OPTICAL SPECTROSCOPY OF BILAYER MEMBRANES

Abstract— The absorption and fluorescence spectroscopy of natural and model bilayer lipid membranes is reviewed. Basic structural features of biological membranes and the relative advantages of black lipid membranes (BLM) and of liposomes are discussed. Theoretical considerations show that the wavelengths of absorption maxima are affected by the refractive index and dielectric constant of the medium surrounding the chromophore. Techniques of obtaining photoelectric action spectra, direct absorption spectra, and reflection spectra of BLM are described. Polarized spectra can give information about the orientation of membrane constituents and show, for example, that the porphyrin ring of chlorophyll in BLM is tilted at 45 ± 5° to the membrane surface. Absorption maxima of chlorophyll in BLM are compared with solution spectra of various chlorophyll adducts and aggregates. It is concluded that chlorophyll in BLM exists largely as solvated monomer and dimer (or oligomer), depending on concentration, and is not coordinated with water. From the theory of fluorescence spectroscopy it follows that aggregation and the polarity of the environment affect the fluorescence yield and lifetime of a membrane component, and also the wavelength of its emission maximum. The microviscosity of the membrane matrix affects the anisotropy of fluorescence. Techniques of steady-state fluorescence spectroscopy and of fluorescence lifetime measurements are reviewed. Examples of the use of fluorescent probes in membrane studies are given. Certain probes such as anilinonaphthalene sulfonate (ANS) preferentially bind to membrane proteins. The location of a probe in a particular membrane region can be pinpointed from its fluorescence yield and emission maximum. The orientation of the hydrocarbon chains of membrane lipids has been found, from fluorescence polarization of certain probes, to be normal to the membrane surface as postulated a priori on the basis of the lipid bilayer model. Anisotropy of fluorescence shows that elongated probe molecules rotate rapidly about their long axes when surrounded by phospholipids but become immobilized when bound to proteins. Changes in intensity and anisotropy of fluorescence as function of temperature have demonstrated the existence of phase transitions and phase equilibria of membrane lipids. Excimer fluorescence has been used as a measure of the available lipid core volume of membranes. Mechanisms of energy transfer between membrane components are reviewed. The theoretical dependence of energy transfer on distance and orientation for several rigid and fluid membrane models is discussed in terms of the structural information it can provide. Fluorescence sensitization resulting from energy transfer within and across bilayer membranes has been demonstrated in various systems. Quantitative measurement of energy transfer efficiency in BLM has shown that such transfer is about five times more efficient than in solutions at comparable donor-acceptor distances. Lipid membranes can be viewed as structures which maintain their components at high concentrations, in a reactive state, and at favourable orientations.     

Determination of Fluorescence Polarization and Absorption Anisotropy in Molecular Complexes Having Threefold Rotational Symmetry

Abstract— The current work concerns investigation of the polarization properties of complex molecular ensembles exhibiting threefold (C₃) rotational symmetry, particularly with regard to the interplay between their structure and dynamics of internal energy transfer. We assume that the molecules or chromophores in such complexes possess strongly overlapped spectra both for absorption and fluorescence. Such trimeric structures are widely found in biological preparations, as for example the trimer of C-phycocyanin (C-PC). Higher order aggregates, e.g. hex-amers and three-hexamer rods, are also investigated and compared with the trimer case. The theory addresses both steady-state and 8-pulse excitation and establishes some links between them. Monochromophoric, bichro-mophoric and trichromophoric molecular complexes are individually examined. For steady-state excitation, analytical formulas are reported for the degree of fluorescence polarization and absorption anisotropy. It is shown that the polarization is dependent on the chromophore inclination relative to the symmetry axis, the relative efficiencies of absorption and fluorescence by chromophores of different spectral types, and the rates of energy equilibration. To assess the validity of the theory, it has been applied to C-PC aggregates. Here it was found that different C-PC aggregates provide practically identical polarization response. For S-pulse excitation we give analytical formulas for determination of the fluorescence depolarization, and also the depolarization associated with absorption recovery, both for a monochromophoric trimer and some particular cases of bichromophoric trimer. More complicated systems are analyzed by computer modeling. Thus it transpires that the initial polarization anisotropy r(t = 0) takes the value 0.4 for all considered aggregates; the long-time limit r(t →∞) has about the same value as is associated with steady-state excitation. We also show that with steady-state excitation the degree of fluorescence polarization is practically equal for various C₃ aggregates of C-PC, and that the major factor determining the polarization is the chromophore orientation relative to the symmetry axis.     

CHLOROPHYLLS IN POLYMERS. I. STATE OF CHLOROPHYLL a IN UNSTRETCHED POLYMER SYSTEMS

Abstract— Model systems for the study of energy transfer processes are useful for the elucidation of the various factors governing the mechanism of energy transfer in photosynthetic systems. Here we describe the characterization of two systems, consisting of chlorophyll a incorporated in anhydrous nitrocellulose and polyvinylalcohol films. First, optical spectroscopy and time-resolved fluorescence techniques are used to characterize the state of the chlorophyll molecules in the films. We find that in nitrocellulose films the state of chlorophyll a depends strongly on the ratio of nitrocellulose to dimeth-ylsulfoxide in the solutions from which the films are cast. The state of chlorophyll a in polyvinylalcohol films does not depend on the amount of polymer originally dissolved in dimethylsulfoxide. In these films the pigment is monomeric at low concentrations of chlorophyll a, but aggregates are formed at much lower concentrations than in nitrocellulose. The latter fact is explained by the existence of pockets in polyvinylalcohol, leading to high local concentrations.
To further test the suitability of the nitrocellulose polymer films as model systems for energy transfer processes, time-resolved fluorescence anisotropy profiles are measured in dependence of the concentration of pigments in the matrix. Fits of the observed decay profiles to the predicted decay show good correspondence, as long as no traps are present. Furthermore, the fitted decay times yield the correct value of the Forster radius R₀ as compared to the value obtained spectroscopically. We thus conclude that the chlorophyll a-nitrocellulose system can be very appropriate for the study of energy transfer processes between photosynthetic pigment, since the pigments are uniformally distributed in the matrix.     

Chlorin e6-liposome interaction. Investigation by the methods of fluorescence spectroscopy and inductive resonance energy transfer

On the basis of spectral fluorescence and polarization measurements and results obtained on the luminescence quenching of the membrane fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) by incorporated chlorin e6 (chl e6) molecules, it is shown that the interaction of the water-soluble pigment with smaller unilamellar lipid vesicles occurs by a mechanism of partition between the aqueous and lipid phases (partition coefficient Kp = 6.7 x 10(3) and provides rigid fixing of chl e6 monomers at the boundary between the polar and non-polar parts of the lipid membrane. In terms of inductive resonance electronic excitation energy transfer between DPH and chl e6 (R0 = 36.2 A), we have analysed data on DPH fluorescence quenching under different conditions of chl e6 localization in the lipid bilayer and have concluded that the incorporation of the pigment molecules into the vesicles from the aqueous phase occurs mainly into the external monolayer.     

Exciton energy transfer between optically forbidden states of molecular aggregates

3-ethyl-2-[3-(3-ethyl-2(3H)-benzoxazolylidene)-1-propenyl]benzoxazolium iodide (dye I) and pseudoisocyanine bromide are employed to form H aggregates as donors and J aggregates as acceptors. The energy of an H band of the H aggregates is higher than that of a J band of the J aggregates. It was confirmed that excitation of the H band does not emit fluorescence by comparison of excitation spectra of dye I H aggregates with that of dye I monomer. Absorption, fluorescence, and excitation spectra of spin-coated films of H aggregates mixed with various quantities of J aggregates have been observed. Excitation spectra probed at the J band are found to have a component of the H band. Fluorescence spectra originated from excitation of the H band are extracted and qualitatively analyzed. It is confirmed that excitation of the H band causes to emit fluorescence of a J band of the J aggregates. These phenomena show that exciton energy can transfer from the lowest energy in electronic states of the H aggregate, which state is optically forbidden, to electronic state of the J aggregate.     

EXCITATION TRANSFER BY CHLOROPHYLL a IN MONOLAYERS AND THE INTERACTION WITH CHLOROPLAST GLYCOLIPIDS*

In mixed monolayers with purified chloroplast glycolipids and other colorless lipids, chlorophyll a fluorescence exhibits a decrease in quantum efficiency with increasing chlorophyll concentration. The fluorescence, which is strongly polarized in dilute films, becomes progressively depolarized as the area fraction of chlorophyll increases, and it is completely depolarized in a pure chlorophyll a monolayer. The observed behavior is consistent with an inductive resonance mechanism of energy transfer among the chlorophyll molecules with a critical transfer distance of 20–90 Å, depending on the model chosen for the energy transfer mechanism. The purified glycolipids–mono-and digalactosyl diglycerides and sulfoquinovodiglyceride–separately form stable, compressible monolayers of the liquid-expanded type on an aqueous subphase and in an atompshere of nitrogen. At maximum compression the three glycolipids occupy areas of 55, 80 and 47 A²-molecule^-1, respectively, in the monolayer. Mixed monolayers of chlorophyll a with, separately, the monogalactolipid and the sulfolipid behave upon compression as two-dimensional solutions. The fluorescence polarization at high chlorophyll concentrations in mixed monolayers indicates that several of the lipid diluents facilitate local ordering of the pigment molecules.     

Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals

Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef‐building coral tissue. We used a streak‐camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (<9 m)–dwelling Acropora spp. and Plesiastrea versipora specimens were collected from Okinawa, Japan, and Sydney, Australia, respectively. The main FP emitted light in the blue, blue‐green and green emission regions with each species exhibiting distinct color morphs and spectra. All corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900–2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (～683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640–760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time‐resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp.     

Energy transfer via guest excitons in isotopic mixed naphthalene crystals

The temperature dependence of the fluorescence spectra of aggregates in naphthalene-perdeuteronaphthalene mixed crystals has been investigated between 1.4 and 70 K and for concentrations up to 50% naphthalene. It is shown that the most abundant traps — the monomer guest molecules — transfer energy like a guest exciton band 48 cm^?1 below the host exciton band. With increasing temperature, the excitation energy is redistributed between the different aggregate traps by thermal activation into the monomer states. The energy transfer constant within the monomer exciton band is measured as a function of concentration. It is suggested that dipole-dipole interaction between the monomer guests is responsible for the energy transfer via guest excitons.     

红藻条斑紫菜R-藻红蛋白中的能量传递过程

本文通过对条斑紫菜R-PE(藻红蛋白)及其α-β-γ亚基的吸收光谱和荧光光谱进行计算机解叠,研究了R-PE内发色团之间的能量传递过程,并对R-PE及亚基内的各发色团进行了“s”和“f”型的指认。发现在亚基中为“f”型的发色团在R-pE(αβ)₆γ中起着“s”型发色团的作用,且将能量传递给最后的“f”型发色团。荧光激发偏振光谱进一步证明了R-PE内的能量转移过程与计算机解叠的结果一致。     

ENERGY TRANSFER FROM CAROTENOIDS TO CHLOROPHYLL a IN BLACK LIPID MEMBRANES

Abstract— Black lipid membranes (BLM) were prepared from extracts of Chlorella and spinach chloroplasts. Excitation spectra of the 730 nm fluorescence of chlorophyll a in the BLM contained peaks identified as due to carotenoids and which therefore indicate sensitization of the chlorophyll fluorescence by them. The efficiency of this energy transfer was evaluated by comparison of the actual excitation spectra with those corresponding to 0 and 100 per cent transfer efficiency. Efficiencies were of the order of 40–50 per cent in BLM, but only 10 per cent in pigment solutions, when the mean distance between pigment molecules was 23 Å in both systems. The fluorescence quantum yield of chlorophyll a in such solutions was only 2 per cent of that found in BLM. Enhancement of energy transfer in BLM is considered to be mainly due to suppression of competing deactivation processes of excited carotenoid states, such as diffusional quenching by ground-state molecules and internal conversion. Favorable orientation of pigment molecules in the BLM constitutes a further enhancement factor.     

PHOTOPHYSICAL PROPERTIES OF PHYCOBILIPROTEINS FROM PHYCOBILISOMES: FLUORESCENCE LIFETIMES, QUANTUM YIELDS, AND POLARIZATION SPECTRA

Abstract— Absorption and fluorescence polarization spectra, as well as absolute fluorescence quantum yields, and lifetimes of phycobiliproteins separated from intact phycobilisomes of Porphyridium cruentum, Nostoc sp. and Fremyella diplosiphon were measured. Two different types of phycoerythrin, in addition to phycocyanin and allophycocyanin, were separated from both Porphyridium cruentum and Nostoc sp. phycobilisomes. They were distinguishable by the shape of their absorption spectra, values of fluorescence quantum yields and their limiting polarization. Phycobilisomes of Fremyella diplosiphon had a type of phycoerythrin that was different from the above kinds. By the use of fluorescence quantum yields and lifetime data, the values of natural lifetimes, the decadic molar extinction coefficients, as well as Förster's critical distances R ₀ for excitation energy transfer, between phycobiliproteins in phycobilisomes, were estimated. The values obtained of Förster's critical distances indicate that for most efficient energy transfer from phycoerythrin to allophycocyanin, the outer layers of Porphyridium cruentum and Nostoc sp. phycobilisomes should be composed of bangiophycean, phycoerythrin and cyanophytan phycoerythrin-II respectively.     

SPECTROSCOPIC STUDY OF CHLOROPHYLL a IN ORGANIC SOLVENTS AND POLYMERIZED ANHYDROUS POLYVINYL MATRIX

In this paper we present a spectroscopic study of chlorophyll a in solutions and in anhydrous polyvinyl alcohol films. Absorption, excitation and emission spectra, combined with fluorescence lifetime and time-resolved anisotropy measurements show that chlorophyll a in anhydrous polyvinyl alcohol films exists in a purely monomeric state. Furthermore, it appears that the monomeric chlorophyll a exhibits an efficient excitation energy transfer in this polyvinyl alcohol matrix. These results are rationalized in terms of a model in which the chlorophyll a molecules are located within pockets, formed by the polymer chains. It is concluded that the chlorophyll a-anhydrous polyvinyl alcohol film is a suitable system for studying energy transfer processes, especially because the factors governing energy transfer such as mutual orientation and separation of the molecules can easily be controlled.     

Fluorescence spectra of naphthalene-perdeuteronaphthalene mixed crystals - energy transfer via guest excitons

The fluorescence spectra of the mixed crystal system naphthalene in perdeuteronaphthalene at concentrations between 0.1 and 50% have been measured with high spectral resolution. Up to 10% the spectra are superpositions of the spectra of monomers, pairs, trimers and some higher aggregates. The relative intensities of these individual aggregate spectra given evidence for trap to trap energy transfer without thermal excitation into the host exciton band. The monomers as the most abundant traps form a dilute exciton band 50 cm^?1 below the host band. By thermal activation into this dilute band energy is transferred between the aggregate states. In the 50% crystal emission from a mixed guest-host exciton band without individual clusters is observed.     

藻胆体核心复合物结构与功能的研究(Ⅱ)──4种变藻蓝蛋白复合物发色团相互作用机制

从螺旋藻藻胆体中分离出4种不同结构和光谱形式的变藻蓝蛋白复合物APⅠ、APⅡ、APⅢ和APB, 利用吸收光谱、荧光光谱比较了三聚体和单体的光谱特性, 通过对吸收光谱的光谱解曾以及各组分的归属, 研究了变藻蓝蛋白复合物内各色团间相互作用的性质和在能量传递中的功能.结果表明, 复合物内色团间的作用关系可以用Forster偶极-隅极作用机制来解释, 由于连接蛋白和同源亚基的存在影响其结构的对称性, 进而影响各色团间相互作用的形式和性质.     

萘和丹酰基修饰的扇形树枝状分子聚集体的能量转移行为

分别对1-3代聚(酰胺-胺)(PAMAM)结构的dendron分子的外端基和focal point进行了修饰,得到了外端基为萘(给体)色团、焦点(focal point)为丹酰(受体)色团的树枝状化合物Dan-ABπ-Nap(n=2,4,8).利用荧光光谱测定了不同浓度下所得一系列树枝状分子在水中的荧光强度,并计算了它...     

Quenching of chlorophyll a singlets and triplets by carotenoids in light-harvesting complex of photosystem II: comparison of aggregates with trimers

Laser-induced changes in the absorption spectra of isolated light-harvesting chlorophyll a/b complex (LHC II) associated with photosystem II of higher plants have been recorded under anaerobic conditions and at ambient temperature by using multichannel detection with sub-microsecond time resolution. Difference spectra (ΔA) of LHC II aggregates have been found to differ from the corresponding spectra of trimers on two counts: (i) in the aggregates, the carotenoid (Car) triplet–triplet absorption band (ΔA>0) is red-shifted and broader; and (ii) the features attributable to the perturbation of the Q_y band of a chlorophyll a (Chla) by a nearby Car triplet are more pronounced, than in trimers. Aggregation, which is known to be accompanied by a reduction in the fluorescence yield of Chla, is shown to cause a parallel decline in the triplet formation yield of Chla; on the other hand, the efficiency (100%) of Chla-to-Car transfer of triplet energy and the lifetime (9.3 μs) of Car triplets are not affected by aggregation. These findings are rationalized by postulating that the antenna Cars transact, besides light-harvesting and photoprotection, a third process: energy dissipation within the antenna. The suggestion is advanced that luteins, which are buried inside the LHC II monomers, as well as the other, peripheral, xanthophylls (neoxanthin and violaxanthin) quench the excited singlet state of Chla by catalyzing internal conversion, a decay channel that competes with fluorescence and intersystem crossing; support for this explanation is presented by recalling reports of similar behaviour in bichromophoric model compounds in which one moiety is a Car and the other a porphyrin or a pyropheophorbide.     

CHLOROPHYLL a AGGREGATES IN HYDROCARBON SOLUTION, A PICOSECOND SPECTROSCOPY AND MOLECULAR MODELING STUDY

Abstract Chlorophyll a aggregates in 3-methylpentane solution have been studied by using picosecond absorption and fluorescence spectroscopy and molecular modeling. Chlorophyll a aggregates give rise to reversible temperature changes in the absorption and fluorescence spectra. Time-dependent anisotropies were used to estimate rotational correlation times of the aggregates. These were indicative of the sizes of the aggregates. The rotational diffusion of the monomer and the two identified aggregates was hydrodynamic over the viscosity range studied (0.29–1.8 cP). Molecular mechanics calculations were used to predict the minimum energy structures of several chlorophyll a dimers suggested earlier in the literature. These structures were used to estimate exciton splitting of the Q_y,0–0 transition and compared to the experimentally observed spectral shifts.     

Resolution of the excitation-emission spectra of FMN in rigid poly(vinyl alcohol) matrices

The excitation-emission spectra of flavin mononucleotide (FMN) were measured in rigid PVA films for concentrations ranging from 6.92 x 10(-4)M to 1.03 M. The theoretical three-linear decomposition of the excitation-emission spectra indicated the presence of two absorption and emission centers corresponding to FMN monomer and dimer, respectively. The component of the fluorescence profile corresponding to the FMN monomer has a large negative part which is the mirror image of the emission band profile of the dimer. The elimination of this part by taking a linear combination of the emission components of the monomer and of the dimer resulted in emission spectrum, which is in a very good agreement with the monomer spectrum measured directly. The appearance of a negative part of the monomer emission profile obtained by trilinear decomposition of the emission-absorption spectra of FMN can be explained in terms of the non-radiative reverse energy transfer from the FMN dimers to the FMN monomers. The presented results confirm that the FMN molecules in rigid PVA form dimers but not higher order aggregates. Moreover, they enable to obtain fluorescence spectra of dimers and suggest that FMN dimers may take part in the process of non-radiative energy transfer occurring in photoreception phenomena.     

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.     

Cyanobacterial chlorophyll as a sensitizer for colloidal TiO2

Chlorophyll has been extracted from cyanobacteria. The adsorption of chlorophyll on the surface of colloidal TiO(2) through electrostatic interaction was observed. The apparent association constant (K(app)) of chlorophyll-TiO(2) obtained from absorption spectra is 3.78x10(4)M(-1). The K(app) value of chlorophyll-TiO(2) as determined from fluorescence spectra is 1.81x10(4)M(-1), which matches well with that determined from the absorption spectra changes. These data indicate that there is an interaction between chlorophyll and colloidal TiO(2) nanoparticle surface. The dynamics of photoinduced electron transfer from chlorophyll to the conduction band of colloidal TiO(2) nanoparticle has been observed and the mechanism of electron transfer has been confirmed by the calculation of free energy change (DeltaG(et)) by applying Rehm-Weller equation as well as energy level diagram. Lifetime measurements gave the rate constant (k(et)) for electron injection from the excited state chlorophyll into the conduction band of TiO(2) is 4.2x10(8)s(-1).