EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue-gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlstein's theory of energy migration devised for a three-dimensional photosynthetic unit. It was also shown that Paillotin's theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.     

NUMBER AND DISTRIBUTION OF CHROMOPHORE TYPES IN NATIVE PHYCOBILIPROTEINS

Abstract— Fluorescence lifetimes and absolute quantum yields of a number of chromatographically pure phycobiliproteins have been determined. In conjunction with absorption, fluorescence emission and polarization spectra, these data were used to calculate the number of different types of chromophore, sensitizing and fluorescing, per chromoprotein.
To characterize the spatial distributions of the chromophores, the observed emission anisotropies were compared with those calculated from models, using the Förster transfer mechanism and the Jablonski 'active sphere' approximation. The experimental values are more consistent with surface locations for the fluorescing chromophores rather than with their distribution throughout the volume.
Theoretical efficiencies of transfer between sensitizing and fluorescing chromophores on the same macromolecule are consistent with those observed. The transfer efficiency from phycoerythrin prosthetic groups to chlorophyll a compared with that for transfer via phycocyanin indicates that the latter process is probably the favoured migration route.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

EXCITATION ENERGY TRANSFER IN THE CHROMATICALLY ADAPTED PHYCOBILIN SYSTEMS OF BLUE-GREEN ALGAE: DIFFERENCE IN THE ENERGY TRANSFER KINETICS AT PHYCOCYANIN LEVEL

Abstract— Excitation energy transfer in chromatically adapted phycobilin system was investigated with the blue-green algae Tolypothrix tenuis and, supplementary, Fremyella diplosiphon with use of time-resolved fluorescence spectrum (Yamazaki et al , 1984). Special attention was paid to the energy migration at the phycocyanin (PC) level in the phycoerythrin (PE)-rich and PE excited system and in the PE-less and PC excited system. The energy transfer from PC to allophycocyanin was far faster in the former than in the latter in both organisms. Such feature was the same as our previous observation for PE-rich system of Porphyridium cruentum and PE-less system of Anacystis nidulans (Yamazaki et al , 1984). Thus, the difference in phycobilisome structure is not a cause for such difference. Based on simulation analysis, we interpreted our observation as that (1) all PC chtomophores do not equally participate to the energy migration within PC compartment but (2) a short transfer path through PC compartment is formed probably by f-type chromophores and (3) the difference in the "length" of this path is a main determinant for kinetic difference between PE-rich and PE-less systems.     

Phycobiliproteins encapsulated in sol-gel glass

Light transducing phycobiliproteins are encapsulated in optically transparent sol-gel matrices. Absorption and fluorescence spectroscopies are used to characterize the effect of the sol-gel encapsulation on the conformation and aggregation states of the three major phycobiliproteins present in phycobilisomes: phycoerythrin, phycocyanin, and allophycocyanin. It is found that the effects of sol-gel entrapment on the spectroscopic properties are significantly different for the three phycobiliproteins. The results indicate that phycoerythrin undergoes only minor change in its native structure when entrapped in sol-gel. However, significant changes in conformation and aggregation state occur when phycocyanin and allophycocyanin are entrapped in sol-gel matrices. A thin film of sol-gel encapsulated phycoerythrin is also coated on an optical fiber surface and strong fluorescence from the evanescent wave excitation is detected. The potential applications of sol-gel encapsulated phycobiliproteins in biosensors are discussed.     

EXCITATION ENERGY TRANSFER IN THE LIGHT HARVESTING ANTENNA SYSTEM OF THE RED ALGA Porphyridium cruentum AND THE BLUE-GREEN ALGA Anacystis nidulans: ANALYSIS OF TIME-RESOLVED FLUORESCENCE SPECTRA

Abstract— Time-resolved fluorescence spectra of intact cells of red and blue-green algae Porphyridium cruentum and Anacystis nidulans were measured by means of a ps laser and a time-correlated photon counting system. Fluorescence spectra were observed successively from various pigments in the light harvesting system in the order of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and chlorophyll a (Chl a ). The spectrum changes with time in the range of0–400 ps in P. cruentum and of0–1000 ps in A. nidulans . The time-resolved spectra were analyzed into components to obtain the rise and decay curve of each fluorescence component. Overall time behaviors of the sequential fluorescence emissions from various pigments can be interpreted with a decay kinetics ofexp(–2 kt ^½). The rate constants of the energy transfer show that the energy transfer takes place much faster in the red alga P. cruentum than in the blue-green alga A. nidulans , particularly in the step PCAPC. Results also indicated that a special form of APC, far-emitting APC, exists in the pigment system of A. nidulans , but it does not mediate a main energy transfer from phycobilisome to Chl a.     

SPECTRAL PROPERTIES OF PHYCOBILISOMES AND PHYCOBILIPROTEINS FROM THE BLUE-GREEN ALGA-NOSTOC SP.*

Abstract— An improved method for phycobilisome isolation from a blue-green alga Nostoc sp. was developed using 1% Triton X-100. The phycobilisome preparations showed little fragmentation and had structures similar in size to those observed in thin sections of the organism. Phycobiliproteins isolated from phycobilisomes and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, had subunits with the following molecular weights: phycoerythrin (PE), 20,000 and 16,900; phycocyanin (PC), 14,700 and 16,300; and allophycocyanin (APC), 14,000. Isoelectric focusing of each phycobiliprotein resulted in major bands isoelectric at the following pH values: PE, 4.43, 4.45; PC 4.32; APC, 4.38. Absorption spectra at -196°c showed maxima at 551 and 566 nm for PE; 598 and 631 nm for PC; and 590, 600, 629 and 650 nm for APC. Concentrated vs dilute difference spectra of phycobiliproteins showed increased absorption at 574 nm (PE), 630 nm (PC) and 651 nm (APC) suggesting that spectral changes resulted from aggregation. Fluorescence analysis of each phycobiliprotein and of intact phycobilisome preparations showed that energy absorbed by phycoerythrin is transferred to allophycocyanin, possibly by a resonance transfer mechanism. These observations support a model where allophycocyanin forms the base of the phycobilisome which is attached to the photosynthetic membrane. The next layer is assumed to be phycocyanin, which in turn is followed by a phycoerythrin layer that is the outermost layer (on the stroma side) of the phycobilisome.     

STUDIES ON ENERGY DISSIPATION IN PHYCOBILISOMES USING THE KENNARD-STEPANOV RELATION BETWEEN ABSORPTION AND FLUORESCENCE EMISSION SPECTRA

Absorption and fluorescence emission spectra were measured at room temperature ( ca. 22°C) for solutions of phycocyanin-1, phycocyanin-2 and allophycocyanin from Phormidium luridum , and also for phycobilisome preparations from various blue-green algae ( Anabaena variabilis, Nostoc muscorum strain A, Nostoc sp. strain Mac, Phormidium luridum ). Kennard-Stepanov (KS) temperatures ( T ) were computed using the Kennard-Stepanov relationship F () = b A () ^-5 exp(-h/ kT ), where F () stands for fluorescence (energy per wavelength interval) as a function of wavelength (), A () is absorbance as a function of wavelength, b a proportionality factor, and h, c and k are Planck's constant, the velocity of light and Boltzmann's constant, respectively.
In most cases experimenta/ data followed the expected relationship, but at low ionic strength allophycocyanin gave a clearly biphasic KS plot, i.e. In ⁵ F ()/ A () vs 1/. This could be due to the presence of both monomers and trimers in the sample at low ionic strength.
For purified allophycocyanin and phycocyanins (PC-1 and PC-2) as well as phycobilisomes from Phormidium luridum , the KS temperatures were only slightly (insignificantly) elevated above the sample temperature. Thus, after absorption of a photon, vibrational and configurational equilibration is essentially completed before emission of the fluorescence photon takes place.
For phycobilisomes from Anabaena variabilis and the two Nostoc species the KS temperatures were moderately elevated. Since there was no correlation between radiation temperature and excitation wavelength, the elevation is not due to excess (undissipated) vibrational energy, but rather to incomplete configurational equilibration.     

PHYCOBILISOMES AND ISOLATED PHYCOBILIPROTEINS. EFFECT OF GLUTARDIALDEHYDE AND BENZOQUINONE ON FLUORESCENCE

Abstract— The fluorescence of the biliproteins C-phycocyanin from Spirulina platensis , B-phycoerythrin from Porphyridium cruentum and of isolated whole P. cruentum phycobilisomes is quenched in the presence of glutardialdehyde (GA) or benzoquinone (BQ). The kinetics of fluorescence decrease thus induced is biphasic. If GA is used as a quencher, the fluorescence can be recovered at 77 K. Contrary to the GA-effect, only a minor recovery takes place with BQ at 77 K, thus demonstrating a different mechanism of action of GA and BQ on biliproteins.     

POLARIZED OPTICAL PROPERTIES OF PHYCOBILISOMES FROM Anabaena, Nostoc and Synechococcus

Polarized absorption, photoacoustic, fluorescence excitation and fluorescence emission spectra of phycobilisomes were measured when embedded in polyvinyl alcohol films. The phycobilisomes were isolated from the following organisms: Anabaena cylindrica, Nostoc punctiforma and Synechococcus elongatus. The ratio of photoacoustic spectra to absorption was taken as a measure of thermal dissipation of excitation energy. The isotropic samples and those oriented by the film stretching were investigated. The stretching of the sample strongly influences the efficiency of excitation energy transfer occurring among biliproteins in phycobilisomes, as is seen from the dramatic changes in the fluorescence and thermal dissipation spectra. The effect of stretching the film depends on the shape of phycobilisomes and on the strength of interactions between biliproteins in phycobilisomes.     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.

     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.     

Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin,Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM

An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with α‐ and β‐subunit of PE and APC occurred after 4 h of UV‐B exposure. After 5 h of UV‐B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV‐A radiation, a severe decrease in fluorescence of all PBPs was observed under UV‐B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15–30 min of UV‐B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV‐B radiation. Moreover, the present study indicates that UV‐B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.     

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.     

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.     

PHOTOPHYSICAL BEHAVIOUR OF 5-METHOXYPSORALEN IN DIOXANE-WATER MIXTURES

Abstract— Triplet-triplet absorption spectra, intersystem crossing quantum yields, fluorescence lifetimes, and fluorescence quantum yields of 5-methoxypsoralen (5MOP) in dioxane-water mixtures are reported. The fluorescence and triplet formation quantum yields depend strongly on the water content in the mixtures, increasing up to maximum values which are, respectively, twentyfold and fivefold larger than in dioxane and then decreasing. This behaviour is essentially due to the variation of the internal conversion rate constant. With these solvent mixtures it is possible to emulate the dielectric response of 5MOP photophysical parameters in other media, such as in biological model systems and in human blood lipoproteins. The biological importance of these results is discussed.     

FLUORESCENCE QUANTUM YIELDS AND LIFETIMES FOR BACTERIOCHLOROPHYLL c

Abstract— Absorption and emission spectra of bacteriochlorophyll c dissolved in a variety of solvents were measured and fluorescence quantum yields determined from the integrated emission spectra. Values for the emission maxima calculated from the positions and bandwidths of the red absorption band using the Stepanov relationship agreed closely with the experimental values. Fluorescence quantum yields varied between 0.10 in methanol and 0.36 in tetrahydrofuran and in dibutylamine. Fluorescence lifetimes were also determined for bacteriochlorophyll c in four of the solvents, and ranged from 2.7 ns in methanol to 7.6 ns in dimethylsulfoxide.     

吩噻嗪-Corrole 镓(III)配合物的合成、荧光及其光断裂DNA性质

合成了吩噻嗪(PTZ)-corrole二元体1-3及其镓(III)配合物4-6.采用稳态吸收与稳态发射及时间分辨的瞬态光谱技术研究了这几种化合物的光物理特性.结合荧光量子产率和荧光寿命计算得到它们的辐射和无辐射速率常数.稳态吸收光谱表明:几种二元体中,corrole镓(III)单元表现出更强的Soret带和Q带.化合物1-3的荧光量子产率分别是0.156、0.134和0.139,辐射速率常数分别为4.02′107、3.47′107和2.89′107s-1.化合物4-6的荧光量子产率分别是0.502、0.443和0.494,辐射速率常数分别为20.90′107、16.78′107和21.11′107s-1.可见,化合物4-6的荧光量子产率和辐射速率常数均高于化合物1-3.然而,化合物4-6的荧光寿命分别是2.40、2.64和2.34ns,低于自由corrole1-3.琼脂糖凝胶电泳实验表明:在光照的条件下,这些吩噻嗪-corrole镓(III)二元体化合物能够把超螺旋DNA(formI)切割成缺刻型DNA(formII).     

Spectroscopy,photophysics and photochemistry of 1,3-diketoboronates: IV: Luminescence spectroscopic investigations of 2-naphthyl-substituted 1,3-diketoboronates

Aryl-substituted 1,3-diketoboronates exhibit high fluorescence quantum yields. In the case of 2-naphthyl-substituted 1,3-diketoboronates, excitation-wavelength-dependent luminescence spectra and luminescence-wavelength-dependent excitation spectra were recorded. In polar solvents the compounds exhibit large Stokes shifts and the fluorescence lifetimes depend on the excitation and fluorescence wavelengths. The multiple fluorescence behaviour is interpreted by assuming the existence of excited non-equilibrated rotamers with different photophysical properties and high dipole moments in the excited state. The relaxation of polar solvents around these high polar species contributes to the broadening of the fluorescence bands and to the fluorescence-wavelength dependence of the lifetimes caused by the fluorescence of incompletely solvent-relaxed species.     

STUDIES ON FLAVINS IN ORGANIC SOLVENTS — III. SPECTRAL BEHAVIOUR OF LUMIFLAVIN

Abstract— The absorption and fluorescence emission spectra of lumiflavin have been studied in nonaqueous solvents of different polarity. The corrected emission spectra show apparent mirror symmetry to the longest wavelength absorption maximum. In view of high molar extinction coefficient of the longest wavelength maximum in absorption spectra, constant polarization of fluorescence across it in dioxane and high values of quantum yields of Buorescence, τ→τ* character of corresponding electronic transition was proposed. Roughly linear correlation was found between transition energies and quantum yields of fluorescence on the one hand, and polarity of solvents expressed in Kosower's 'Z' values (excepting acetic acid) on the other.
In preliminary experiments it was found that lumiflavin in dioxane and in acetic acid is strongly photodecomposed by the monochromatic(436 nm) light.