System II and I trapping centers. Brief illumination ( t _1/2˜ 2–3 min) of dark-grown cells resulted in incorporation of Mn into the O₂-evolving centers and the capacity to evolve O₂ (photo-activation). Kinetic data from flashing or continuous light showed that the photoactivation is a multi-quantum process involving several rate limitations and a photosensitive state of limited stability. Measurements of oxygen-yield oscillations indicated that throughout the photoactivation process each newly formed oxygen-yielding center was independent of its neighbors. It is concluded that photoactivation of the Mn-containing oxygen-yielding catalyst is a fundamental process in all photosynthetic oxygen-evolving tissue.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

Alterations in Skin Immune Response Throughout Chronic UVB Irradiation—Skin Cancer Development and Prevention by Naproxen

Skin exposure to UVB radiation has been reported to produce both a significant inflammatory response and marked immunosuppression. This work was aimed to evaluate whether the response of murine skin to an acute UVB dose was modified by pre-exposure to chronic UVB irradiation and by topical treatment with naproxen, a nonsteroidal anti-inflammatory drug. Moreover, the effect of naproxen on the incidence of UV-induced skin tumors was studied. Prostaglandin E₂ (PGE₂) and tumor necrosis factor alpha (TNF-α) levels were increased 96 h post-UVB in acutely irradiated animals and both mediators were modified by topical naproxen application—PGE₂ was decreased while TNF-α was increased. Such inflammatory response was suppressed when mice were chronically irradiated. Naproxen application on chronically irradiated mice reduced the incidence of tumor lesions. Taken together, our data suggest that chronic UVB irradiation generates an immunosuppressive state that prevents skin cells from responding normally to an acute irradiation challenge, thus impairing the protective effect of TNF-α against skin tumor development. Furthermore, reduction in the incidence of tumor lesions by naproxen may be due to its ability to increase TNF-α levels as well as to decrease PGE₂.     

THE PHOTOSYNTHETIC APPARATUS OF Acetabularia mediterranea GROWN UNDER RED OR BLUE LIGHT. BIOPHYSICAL QUANTIFICATION and CHARACTERIZATION OF PHOTOSYSTEM II and ITS CORE COMPONENTS

Abstract— The photosynthetic activity of white light-grown Acetabularia mediterranea Lamouroux (= A. acetabulum (L.) Silva) decreases under continuous red light to less than 20% within 3 weeks. Subsequent blue light reactivates photosynthesis within a relatively short period of 3 days. In a former publication (Wennicke and Schmid, Plant Physiol. 84 ,1252–1256, 1987) we have shown that the regulated rate limiting step, which is an immediate light driven reaction, is part of photosystem II (PS II). The following biophysical properties of PS II were analyzed in thylakoids isolated from algae grown 3 weeks under either blue or red light with or without subsequent 3 days of blue light illumination: (a) fluorescence induction in the short time domain dominated by Q_A reduction, (b) the slow fluorescence decline reflecting pheophytin photoaccumulation, (c) absorption changes at 320 and 830 nm under repetitive flash excitation as indicator for the turnover of Q_A and P₆₈₀, respectively, (d) oscillation pattern of the oxygen yield by a flash train in dark adapted samples and (e) the binding capacity for atrazine. None of these PS II functions were severely affected, but a minor impairment of20–30% was observed in the thylakoids from algae grown for 3 weeks in red irradiation. The changes do not fully account for the drastic reduction of the electron transport through PS II which was 80% after red light treatment. Therefore, the regulated rate-limiting step appears to not be mainly located in the PS II core complex itself. It seems likely that the regulation process predominantly comprises the antenna system.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.     

COMPARATIVE EFFECT OF UVA AND UVB ON CULTURED RABBIT LENS

Abstract Effects on lens physiology of UVB and UVA used separately and sequentially were investigated using 4 week old rabbit lenses in organ culture. Narrowband UVB at 0.3 J/cm²= joules/lens (1 h exposure) has little effect on sodium and calcium concentrations in the lens interior or transparency of lenses subsequently cultured for 20 h after a 1 h exposure. With an incident energy of 3 J/cm² of broadband UVB (295–330 nm), lenses become opaque and slightly swollen with significant ion imbalances during culture over a 1 day period. In contrast, lenses exposed to approximately 6–24 J/cm² of UVA (330–400 nm) remain transparent after 1 day of culture. Extended culture up to 4 days reveals no signs of opacification. Ion homeostasis and normal lens hydration are also maintained in UVA-irradiated lenses. The presence of 95% oxygen during UVA irradiation is also without effect. Broadband UVA irradiation is damaging, however, if lenses are first exposed to subthreshold doses of narrowband UVB (307 ± 5 nm) irradiation, viz . 0.3 J/cm². Thus, sequential UVB/UVA irradiation at subthreshold doses causes impaired active cation transport and accumulation of sodium and calcium accompanying lens opacification.     

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60-120 MW/cm²) of an oxygen-free aqueous solution of pyranine (8-hydroxypyrene-l,3,6-tri-sulfonate) by the third harmonic frequency of an Nd-Yag laser (355 nm) drives a two successive-photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ⁺). The oxidized product, φ⁺, is an intensely colored compound (Λ_max= 445 nm, ε= 43 000 ± 1000 M ⁻¹ cm⁻¹) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ⁺ is stable, having a lifetime of -10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H⁺. In alkaline solution the favored electron acceptor is the ground-state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M^-l cm⁻¹. The oxidized radical can be reduced either by φ^- or by other electron donors. The apparent second-order rate constants of these reactions, which vary from 10⁶ up to 10⁹M⁻¹ s⁻¹, are slower than the rates of diffusion-controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.