Showing papers in "Photochemistry and Photobiology in 1982"

Erythema and melanogenesis action spectra of normal human skin

Abstract: The action spectra for delayed erythema and melanogenesis in Caucasian human skin are determined for wavelengths between 250 and 435 nm. The untanned skin of very fair volunteers was observed after single exposures to a range of fluences of monochromatic radiation. At wavelengths longer than 300 nm the two action spectra are indistinguishable, and at wavelengths shorter than 300 nm, they are of similar shape despite a distinct separation. This suggests a common or similar chromophore for initiation of the vascular and pigmentary responses to UV. A broad minimum in the action spectra occurs near 280 nm, a maximum near 296 nm, and for wavelengths longer than 300 nm, increasingly larger fluences of radiation are required to induce delayed erythema and melanogenesis. Between 304 and 334 nm both action spectra exhibit a rapid decrease of almost three orders of magnitude. In contrast, between 334 and 405 nm the spectra decrease by only one order of magnitude, and there is a suggestion of a small maximum at or near 365 nm. Different chromophores, sites of damage, or response mechanisms may be responsible for induction of delayed erythema at these longer wavelengths. After spectral corrections for the optical effects of the stratum corneum, the shape and magnitude of the action spectra are grossly consistent with a mechanism in which DNA is the primary chromophore initiating the response pathways for wavelengths less than 313 nm. Whatever the actual basis for these action spectra may be, they are of practical significance in predicting skin response to sources of ultraviolet radiation.

THE Py SCALE OF SOLVENT POLARITIES. SOLVENT EFFECTS ON THE VIBRONIC FINE STRUCTURE OF PYRENE FLUORESCENCE and EMPIRICAL CORRELATIONS WITH ET and Y VALUES

Abstract: — Pyrene fluorescence spectra have been run in 62 solvents of widely differing solvent polarity. As has been noted previously, the intensity ratio of the first (the 0–0 band) and third bands in vibronic fine structure of these spectra are very sensitive to solvent polarity. These I1/I3 values, however, are not sensitive to hydrogen bonding aspects of solvent-solute interactions. Correlations are reported with Winstein's Y values and with Dimrotb's ET values. On this basis the I1/I3 values for pyrene fluorescence are suggested as the basis for a new empirical scale of solvent polarity, called the Py scale, which offers certain conveniences over other scales of solvent polarity.

Spectral perturbations of the aequorea green-fluorescent protein

Abstract: — In the jellyfish Aequorea, the green-fluorescent protein (GFP) functions as the in vivo bio-luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has indicated that the Aequorea GFP is a relatively inflexible protein. Present evidence, however, indicates that the chromophore environment is readily accessible to a variety of external perturbants. Native Aequorea GFP has an absorbance maximum at 395 nm and a shoulder at 470 nm. In low ionic strength buffer at neutral pH and room temperature the 395/470 nm absorbance ratio is about 2.0. We show that this ratio is highly variable depending upon temperature, ionic strength, protein concentration, and pH. A maximum ratio of 6.5 (at a protein concentration of 18.6 mg/m/) and minimum of 0.42 (at a pH of 12.2) have been measured. In the latter case, the resulting absorption and excitation spectra resemble those of Renilla GFP in spectral shape (but not wavelength maximum). In all cases as the perturbant is varied the resulting spectra pass through a sharp isosbestic point, suggesting a relatively simple two-state mechanism. These spectral perturbations are fully reversible. On the basis of these results, we suggest that the chromophore binding site is conformationally flexible. pH-Dependent changes in the near-UV and visible circular dichroism spectra plus spectrophotometric titration of tyrosine residues lend additional support to this hypothesis.

Design and performance of the Okazaki Large Spectrograph for photobiological research.

Abstract: A computer-operated spectrograph was recently built at Okazaki, Japan. Different specimens can be placed on a horseshoe-shaped focal curve (10 m long) covering a wavelength range of 250 to 1000 nm so they can be irradiated simultaneously. The linear dispersion is about 0.8 nm/cm. The photon fluence rate on the focal curve is 5 x 1015. photons x cm-2 x s-1 at 300nm and 1 x 1016 photons x cm-2 x s-1 at 600 and at 900 nm. The spectral half width is 5.5 nm or less on the focal curve. The stray light content is about 10-5 of the main peak at the peak wavelength ± 100 nm. Specimens are set in microcomputer-controlled threshold boxes so that wavelengths, photon fluence rates, photon fluences and timing of irradiations are controlled automatically according to a pre-programmed schedule. An optical fiber system is also provided for remote irradiations.

EFFECTS OF SOLVENT ON THE FLUORESCENCE PROPERTIES OF BACTERIOCHLOROPHYLL a

Abstract: Fluorescence lifetimes (τf) of bacteriochlorophyll a (BChl a) have been measured by the method of time-correlated single-photon counting on dilute (1 μM) solutions of the pigment in 15 solvents. There is a pronounced dependence of τf on the nature of the solvent. Specifically, τf, is longer when the central magnesium is hexacoordinated than when pentacoordinated and shorter when the macrocycle is hydrogen-bonded than when it is not, but the latter effect is more pronounced. Both trends were confirmed by parallel studies on bacteriopheophytin a (BPheo a). Because of the short lifetimes (˜ 2.2–3.6 ns), quenching of fluorescence by molecular oxygen is not a significant factor in aerated solutions of the bacterial pigments. However, reabsorption artifacts are non-negligible, which necessitates studies on dilute solutions. Fluorescence quantum yields (of) have been estimated for BChl a in 13 solvents by comparing the observed fluorescence lifetimes with the radiative lifetimes calculated from the integrated absorption spectra.

A STUDY OF THE KINETICS OF INCLUSION OF HALONAPHTHALENES WITH ß-CYCLODEXTRIN VIA TIME CORRELATED PHOSPHORESCENCE

Abstract: — The phosphorescence of 1-bromonaphthalene and 1-chloronaphthalene is readily observable in nitrogen purged aqueous solutions containing s-cyclodextrin. Addition of acetonitrile increases both the phosphorescence intensity and lifetime. The quenching of halonaphthalene phosphorescence in aqueous solution by nitrite is substantially inhibited upon addition of s-cyclodextrin, as a result of a guest-host complex. The rate constants for formation and dissociation of the l-bromonaphthalene/s-cyclodextrin complex are evaluated from an analysis of the dependence of phosphorescence lifetimes on nitrite concentration.

Exciton migration and trapping in photosynthesis

Abstract: — Mobile electronic excited states, excitons, undergo random walks through the antenna chlorophyll arrays of photosynthetic organisms. The time interval from exciton creation, by photon absorption, until its first arrival at a reaction center (RC) is called the “first passage time” (FPT) of the random walk. A theory of exciton migration and trapping presented here predicts that the exciton lifetime, as measured from chlorophyll fluorescence decay in chromatophores or P700 complexes, is a linear function of the fractional number of quanta absorbed directly by the antenna, not by the RC. The slope of this line is the FPT, and its intercept is the exciton's lifetime as limited only by photoconversion at the RC. This photoconversion-limited lifetime is simply related to the in situ photoconversion rate constant via two parameters, each of which is experimentally accessible. It is also possible to obtain values of individual FoUrster rate constants, at least approximately, from measurements of exciton lifetime as functions of temperature and excitation wavelength. This new theory, based on lattice random walk models, receives some support from fluorescence measurements done on Rhodopseudomonas sphaeroides R26 chromatophores. In its present form the theory is only applicable to one-antenna-component systems, like Rp. sphaeroides R26 or Rhodospirillum rubrum chromatophores or P700 complexes, but should be readily extendible to multi-antenna-component systems including whole chloroplasts.

The effect of blue light on plants and microorganisms

Abstract: The growing interest in blue light-induced phenomena in plants and microorganisms is reflected by the increasing research activity in this field. This review tries to cover the publications after the recent appearance of the Proceedings of the “1st International Symposium on the Effect of Blue Light in Plants and Microorganisms” [lo91 and the reviews on “Blue Light Receptors: Primary Reactions and Subsequent Metabolic Changes” [l lo] and “Physiological Blue Light Reception” [loo]. Research on blue lightinduced phenomena focusses on a wavelength region between 350 and 500 nm but sometimes overlaps with the widely studied effects of UV on the one and the more recently discovered induction of morphogenetic activity by green light on the other side of the spectrum.

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.

Microspectrofluorometric approach to the study of free/bound nad(p)h ratio as metabolic indicator in various cell types

Abstract: Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δIf) due to microinjection of glucose-6-phosphate (G6P) into EL2 ascites cells. Above 510 nm, δIF is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δIF level. The difference between δIf and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δIF can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component. The questions raised by the lack of such a component are answered using a new microspectrofluorometer, which aiiows correlated monitoring of NAD(P)H and flavins with excitations at 365 and 436 nm, respectively. As detected by excitation at 436 nm, injections of G6P, malate, ADP, and treatments with azide, cyanide or partial anaerobiosis, all indeed show a redox change of flavins, in the sense of decreased emission. It is understandable, however, that such a change which is not very large even using 436 nm excitation should remain undetected when flavins are excited at 365 nm, i.e. using the tail of their excitation spectrum. In contrast to the increased δIF spectrum recorded in response to injected substrate, the initial spectrum (If) of the cell prior to a metabolic perturbation reveals a third component, even with 365 nm excitation. The position and reactivity of this component shows flavin-like properties. The structural resolution attainable makes it possible to obtain the evaluation of free vs. bound NAD(P)H and flavin fluorochromes in the mitochondrial and cytosolic compartments of the intact cell.

SINGLE‐STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract: — The induction of single-strand breaks (alkali-labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far-UV (below 320 nm) component and a minor near-UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near-UV (above 320 nm) wavelengths faster than the wild-type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali-labile bonds induced by 365-nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali-labile bonds, probably apurinic and apyrimidinic sites.

Chloroplast membrane protein phosphorylation

Abstract: Photosystem (PS)*II and PS I operate in series during noncyclic photosynthetic electron transport in order that the chloroplast can generate both ATP and NADPH. For this to occur at optimal quantum efficiency, in sub-saturating light, the two photosystems must operate at approximately the same turnover rate. Chloroplasts have a mechanism which corrects for imbalance in excitation of PS I and PS I1 [20. 25, 51, 67, 681; these adaptive changes are referred to as “State” transitions of the pigment bed [52]. State I is the adaptive conditon which results from over-excitation of PS I; State I1 results from over-excitation of PS 11. The biochemical events underlying State I-State I1 transitions have, until recently, been poorly understood. Approximately 60% of the total thylakoid chlorophyll (Clil) is associated with the lightharvesting pigment-protein complex with serves photosystem 11. This is designated as LHC-I1[39]. It has been estimated that one LHC-I1 structural unit (see Fig. 1D) consists of 4-7 individual polypeptides of 25-29 kdaltons, each of which binds 6-13 Chl a + b in the native complex [21,44,49]. A surface-exposed segment of the LHC-I1 (amino-acid sequence shown in Fig. 1D) [50] is required for the formation of grana stacks [22, 57, 581 and regulation of State I-State 11 transitions. When intact chloroplasts are incubated in the light with [32P]-orthophosphate, the isotope is incorporated into several membrane polypeptides [14, 15, 561. Among the most heavily labelled polypeptides were those of LHC-11. This labelling was found to be reversible [15, 171. From these initial observations on light-induced protein phosphorylation, Bennett [ 151 hypothesized probable effects of this covalent protein modification upon excitation energy tranfer and electron transport. A hypothesis explaining the biochemical basis of state changes was recently presented [18,32]: LHC-I1 phosphorylation was suggested to cause State I-State I1 transitions. In this review we present a summary of the evidence which has accumulated in support of this concept. We will also described the activation control of the protein kinase which

Photoionization of melanin precursors: an electron spin resonance investigation using the spin trap 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (dmpo)

Abstract: The photoionization of 3,4-dihydroxyphenylalanine (dopa) and catechol has been studied by electron spin resonance spectroscopy using the free radical scavenger 5,5-dimethyI-1-pyrroline-1 -oxide as a spin trap for hydrated electrons and hydrogen atoms. The photochemistry of these materials is shown to resemble tyrosine in that both photoionization and photohomolysis (to give H) occur, with photoionization predominating (by a factor of 2.6 for dopa). Ionization of one of the phenolic hydroxyl groups increases the yield of radicals by a factor of 2. Action spectra and quantum yields for radical production are reported.

FLUORESCENCE LIFETIMES OF CHLOROPHYLL a: SOLVENT, CONCENTRATION AND OXYGEN DEPENDENCE

Abstract: Fluorescence lifetimes (τf) of chlorophyll a (Chi a) have been measured by the single-photon-counting technique over a wide range of concentrations (˜10-7˜10-4M) in deoxygenated pyridine, diethyl ether, toluene and methanol. At pigment concentrations ˜1 μM, reabsorption of fluorescence induces significant artifacts on measured values of τf which are dependent on detection wavelength and the specific geometry of the experiment. There is a clear dependence of τf on the nature and degree of solvation, including both coordination of the central magnesium and hydrogen-bonding of the solvent (viz. alcohols) to the macrocycle. Quenching of the excited singlet state by molecular oxygen was measured quantitatively in ether, and a bimolecular rate constant markedly slower than the diffusion-controlled limit was obtained.

Membrane lysis in chinese hamster ovary cells treated with hematoporphyrin derivative plus light

Abstract: Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [3H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.

Lethality and the induction and repair of DNA damage in far, mid or near UV-irradiated human fibroblasts: comparison of effects in normal, xeroderma pigmentosum and Bloom's syndrome cells.

Abstract: Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation. In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.

Ultraviolet light: photosensitivity and other effects on the visual system.

Abstract: It is generally stated in elementary lectures on sensory systems that the limits of the visible spectrum are 400 nm (violet) and 700 nm (red), although there are several exceptions to this generalization. Recently, the properties of ultraviolet (UV) vision in invertebrates, as well as vertebrates, have been better characterized, and it is worthwhile to examine visual phenomena in the UV region of the spectrum. The purpose of this review is to integrate what is known about UV vision in invertebrates, which have been particularly well studied, with a growing literature on UV sensitivity and UV effects in the visual systems of humans and other vertebrates.

PIGMENT ORGANIZATION IN THE LIGHT‐HARVESTING CHLOROPHYLL‐a/b PROTEIN COMPLEX OF LETTUCE CHLOROPLASTS. EVIDENCE OBTAINED FROM PROTECTION OF THE CHLOROPHYLLS AGAINST PROTON ATTACK and FROM EXCITATION ENERGY TRANSFER

Abstract: — The light-harvesting Chl-a/b protein complex (LHC) of Lactuca sativa L. was examined for pigment content, excitation energy transfer and behavior under acidic conditions: (1) Lettuce LHC contains Chl-a, Chl-b and xanthophylls (lutein, neoxanthin, lactucaxanthin, viola-xanthin) at a molar ratio of 6:4:3; their contribution to the absorbance of the LHC between 390 and 530 nm is estimated to be about 31% (Chl-a), 26% (Chl-h) and 43% (xanthophylls). (2) Energy transfer from xanthophylls and Chl-fe to Chl-a takes place at 100% transfer efficiency. (3) LHC exhibits an unusual acid stability: in contrast to complexes of photosystem I or II, LHC-bound chlorophylls are not converted to phaeophytin and LHC apoprotein is not denatured at pH 1.5; also, energy transfer is maintained. (4) Pronase or trypsin treatment do not affect acid stability and energy transfer. (5) Treatments that break down acid stability (heat, urea or TritonX–100) also inhibit energy transfer. The coincidental breakdown of energy transfer and acid stability points at one underlying process, namely, the breakdown of a structure that enables protection of chlorophylls from proton attack and close contiguity of xanthophylls and chlorophylls as required for energy transfer. Dense packing of xanthophylls and chlorophylls within lipophilic crevices of the LHC is suggested.

ANALYSIS OF Pfr DESTRUCTION IN AMARANTHUS CAUDATUS L EVIDENCE FOR TWO POOLS OF PHYTOCHROME

Abstract: — Kinetics of the destruction of the far red absorbing form of phytochrome (Pfr), measured by in vivo spectroscopy, show two phases: after a saturating red light pulse, rapid first order decay results in the loss of most, but not all, of the detectable Prr; decay of the rest is much slower. The concentration of the more stable Pfr is positively correlated to the concentration of the total Pfr established at time zero. The linear relationship between total and ‘stable’ Pfr exludes the existence of a threshold level of Pfr for fast destruction. Photoconversion of the Pr (red absorbing form of phytochrome) present during the slow decay, by exposure to a second light pulse, is followed by fast destruction of most of the newly formed P,r, whereas some Pfr formed by the first pulse still remains. The experiment suggests that not all Pfr molecules are accessible to the same destruction mechanism, i.e. there are two populations of PfI.

A climatology of sunburning ultraviolet radiation.

Abstract: — Data are presented from 14 sites where continuous measurements of the sun's shortest ultraviolet radiation reaching the earth's surface have been made for four or more years. Average daily dose per month and its variability from year to year is shown for each station. Some of the many influences affecting these measurements can be discerned by station intercomparisons. No consistent long term change in solar UV-B radiation reaching the ground is evident.

ANALYTICAL CHARACTERIZATION OF SPECTRAL ACTINIC FLUX and SPECTRAL IRRADIANCE IN THE MIDDLE ULTRAVIOLET

Abstract: — We present an analytic characterization of upward and downward diffuse spectral irradiance for the wavelength range 280–380 nm, solar zenith angle range from 0 to 86, altitude range from 0 to 5 km and for non-zero surface albedo. This work is a modification and extension of the previous work of Green, Cross and Smith based upon the radiative transfer calculations of Braslau, Dave and Halpern. Guided by these irradiance systematics we develop an analytic characterization of diffuse spectral scalar irradiance or actinic flux also broken down into upward and downward components for the above wavelengths, solar zenith angles and altitudes, for non-zero surface albedo utilizing the actinic flux calculations of Peterson.

Mechanisms for dye‐mediated photodynamic action: singlet oxygen production, deoxyguanosine oxidation and phage inactivating efficiencies

Abstract: The production of singlet oxygen (1O2) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6-tetramethyl-4-piperidone as 1O2 acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of 1O2 production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of 1O2 was not observed with 9-aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the 1O2 production and (ii) the bacteriophage OX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated 1O2. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.

PHYTOCHROME‐MEDIATED PHOTOTROPISM AND DIFFERENT DICHROIC ORIENTATION OF Pr AND Pfr IN PROTONEMATA OF THE FERN ADIANTUM CAPILLUS‐VENERIS L.

Abstract: — Single-celled protonemata of Adiantum capillus-veneris were cultured under continuous red light for 6 days and then in the dark for 15 h. Brief local exposure of a flank (5 times 20 /mi) of the subapical region of a protonema to a microbeam of red light effectively induced a phototropic response toward the irradiated side. The degree of the response was dependent upon the fluence of the red light. Red/far-red reversibility was typically observed in this photoreaction, showing that phytochrome was the photo-receptive pigment. When the flank was irradiated with a microbeam of linearly polarized red and far-red light, red light with an electrical vector parallel to the cell surface was most effective. However, the far-red light effect was most prominent when its electrical vector was normal to the cell surface. These polarized light effects indicate the different dichroic orientation of Pr (red-light-absorbing form of phytochrome) and Pr (far-red-light-absorbing form of phytochrome) at the cell flank.

Photooxidation and singlet oxygen sensitization by protoporphyrin ix and its photooxidation products

Abstract: Protoporphyrin IX and its various ester derivatives have been previously shown to undergo self-sensitized photooxygenation to yield hydroxyaldehydes (photoprotoporphyrin) and mono- and diformyl deuteroporphyrin derivatives. In the present study the photoreactions of these products in the presence of oxygen have been investigated. All of the photooxidation products are themselves good sensitizers of singlet oxygen. In addition spin trapping experiments indicate these products can produce superoxide in low-to-moderate efficiency by an excited state electron transfer process. The photo-products themselves are somewhat more stable to photooxidation than protoporphyrin IX itself. The two monoformyl-monovinyl deuteroporphyrins have been found to undergo further photooxidation at the vinyl groups to yield primarily monoformyl hydroxyaldehydes in a reaction mainly involving singlet oxygen analogous to the initial reaction of protoporphyrin IX.

Chlorophyll fluorescence in green plants and energy transfer pathways in photosynthesis

Abstract: The photosynthetic apparatus consists of several pigment-protein complexes which are involved in light absorption and subsequent transfer of excitation energy to the reaction centers where photochemical charge separation takes place. In green plants chlorophylls (Chl)* are the major pigments of these complexes and are directly involved in light absorption, energy transfer and photochemistry. Obviously Chl fluorescence has become an important probe in understanding various photosynthetic processes. Some of the Chl fluorescence characteristics (emission spectrum and fluorescence induction during illumination) have been studied in several laboratories and a tripartite model of the photosynthetic apparatus has been proposed [20]. According to this model a light-harvesting protein-pigment complex (LHCP) absorbs the incident light and distributes between the two photosystems (PS I1 and PS I) , each of which consists of a reaction center and antenna Chl complexed with proteins. The experimental and theoretical approaches to this model have been reviewed [18, 191. Chlorophyll fluorescence is used to monitor a wide variety of phenomena and properties; it is not possible to discuss all aspects within the limit of this review. In this review, discussion will be restricted to two aspects of Chl fluorescence; (i) characterization of the emission bands in terms of the pigment-protein complexes involved in emission or in energy transfer, and (ii) picosecond fluorescence kinetics related to energy transfer between pigment-protein complexes. The final goal of the review is to convey that the experimental approaches involved in the two above-mentioned aspects of Chl fluorescence studies should be combined in future research for a better understanding of the role of the pigment-protein complexes in energy transfer.

Determination of parameters of excited states of DNA and RNA bases by laser UV photolysis

Abstract: — The mechanism of photodecomposition of nucleic acid bases in a neutral aqueous solution upon two-step excitation of high-lying electronic states by a powerful laser UV radiation is discussed. Experimental dependences of photodecomposition efficiency versus UV radiation intensity are measured both under picosecond and nanosecond laser UV irradiations. By comparison of experimental dependences with a theoretical model, we obtain some characteristics of excited states, such as lifetime t1 of the first electronic excited state S1 intersystem crossing yield φ, photosensitivity from an intermediate excited state and others for all five nucleic acid bases.

Direct recording of the initially excited and the solvent relaxed fluorescence emission spectra of tryptophan by phase sensitive detection of fluorescence.

Abstract: Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent-relaxed emission spectra of N-acetyl-L-tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation. The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.

Photodynamic effects induced by furocoumarins on a membrane system. Comparison with hematoporphyrin.

Abstract: Isolated rat liver mitochondria have been used as a model to investigate the photodynamic action of psoralen (Ps) and 4,5′,8-trimethylpsoralen (TMP) on cellular membrane systems in comparison with hematoporphyrin (Hp). Oxidation was detected by the consumption of free oxygen as measured polarographically in the respiratory chamber when irradiated with UV light (320-380 nm). In the presence of Ps, singlet oxygen was produced in the respiratory medium but neither the respiration nor the oxidative phosphorylation were affected. On the contrary the hydrophobic derivative TMP impaired the respiration with rapid uncoupling of oxidative phosphorylation as did Hp. The ineffectiveness of Ps as well as the effectiveness of TMP vs. Hp is explained on the basis of photophysical properties of the molecules and their partition coefficient. These results may indicate that, in the photochemiotherapy of skin diseases, furocoumarins can drive photodynamic reactions at various subcellular levels according to their hydrophobicity.

Model studies on photodynamic cross‐linking

Abstract: — A model system is described for studying photodynamic protein cross-linking. It appeared that several different types of cross-links are possible between photooxidized amino acid residues—especially histidine—and functional groups in proteins.

MODE OF ACTION OF α‐TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

Abstract: The photodynamic effects of sc-terthienyl (scT) in near-UV light (UV-A) on Escherichiu coli showed close agreement with the light absorption of scT at different wavelengths suggesting that rT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of otT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. co(i and a more resistant bacterium, Pseudomonas aeruginosa, was absolutely dependent on the presence of oxygen. The rate of killing by otT/UV-A showed a rather small dependence on preincuba- tion temperatures. with quite rapid killing at 5°C. suggesting that the movement of otT across the cytoplasmic membrane of E. c-oli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30 min of treatment with rT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that rT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.