Characterization of Two Light-Harvesting Subunits Isolated from the Brown Alga Pelvetia canaliculata: Heterogeneity of Xanthophyll Distribution

The main light-harvesting fraction from Pelvetia canaliculata was isolated on a sucrose density gradient from digitonin-solubilized chloroplasts. After further solubilization by dodecyl maltoside, the bulk fraction was separated into two subunits by preparative isoelectric focusing. The more acidic brown fraction was mainly composed of 22 kDa polypeptides having an apparent pI of 4.55. Its pigment composition was very simple, containing chlorophyll (Chi) a, Chi c and fucoxanthin. The in vivo spectral properties of fucoxanthin, namely a shift in light absorption to the green and efficient energy transmission to Chi a, were conserved in this subunit. No xanthophyll associated with photoprotection was found in this band, even when obtained from photoinhibited thalli. The less acidic green band contained predominantly 22 kDa polypeptides that were resolved into numerous components by denaturing isoelectric focusing. Its pigment composition was more complex, containing, in addition, pigments of the so-called xanthophyll cycle. In photoinhibited thalli, about half of the violaxanthin was converted into antheraxanthin and zeaxanthin. All the pigments of the xanthophyll cycle were specifically associated with this subunit, and it may thus have a central role in the thermal dissipation of the absorbed light energy as postulated for light-harvesting complex II isolated from green plants.     

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light-induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light-induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.     

PICOSECOND FLUORESCENCE FROM PHYCOCYANIN 612

The biliprotein, phycocyanin 612, was purified from a cryptomonad, Hemiselmis virescens. The protein, which is an α₂β₂ dimer having four spectrally different tetrapyrrole chromophores, was studied using picosecond fluorescence by exciting the various chromophores at three wavelengths, 565, 585 and 615 nm. These wavelengths were chosen to excite selectively the three highest energy chromophores. Decay times were measured as the excitation energy migrated from each of the three excited chromophores to the lowest-energy chromophore. The ps decay times were found to be 9, 13, and 12 ps for excitations at 565, 585, and 615 nm, respectively. A comparison is made between phycocyanin 612 and phycocyanin 645 with regard to the causes of their differing absorption maxima.     

Videofluorometric Analysis of Aging Human Collagen

Abstract

The development of fluorescent pigments in aging human collagen has been observed, but neither the source of these compounds nor their nature has been described. Recently two distinct fluorophores were isolated from aging insoluble human collagen rich tissue following a sequence of proteolytic digestions and chromatographic separations. Using the videofluormeter, which monitors the fluorescence intensity of a sample as a function of several excitation and emission wavelengths, the fluorescence of the collagen rich tissue at various stages of the separation process was analyzed to determine the number of fluorescent components in each of the samples and estimate their fluorescence spectra. The analysis indicated that the isolated fluorophores were indeed single-component samples and that the insoluble collagen-rich fraction contains two major fluorophores whose spectra are consistent with the spectra of the isolated compounds.     

A NOVEL BACTERIORHODOPSIN ANALOGUE WITH CONFORMATIONALLY 6-s-cis FIXED RETINALS

Abstract— The artificial pigments of bacteriorhodopsin (bR) were synthesized with retinal analogues which have 6-s-cis fixed conformation. In order to study the shape of binding pocket around β-ionone ring of retinal, we used three different 6-s-cis fixed retinals (1–3), which have a gem-dimethyl group at a different position of the ring structure. These three 6-s-cis fixed retinals produced bR analogue pigments with absorption maxima at 574, 594 and 584 nm, respectively. These analogue pigments showed proton pumping activities upon incorporation into vesicles, indicating that 6-s-trans structure of retinal is not necessary to pump protons. Although these retinal analogues possess the same conjugated π-electron systems, the wavelengths of maximum absorbance of the analogue pigments were different from each other, indicating a distinctly steric interaction between retinal ring portion and apoprotein. The analogue pigments were also different from each other in regeneration rate and the number of the final products. These differences resulted from a difference in the position of the dimethyl substituent at the ring structure. Based on the results the molecular structure around β-ionone ring of retinal-binding pocket was discussed.     

EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS—XI PROTOCHLOROPHYLL(IDE) AND ITS PHOTOCONVERSION*

Abstract— The small amounts of chlorophyll precursors in dark-grown cells of Euglena gracilis Klebs var. bacillaris Pringsheim and the presence of contaminants which interfere with their purification have made their isolation and characterization difficult. We now extract cells with acetone: 0.1 M NH₄OH (9:1 v/v). Protochlorophyll is obtained by extracting this solution with petroleum ether (30–50° b.p.) and extracting this petroleum ether fraction with 80% acetone to remove substances which interfere with subsequent chromatography. Protochlorophyllide remaining in the original acetone: NH₄OH fraction is extractable into diethyl ether after adjusting the pH to approximately 5.5. Both pigments are verified and further purified by chromatography on Whatmann 3 MM paper using benzene:petroleum ether:acetone (30:10:1) or cellulose MN 300 thin layers with methanol:methylene chloride:water (100:18:20). These pigments resemble their well-described barley counterparts in solubility properties, spectral absorption maxima in ether (432, 530, 571 and 623 nm) and chromatographic behavior. Substantial amounts of protopheophytin and protopheophoribide are also found along with the Mg²⁺— containing pigments in cell extracts even when precautions are taken to avoid loss of Mg²⁺ during extractions and purification. Using heated cells before and chilled solvents after illumination to preclude enzymatic modification of pigments, chlorophyll and very small amounts of chlorophyllide are found as products of the photo-conversion of the protopigments suggesting that both protochlorophyll and, to a much smaller extent, protochlorophyllide are photoconvertible in these organisms. These properties join several others which suggest that the Euglena chorophyll-forming system more closely resembles that found in young bean leaves rather than the chlorophyll-forming system of the older leaf material usually studied.     

Extended cooperative electronic effects in poly((E,E)-[6.2]paracyclophane-1,5-diene)

Fluorescence spectroscopy of poly((E,E)-[6.2]paracyclophane-1,5-diene) shows the existence of several emission maxima which appear at wavelengths longer than that of the [3.2]paracyclophane repeat-unit excimer. These maxima appear to be emission bands due to “extended excimer” fluorescence from multiples of electronically interacting repeat units. Oxidation of the polymer by exposure to iodine vapor results in a material which exhibits a strong, asymmetric electron-spin-resonance spectrum with g = ca. 2.00 and a DC conductivity of 5 X 10^—4 S-cm^—1. These results are interpreted by a model in which segments of interacting radical cation salts occur pendant to and along the polymer chains but are of random length and orientation in the bulk polymer. Similar results were obtained for a structurally related polymer containing [3.3]paracyclophane rings.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

Comparison of the action spectra and quantum requirements for photosynthesis and photo-reduction of Scenedesmus

Abstract -The action spectrum and the quantum requirement of photoreduction (P.R) by hydrogen-adapted Scenedesmus have been determined under conditions where photosystem II is inoperative. The action spectrum of P.R., when compared to that of photosynthesis, shows approximately a 20 nm shift in the long wavelength maxima and an apparent decreased utilization of light in the wavelength region 600–650 nm. The quantum requirement for P.R. is high at wavelengths below 650 nm and decreases to a minimum between 705–720 nm; the best value obtained was 10. These observations indicate that this process is exclusively a photosystem I reaction and furthermore offer additional evidence for a lack of appreciable transfer of excitation energy between the pigments of photosystems I and II. The relatively high requirement photophosphorylation or to the inadequacy of H₂-hydrogenase system as the electron donor mechanism for photosystem I.     

THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.     

Viscosity of Aqueous Solutions of n-butylamine,sec-butylamine and tert-butylamine

Abstract

Viscosities of the systems, water (W) + n-butylamine (NBA), W + sec-butylamine (SBA) and W + tert-butylamine (TBA) have been measured in the temperature range 298.15–323.15K. The viscosities (η) and excess viscosities (η^E) have been plotted against mole fraction of amines (X ₂). On addition of amines to water, viscosities first increase rapidly, then pass through maxima at 0.2 mole fraction of amines and then decline continuously as the addition of amines is continued. η^E show large positive values, with maxima also at 0.2 mole fraction of amines. The maxima of the curves of η and η^E vs. mole fraction of butylamines follow the order, W + TBA > W + SBA > W + NBA. The ascending part of the η vs. X ₂ curves in the water-rich region is explained by the hydrophobic hydration caused by the hydrocarbon tails and the hydrophilic effect due to — NH₂ group of amines. Following the maxima, amine - amine association is preferred, which accounts for the steady decrease of viscosity up to the pure state of amines.     

ACTION SPECTRA FOR HYPHAL AGGREGATION,THE FIRST STAGE OF FRUITING,IN THE BASIDIOMYCETE Pleurotus ostreatus*

Abstract. Action spectra were determined for hyphal aggregation in Pleurotus ostreatus at wavelengths between 360 and 600 nm. The action spectrum for a 50% response had two maxima, one at 370 nm in the near-UV and the other a broad peak at 440–450 nm in the blue. Both were approximately of the same magnitude. A minimum was present at 400 nm and wavelengths greater than 530 nm invoked no response. Action spectra for a range of responses, 10–90%, were also determined which showed gradual changes in the peaks in the blue region. It was concluded that hyphal aggregation in P. ostreatus is under the control of a cryptochrome-like photoreceptor system.     

Ring oxidized retinals form unusual bacteriorhodopsin analogue pigments.

Three ring oxidized retinal analogues have been isolated from the exhaustive oxidation of all-trans retinal. All-trans 4-oxoretinal and 2,3-dehydro-4-oxoretinal have similar absorption maxima to that of all-trans retinal and have been shown to be in the 6-s-cis conformation in solution. Pigments formed with bacterioopsin exhibit absorption maxima (520 nm) blue-shifted from that of bacteriorhodopsin (bR), indicating a disturbance of the external point charge by the electronegative carbonyl moiety at the 4 position. The third analogue contains a ring contracted to a cyclopentenyl-alpha,beta-dione. Unlike the majority of retinals, this analogue displays a 6-s-trans conformation in solution and has a red-shifted absorption maximum at 435 nm. The resulting bR analogue pigment (515 nm) is formed five times faster than the other oxoretinal pigments. All three oxoretinal pigments show an irreversible 20 nm blue shift upon exposure to white light. The 4-oxo and 2,3-dehydro-4-oxoretinal pigments, after irradiation, undergo a small reversible blue shift (4-8 nm) on dark adaptation. These two pigments pump protons, although with slowed photocycle kinetics, demonstrating that these structural changes (addition of the carbonyl at the C-4 and insertion of a double bond in the ring) do not block the function of the pigment. Extraction of the C-15 tritiated analogue retinals from illuminated and non-illuminated pigments of all three oxoretinals yield identical results. Therefore, any crosslinking of these oxoretinals to the protein is by linkages which are unstable to the extraction procedures.     

PHOTOSYNTHETIC ACTION SPECTRA AND LIGHT-HARVESTING IN GRIFFITHSIA MONILIS (RHODOPHYTA)

Abstract— In cells of the red alga Griffithsia monilis the action spectrum of photosynthetic oxygen production at low light intensity shows that the phycobilins (including allophycocyanin) are the major light-harvesting pigments. As the light intensity is increased carotenoids and chlorophyll a contribute proportionately more to the spectrum, since the phycobilin activity becomes light-saturated. When action spectra are performed against a background light of various monochromatic wavelengths it can be shown that chlorophyll a increases in its light-harvesting activity. Nevertheless light absorbed at a single wavelength (487 nm) by phycoerythrin (and possibly a carotenoid) still shows the highest photosynthetic activity. Fluorescence measurements at 77K indicate that a chlorophyll a fluorescence is small and that the amount of chlorophyll a _ll (f 693) is very low. A model is proposed in which the phycobilins, in phycobilisomes, pass on absorbed light energy to either photosystem, whereas light absorbed by chlorophyll is passed on mainly to photosystem I.     

A Combined Experimental and Theoretical Approach toward the Development of Optimized Luminescent Carbostyrils

The synthesis and photophysical data of new carbostyrils (quinoline‐2(1H)‐ones) with the longest hitherto observed absorption‐ and emission wavelengths are described. Introduction of 6‐amino, 7‐MeO, and 4‐(CF₃) substituents enabled us to rise the absorption and fluorescence maxima up to 414 and 557 nm, respectively. Supported by semi‐empirical and ab initio calculations, the 6,7‐(1,4‐diazine)‐fused carbostyril 23b displayed absorption maxima at up to 440 nm, with quantum yields of up to 0.9 and large Stokes shifts (>100 nm), comparable to the best coumarin chromophores known. The new fluorophore is neither pH‐sensitive between pH 6 and 10 nor susceptible to O₂ quenching. At pH 3, the emitted light appears greenish‐white, which arises from three different stages of protonation.     

ACTION SPECTRUM FOR ENHANCEMENT OF PHOTOTROPISM BY Arabidopsis thaliana SEEDLINGS

Fluence-response relationships have been measured at wavelengths from 350 to 760 nm for the enhancement of phototropism in Arabidopsis thulium L. (Heynh) strain “Estland” by an irradiation at each of these wavelengths, given 2 h prior to a 450 nm inductive unilateral irradiation. Action spectra have been constructed from these fluence-response relationships based on: (i) the fluence required to obtain a curvature of 25° (corresponding to an enhancement of 15°), (ii) the fluence required to obtain 50% of the maximum enhancement and (iii) the fluence threshold for enhancement by a pre-irradiation. The action spectra exhibit two maxima, one at 669 nm and a second at 378 nm. The height of the maximum at 669 nm is approximately 4 times the height of the maximum at 378 nm. Based on the action spectra, it is concluded that the enhancement of phototropism in A. thaliana is mediated by phytochrome.     

The Use of High Pressure Liquid Chromatography for the Identification and Preparation of Pigments Concerned in Photosynthesis

Abstract

The chlorophylls and carotenoids present in preparations from chloroplasts of marine algae can be extracted and separated by high pressure liquid chromatography (H.P.L.C.). The reverse-phase columns; Partisil 10 ODS (Whatman), μ Bondapak C18 and μ Bondapak CN (Waters Associates) gave good separation of the different pigments. Addition of the ion-pairing agent tetrabutylamnonium phosphate to the methanol/water solvents gave improved separations with complex mixtures and identification was facilitated by examination of the column eluant at 440nm where a ll the pigments absorbed and at 650nm where only chlorophylls absorbed. The method allowed the isolation of individual pigments for further study.     

Photochemistry of Biliverdin IX δ as a model for the study of the photoproducts from natural biliverdin IXγ (pterobilin)

The configurations of biliverdin-IX_γ and -IX_δ dimethyl esters 1 and 2 in solutions, have been studied using Nuclear-Overhauser-Effect (NOE) experiments. Irradiation (500-700 nm) of biliverdin IXδ gave syn-Z → anti-E isomerization of the central methine bridge and in aerated polar solutions, four new cyclized pigments were isolated for which structures 3, 4, 5 and 6 are proposed. Neobiliverdin IXδ 3 is also formed in degassed solution (Φ=4.10^?5) but pigments 4, 5 and 6 arise from photo-oxidation with O₂. Biliverdin IXδ appears to be a good model for the study of photo-reactions occurring on the vinyl groups of the natural biliverdin IXγ (pterobilin).     

Ion recombination luminescence in polymer glasses induced by electron irradiation. I. High-vinyl polybutadiene

The luminescence of 1,2-polybutadiene was measured during and following irradiation with 1.4-MeV electrons at 90 °K. Measured under isothermal conditions, the light intensity at first increases sharply on start-up of irradiation, and then reaches a plateau after several seconds. The equilibrium value was found to be linearly dependent on the dose rate employed. The postirradiation luminescence decay measured at 90°K follows second-order kinetics. The light emitted by the irradiated sample during warm-up to room temperature is characterized by the appearance of four intensity maxima at 118, 168, 223, and 261 °K. The first three peaks are attributable to the onset of local motion, whereas the peak at 223 °K is caused by the long-range motion occurring at the glass-to-rubber transition. A preirradiation of the polymer in vacuo causes an increase in these four peaks as well as the formation of a new maximum at 143 °K. Intermittent exposure of the irradiated sample to light of wavelengths λ > 450 nm causes the isothermal luminescence decay to be interrupted by a burst of light emitted by the sample during and shortly following the light exposure. This treatment also results in considerably reduced intensity maxima during warm-up to room temperature. An interpretation of the findings reported is given on the basis of reactions involving primary and secondary transient species produced during irradiation and on the molecular parameters of the polybutadiene.     

Viscosity of aqueous solutions of some diamines

Viscosities of the systems, ethylenediamine (ED) + water (W), 1,2-diaminopropane (DAP) + W, trimethylenediamine (TMD) + W and N,N-dimethyltrimethylenediamine (DMTMD) + W, have been measured for the whole range of composition at temperatures ranging from 303.15 to 323.15?K. The viscosities have been plotted against the mole fraction of diamines, showing some common characteristics for all the systems. On addition of diamines to water viscosities rise up very rapidly and pass through maxima. Following the maxima viscosities decline quite rapidly and monotonously up to about 0.6 mole fraction of diamines, which then fall down rather slowly. The hydrophobic and hydrophilic effects are considered responsible for the ascending part of viscosities in water-rich region, although the former is thought to be much more predominant than the latter. The height of viscosity maxima and their shifting to more water-rich region have been explained preferentially in terms of the hydrophobic effect. The declining part of viscosities is thought to be due to continuous destruction of cage structures formed by hydrophobic interaction. The excess viscosities have been calculated and plotted against mole fractions of diamines. The characteristics of excess viscosities have been explained by the same reasonings as used to explain viscosities.