D. Frąckowiak

THE ORIENTATION OF BACTERIOCHLOROPHYLL C IN GREEN BACTERIA CELLS AND CELL FRAGMENTS

Abstract Green bacteria cells, their fragments and isolated pigment molecules were oriented by embedding them in anisotropic polymer film. Two methods of film deformation were used: uniaxial stretching, and skew stretching in which a previously uniaxially elongated film was again stretched along the axis, forming a 40 ° angle with the previous direction of stretching. Photographs of samples in transmitted light and under a polarized fluorescence microscope were taken. Polarized absorption and fluorescence spectra were measured, and order parameters were calculated and discussed. In the 670–680 nm region absorption and emission of disaggregated bacteriochlorophyll c from chlorosomes and bacteriopheophytin c is observed. The decrease of mutual pigment interactions caused the spectral shift of the absorption band from 750 to 670 nm; however, disaggregated pigment is still located in chlorosomes and preserves the orientation. The film stretching diminished the absorption of bacteriochlorophyll c in chlorosomes (at about 750 nm) but only slightly increased the absorption of disaggregated bacteriochlorophyll c (670 nm). This suggests that as a result of stretching, part of the transition moments of the chlorosome bacteriochlorophyll c red band became located outside of the plane of the electric vector of the spectrophotometer light beam. In the case of a large sample such as whole bacteria or their fragments, the axis of orientation is closer to the direction of the first stretching axis than to the second skew axis. Separated pigment molecules are oriented along the second axis of stretching. The dimensions of objects seen under the fluorescence microscope are larger than those of bacteria, therefore they are clusters of bacteria or bacteria fragments. Results obtained from microscopic investigations and from polarized spectroscopy suggest that two pools of clusters of bacteria or fragment clusters exist with different angles of orientation (one under about 15 °, the second almost parallel to the film axis), and different average length. It was shown also that the pigment orientation depends strongly on the conditions of bacteria culture and sample preparation.

Photoelectrical properties of green bacteria cells and cell fragments located in electrochemical cell

Abstract Photopotential action spectra as well kinetics of their generation and decay for green bacteria Prosthecochloris aestuarii cells and cell fragments were measured. The sample was located between two transparent electrodes, both semiconducting or one semiconducting and second one metallic. The electric properties of the cells in darkness and under illumination were also established. In the photopotential action spectrum the maximum about 750 nm, responsible for the bacteriochlorophyll c oligomers absorption, is absent. It shows that bacteriochlorophyll c molecules located in chlorosome oligomers are not taking part in the photopotential generation. From the action spectra it follows that predominantly desaggregated bacteriochlorophyll c and bacteriopheophytin, and may be also some chlorophyll a -like pigments are responsible for a photopotential generation. In the 670 nm region the desaggregated bacteriochlorophyll c as well the bacteriopheophytin absorption are located, whereas the Soret band is characteristic rather for the bacteriopheophytin, but some contribution from the desaggregated bacteriochlorophyll c cannot be excluded. With the sample pheophytinization the photopotential amplitudes increase. The complex kinetic of the photopotential generation suggests the superposition of at least two processes: the fast one, occurring just after light absorption, which probably is due to charge separation in the reaction centers and the second one—slower—may be related to some diffusion of the charge generated in the antenna pigments through the cell membrane. Due to slower photopotential decay and higher concentrations of the antenna pigments than that of the reaction centers, the contributions from the desaggregated antenna pigments, at used in the experiments frequencies of light modulation, predominant in the photopotential action spectra. For each sample the sign of the photopotential signal changes with the change of the side of illumination of the electrochemical cell.

Spectral properties of stilbazolium merocyanines oriented in stretched polymer films and Langmuir-Blodgett monolayers

Abstract Three stilbazolium merocyanines: 1-(6′-hydroxyhexyl)-4-[(4-oxocyclohexa-2,5-dienylidene)ethylidene]-1,4-dihydropyridine; 1-(11′-hydroxyhexyl)-4-[(4-oxocyclohexa-2,5-dienylidene)ethylidene]-1,4-dihydropyridine; 1-(10′-carboxydecyl)-4-[4-oxocyclohexa-2,5-dienylidene)ethylidene]-1,4-dihydropyridine salt HCl located in Langmuir–Blodgett monolayers and deposited on quartz plate as well as embedded in isotropic or stretched polyvinyl alcohol films were investigated. Dyes occur in protonated and free-base forms. The concentration ratio of these forms depends on pH of subphase (for monolayers) or resin addition (in polymer film). The polarized absorption and fluorescence spectra were measured and coefficients of absorption and emission anisotropy were calculated. Dye molecules were in both matrices oriented, but degrees of orientation of various forms of dye were different. The anisotropies of absorption and emission are also different which strongly suggests the occurrence of dye forms with different yield of fluorescence and various orientations. The formation of mixed aggregates of protonated and free-base form is suggested. The orientation of stilbazolium merocyanines in anisotropic matrix is important in the application of these dyes as the sensors for measurements of the local electric field in biological membranes as well as for the formation polarizing absorption polymer film used in some types of two-colours liquid crystal displays.

Incorporation of Dye in Resting and Stimulated Leukocytes

Methods of investigating the incorporation efficiency of dye molecules into leukocytes and other cells are reviewed. The application of polarized light spectroscopy into the establishment of changes in the structure of biological membranes due to cell stimulation is reported. Results obtained by polarized light absorption, fluorescence, delayed luminescence and photoacoustic spectra and flow cytometry applications are described. The incorporation of two types of dyes: stilbazolium merocyanines and porphyrins are discussed on the basis of the authors’ results.

Interactions between photosynthetic pigments in organisms and in model systems

The review summarizes results concerning photosynthetic systems with chlorophylls and carotenoids obtained by means of spectral methods such as polarized radiation, photoacoustic spectroscopy, delayed luminescence, thermal deactivation, and leading to construction of model systems.

Photoreaction and thermal deactivation of excitation in purple bacteria light harvesting complexes (LH2) with and without reaction centres

Light harvesting peripheral complexes (LH2) and carotenoids free-reaction centres were isolated from purple photosynthetic bacteria Rhodospirillum molischianum and Rhodobacter sphaeroides, respectively. The LH2 complexes without and with various amounts of reaction centres were immobilized in polyacrylamide gel. The absorption and photoacoustic spectra of such samples were measured. The changes due to continuous light illumination in absorption and photoacoustic spectra were investigated. The changes in absorption generated by flash were also measured. The conclusions concerning the photoreactions in antenna and reaction centre chromophores, the storage of energy in reaction centres as well as the protection of LH2 chromophores against overexcitation by heat emission and excitation transfer to reaction centres are discussed.

Two forms of stilbazolium merocyanine on Langmuir-Blodgett monolayers

Abstract Monolayers of stilbazolium merocyanine were prepared at the air-water interface, and were transferred onto a hydrophilic quartz surface by the vertical dipping method. The molecular forms in the films were studied for two types of merocyanine: A) 1-(6′-hydroxyhexyl)-4[(3,5-di- t -butyl-4-oxocyclohexane-2,5-dienylidene)ethylidene]-1,4-dihydropyridine and B) 1-(hexadecyl)-4[(3,5-di- t -butyl-4-oxocyclohexane-2,5-dienylidene)ethylidene]-1,4-dihydropyridine both with reference to the pH of the buffer. The protonated form gave a more stable monolayer film than the free-base form. Also the molecules of protonated merocyanine did not show degradation, but the free-base was more fragile. Both forms could be transferred onto a hydrophilic substrate building a multilayer structure. Using UV-Vis absorption spectroscopy for one monolayer on quartz, the orientation of molecules relative to the film surface was found to depend on the form of merocyanine. The polarized components of the absorbance spectra indicate that the free-base form of merocyanine is preferentially oriented parallel to the quartz substrate, in contrast to the protonated form which has a more out-of-plane orientation.

Spectral Properties of Bacteriochlorophyll c in Organisms and in Model Systems

Polarized absorption and fluorescence spectra of bacteriochlorophyll c and green photosynthetic bacterium Prostheecochloris aestuarii cells and cell fragments embedded in stretched polymer film were measured. In pigment samples the artificial oligomers of bacteriochlorophyll c (with absorption about 750 nm) and other forms of this pigment and bacteriopheophytin (with absorption at 670 nm) were present. In bacteria samples, embedded in polymer, oligomers were in high degree disaggregated and as a result the absorption about 670 nm was observed. Previously for similar sets of samples the decay of fluorescence excited only at one wavelength was analyzed on three exponential components, but exact lifetime values of these components for various samples were different. The aim of present paper was to check if these differences occur because of various contributions to decay from three well defined forms or if they were related to the existence of several pigment forms with slightly different lifetimes. The global analysis of data obtained for various excitation and observation wavelengths of fluorescence were done. From this analysis it follows that the second situation occurs. For a model system containing artificial oligomers the largest component of decay has a τ4 of about 0.183 ns or 0.136 ns depending on observation wavelength. For the bacteria sample, in which the emission at 680 nm is the superposition from various pigments, global analysis done for various excitation wavelengths shows also that the τ values differ depending on the regions of fluorescence observation. From polarized spectra, it follows that in the model system the pigments absorbing at 670 nm are randomly distributed whereas oligomers are highly oriented. In bacteria fragments absorbing at 670 nm pigment molecules can be divided into two groups: one oriented along the axis of film stretching and the second practically randomly distributed. In living organisms, under some conditions, small amount of 670 nm pigments can be present and can work as excitation energy traps or as antenna transferring the excitation. Present results show that the role of various pools of 670 nm absorbing pigments can be different because of their differing orientation.

Polarized Photoacoustic Spectra of the Cyanobacterium Synechococcus Oriented in Polymer Film

Photoacoustic spectra (PAS) were obtained for the cyanobacterium Synechococcus (Anacystis nidulans) cells embedded in isotropic and stretched polyvinyl alcohol films. The polarized radiation with the electric vector changing in 30° intervals with respect to given direction in a sample plane was used. Two cyanobacterium strains, one with very low biliprotein content, second with normal amount of biliproteins were investigated. The polarized absorption and fluorescence spectra were also measured. Conclusions were drawn about the thermal deactivation occurring in differently oriented pools of chromophores and about mutual orientation of their transition moments. Thermal deactivation in carotenoids (Cars) of both strains was different. The ratio of Car thermal deactivation to the thermal deactivation of chlorophyll (Chl) was higher in cyanobacteria with lower content of biliproteins than in the strain with normal amount of these complexes. Hence biliproteins can play the role in excitation energy transfer from Cars to Chls. For complex biological samples, polarized PAS can be a more sensitive method to investigate the directions of the absorption transition moments than the widely used polarized absorption spectra.

Photothermal Spectra of Thylakoids Isolated from Cucumber Cotyledons at Various Stages of Greening

The character of interaction between carotenoids (Cars) and chlorophylls (Chls) in thylakoids isolated from cucumber cotyledons at three stages of greening (3, 6, and 24 h of irradiation with 120 µmol m−2 s−1) was studied. The shapes of the steady state photoacoustic spectra were changed with the change in time of greening and with the frequency of radiation modulation. The shapes show that changes not only in the contents of various pigments but also in pigment interactions with surrounding occur and that processes of thermal deactivation characterised by different kinetics take place. Slow processes of thermal deactivation are in most cases due to deactivation of triplet states. Long living triplet states are very often engaged in photochemical reactions that can destroy the tissue. Analysis of the time-resolved photothermal spectra shows that at later stage of greening, the chlorophyll (Chl) molecules are better shielded against photo-destruction because Cars more efficiently quench their triplet states. The yield of formation of the pigment triplet states measured by the time resolved photothermal method, always at the same energy absorbed by pigment mixture, declined during sample greening. The decay time of the slow component of pigment thermal deactivation, due predominantly to deactivation of the triplet state of Chl, decreases with the increase of time of greening from 6.2 µs for the 3-h sample to 1.5 µs for the 24 h sample. The energy taken by Cars from Chls is dissipated into heat, therefore the steady state and quick thermal deactivation values increased during the greening process. The Cars/Chls ratio in the thylakoids decreased during greening approximately 2 fold. Hence at a later phase of greening the Cars can quench the triplet states of Chls more efficiently than at an earlier phase of greening.