ENERGY TRANSFER AND ENERGY LOSSES IN BILAYER MEMBRANE VESICLES (LIPOSOMES)

Abstract. The efficiency of singlet-singlet energy transfer was studied in bilayer lipid membrane vesicles (liposomes) for the following donor-acceptor systems: (1) p -terphenyl (TP) and diphenyloctatetraene (DPO); (2) DPO and chlorophyll a (Chl a ); and (3) β-carotene and Chl a. The energy transfer efficiency φ_DA was measured by sensitized fluorescence of the acceptor. Fractional quenching of the donor φ_Q was found from the donor fluorescence in absence and presence of the acceptor. For TP-DPO and for DPO-Chl a , the transfer efficiency increased with increasing acceptor concentration but was essentially independent of the donor concentration. No energy transfer from β-carotene to Chl a could be detected. In liposomes, φ_DA differed only slightly from φ_Q at all donor and acceptor concentrations, thus demonstrating the absence of any appreciable energy losses. For solutions of the same donor-acceptor pairs in cyclohexane φ_Q was considerably larger than φ_DA. The difference represents energy lost, principally by internal conversion, due to collisional quenching. The principal function of the lipid membrane appears to be the suppression of such losses. In addition, the rate of energy transfer in lipid membranes is about double that in solutions (at the same intermolecular distance) due to more favorable orientation.     

ELECTRON TRANSFER FROM CHLOROPHYLL a TO QUINONE IN MONO- AND MULTILAYER ARRAYS

Abstract— Mono- and multilayers of chlorophyll a (Chl a )– lecithin have been prepared on quartz slides, by means of the Blodgett-Langmuir technique, for fluorescence studies. Self-quenching of the Chl a fluorescence has been observed in Chl a -lecithin single layer excited with a laser light at 632.8 nm. The fluorescence yield is reduced by 50% at a concentration of 7 ± 10¹² Chl a molecules cm⁻². Chl a fluorescence quenching, by adding N,N -distearoyl-1,4-diaminoanthraquinone (SAQ), has been studied. in a single layer, in pure Chl a and also at various dilutions of Chl a in lecithin. The results are explained in terms of a dynamic quenching rather than in terms of a permanent complex formation, at the ground state, between Chl a and SAQ. The fluorescence quenching has been interpreted as the result of an electron transfer from excited Chl a to SAQ, and rate constants of 8.3 ± 10⁻⁵ cm² molecule⁻¹ S⁻¹ and 2.4 ± 10⁻⁴ cm² molecule⁻¹ s⁻¹ have been found for pure diluted Chl a , respectively. Ten per cent of the diluted Chl a fluorescence always remains unquenchable and independent of the quinone concentration. In multilayers, where SAQ and Chl a are in different layers, there is no fluorescence quenching for pure or diluted Chl a even when the chromophores are in two adjacent layers. This happens only if SAQ is not able to diffuse from one layer to another. A minimum value of 22.4 nm has been found for the singlet exciton diffusion length in pure Chl a multilayers.     

PICOSECOND LASER SPECTROSCOPY AND OPTICALLY DETECTED MAGNETIC RESONANCE ON A MODEL PHOTOSYNTHETIC SYSTEM

Fluorescence-detected magnetic resonance of triplets in zero magnetic field (FDMR), fluorescence fading (FF) due to triplet-formation, both at 4.2 K, and prompt fluorescence decay kinetics (FDK) at room temperature have been measured for free pheophorbide- a (f-Pheo) and bound (b-Pheo) to a synthetic polypeptide (L-L ys -L-A la -L-A la )_n, dissolved in dimethylformamide (DMF). Fluorescence decay kinetics measurements of f-Pheo in DMF yielded 1-5 ns lifetimes, for b-Pheo in DMF a ~ 50 ps decay-component was found emitting at 730–750 nm. Zero-field splitting parameters |D| and |E| of the lowest triplet state T₁ were determined from FDMR spectra as (337 and 24) 10^-4 cm^-1 for f-Pheo and (359 and 25) 10^-4 cm^-1 for b-Pheo, both in DMF. Decay rate constants of the three spin levels of T¹ of b-Pheo ( K _x= 1200 50 s^-1, k _y= 440 25 s^-1, k _z= 80 5 s^-1) and relative steady-state populations (N_x= 28 2%, N_y= 47 2%, N_z= 26 2%) determined from FF curves predict a fluorescence decrease at the D–E and D + E FDMR transitions, whereas experimentally a fluorescence increase is observed. The FDMR sign-inversion results from singlet-singlet energy transfer from b-Pheo monomers to their aggregates, followed by fast intersystem crossing to T₁. These results indicate that aggregates are formed by two or more b-Pheo molecules at different positions on the folded polypeptide chain. This situation resembles that in chlorophyll-proteins, containing low-lying traps, resulting from interaction of chromophores with other chromophores and with the protein environment.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

A HIGH RESOLUTION FLUORESCENCE DECAY AND DEPOLARIZATION STUDY OF HUMAN PLASMA APOLIPOPROTEINS

Abstract— Human plasma apolipoprotein A-I (apoA-I) and apolipoprotein C-I (apoC-I) were investigated by time-resolved fluorescence decay and depolarization. The tryptophyl fluorescence of apoA-I undergoes a double-exponential decay with lifetimes of 1.07 and 3.43 ns which remain unchanged over the range of apoA-I concentration studied.
The time-resolved fluorescence of both native and denatured forms of apoC-I exhibits an unusual tryptophyl fluorescence decay that was best fit to a triexponential function with lifetimes at 3.7 ± 0.2, 1.1 ± 0.1 and 0.1 ns at 2°C. The native and denatured forms of apoC-I had rotational correlation times of 1.42 and 1.19 ns at 20°C respectively. A shorter rotational correlation time associated with the internal tryptophan motions was not observed or resolved.
The decay of tryptophyl fluorescence in apoC-I/DPPC/cholesterol complex at 20°C is also triexponential with lifetimes at 4.94, 1.28 and 0.21 ns, which are longer than those of the uncomplexed forms. Two rotational correlation times of 28.32 and 0.59 ns at 20°C were resolved by fluorescence depolarization measurements. The long rotational time remained constant with temperatures above 30°C. Also, the temperature dependence of the order parameter, S², resembled a lipid phase transition curve with a transition midpoint at 38°C. The tryptophan and thus apoC-I are found to be affected by the bulk changes in the lipid.     

ENERGY TRANSFER IN A LIGHT-HARVESTING CAROTENOID-CHLOROPHYLL c-CHLOROPHYLL a-PROTEIN OF PHAEODACTYLUM TRICORNUTUM

Abstract— The marine diatom Phaeodactylum tricornutum was readily disrupted in 0.1 N Tris-HCl buffer, pH 7.8, in a Braun Model MSK cell homogenizer at 0-5°C. Treatment of the suspension with sodium lauryol sarcosinate (3 molecules per 10000 daltons of protein) at 5°C in the dark and subsequent centrifugations produced a pigmented, protein fraction whose excitation spectrum exhibited energy transfer from carotenoids to chlorophyll a (Chi a ). Disruption of the pigment-protein complex by heating in 1% sodium dodecylsulfate resulted in loss of energy transfer. For each Chi a molecule this fraction had 1 Chi c , 4 fucoxanthin, and 6.7 accessory pigment molecules. Presence of the accessory complex of Photosystem II in this preparation is suggested by the high xanthophyll content. Further, based on Chl a concentrations, this fraction had about 18 times more apparent fluorescence emission at 680 nm when excited at 470 nm than the intact cells.     

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.     

CAROTENOID TRIPLET STATE AND CHLOROPHYLL FLUORESCENCE QUENCHING IN CHLOROPLASTS AND SUBCHLOROPLAST PARTICLES

Abstract— Absorption changes attributed to the triplet state of carotenoids and to primary electron donors (P-700. P-680)^: and fluorescence quenching at several wavelengths have been measured with a single apparatus. following flash excitation with a dye or a ruby laser. Spinach chloroplasts as well as subchloroplast particles enriched in Photosystem-1 (F₁), Photosystem-2 (F₁) or the light-harvesting Chl a/h (F_III) have been examined at temperatures varying between 5 and 294 K.
The triplet state of carotenoids has been identified on the basis of its difference spectrum (having a peak at 515 nm) and decay kinetics (⋍ 7 µs at low temperature; accelerated by O₂ at 294 K). It is formed in all of the materials studied. The quantum yield of carotenoid triplet formation in chloroplasts increases at low temperature, but less than the fluorescence yield.
In most cases the fluorescence quenching recovers approximately with the same kinetics as the decay of the carotenoid triplets. The fluorescence recovery is, however, significantly faster for chloroplasts at 730 nm. Fluorescence quenching occurs in all types of materials. The ratio of fluorescence quenching to the concentration of carotenoid triplets varies with the material, being maximum in chloroplasts and minimum in F_m particles.
We conclude that the formation of the carotenoid triplet state is not limited to a few sites in the chloroplast and that a carotenoid triplet is a quencher of chlorophyll fluorescence. A detailed comparison of carotenoid triplets and fluorescence quenching gives some information concerning the organization of the pigments in the photosynthetic apparatus.     

PROTECTION OF CHLOROPHYLL a BY CAROTENOID FROM PHOTODYNAMIC DECOMPOSITION

Abstract— The order of inhibition of the photooxidation of chlorophyll a in ethanol and ethanol-benzene is as follows: β-carotene, α-tocopherol, benzoquinone, DABCO, menadione, cholesterol and KI. The quenching of singlet oxygen by β-carotene occurs by a collisional quenching mechanism with a diffusion-controlled rate of 1.7 × 10¹⁰ M ^-1 s^-1. Photodecomposition of Chi a is faster in ethanol-D₂O than in ethanol-H₂O. Photoirradiation (660 nm) of the peridinin-Chl a -protein complex, a photosynthetic light-harvesting pigment isolated from marine dinoflagellates, did not show any photo-decomposition of its Chi a in H₂O or D₂O, even after an extended period (12 h) of irradiation. However, the carotenoid, peridinin, in the photosynthetic antenna pigment was photobleached (ca. 10%) during the irradiation. We conclude that the singlet oxygen formed as a result of the Chi photosensitization is immediately quenched by the low-lying triplet state of four peridinin molecules (per Chl a ) bound within the same protein crevice. The carotenoid thus effectively protects Chl a from photodynamic damage, providing a direct proof for the protective role of carotenoids in the photosynthetic pigment complex.     

PROPERTIES OF CHLOROPH'YLL-LECITHIN VESICLES: ULTRACENTRIFUGATION, ABSORBANCE, EMISSION AND PHOTOBLEACHING

Abstract— Egg lecithin vesicles (liposomes) containing up to one Chl a per 11 lecithin molecules were prepared without sonication. Their absorbance and emission properties indicate that Chl is dispersed in the lecithin, that the vesicles are dispersed in the medium and that there is a strong quenching of fluorescence.
Their photobleaching and chemical bleaching show that there is a negligible amount of colloidal Chl present. In vesicles with a Chl:lecithin molar ratio higher than 0.05, there are probably some small aggregates of Chl a in the bilayer.
The ultracentrifugation of such vesicles shows that their sedimentation constant increases, though not regularly, with their relative Chl content     

THE PHOTOPHYSICS OF MEROCYANINE 540. A COMPARATIVE STUDY IN ETHANOL AND IN LIPOSOMES

Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> ø_T > 0.02 in ethanol and 0.01 > ø_T- > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.     

TRIPLET STATES AND cis-trans PHOTOISOMERIZATION PROCESSES IN THE SCHIFF BASES OF RETINAL ISOMERS

Abstract —The triplet states of the n -butyl-amine Schiff bases of 11- cis , 9- cis , 13- cis and all- trans retinal are produced via triplet-triplet energy transfer. Their absorption spectra, peaking around 435 nm, and their decay kinetics are recorded using pulsed-laser photolysis. Direct-excitation (φ^D_ISO) and triplet-sensitized (φ^T_ISO) photoisomerization yields, determined using steady irradiation methods, are found to be: φ^T_ISO (9- cis ) = 0.06, φ^T_ISO (11- cis ) = 045, φ^T_ISO (13- cis ) = 008, φ^T_ISO (all- trans ) = 0.02-0.05, φ^D_ISO (11- cis , = (4 ± 1) × 10^-3, φ^D_ISO (all- trans ) = (2 ± 1) × 10^-3. The possible role of the triplet state in the isomerization of rhodospin is discussed.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE-1-ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet-and singlet-related photoprocesses of pyrene-1-aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time-correlated determination of fluorescence lifetimes (τ_F) and steady-state photochemical and absorption-emission spectral measurements. In benzene, the lowest triplet of PA (43 < E_T < 46 kcal/mol) has a lifetime of about 50 µs (τ_T) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (ε_max) of 21000 M ^-1cm^-1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (ε_max≥ 25000 M ^-1cm^-1). The quantum yields (φ_T) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φ_F) of fluorescence. Quantum yields (φ_PC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φ_PC is in the range 0.1–0.2 in electron-rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τ_F, τ_T, φ_F. φ_T and φ_PC as well as the effects of 1,2,4-trimethoxybenzene and 2,5-dimethyl-2,4-hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.     

ON THE NATURE OF INTERACTION BETWEEN PROFLAVINE and DNA

Abstract— The fluorescence decay of the mutagenic drug proflavine (PF) bound to DNA at a phosphate-to-drug ratio of 420 is found to be a non-exponential function of time. The deviation from exponentiality is shown to increase with increasing GC content of DNA. Thus, heterogeneity in the high affinity binding sites is clearly demonstrated. The nucleotides guanosine-5'-monophosphate (GMP) and cyti-dine-5'-monophosphate (CMP) are shown to form complexes with PF in 10^-3M sodium cacodylate buffer at pH 6.6. In addition, GMP quenches substantially the fluorescence of PF while CMP enhances it slightly. The fluorescence decay curves for 5 × I0^-6 M PF in the presence of GMP concentrations greater than 10^-2 M exhibit a deviation from exponentiality which parallels that exhibited by the fluorescence decay curves for PF bound to DNA of increasing GC content. It is inferred that (a) guanine is responsible for the quenching of the fluorescence of PF on binding to DNA; and (b) the forces involved in the interaction between PF and DNA are specific in nature. The implications of these findings concerning the mutagenic properties of acridine derivatives are discussed. Nanosecond depolarization studies reveal a quite fast depolarization of the fluorescence of bound PF; for the PF- Cl. perfringens DNA complex the rotational correlation time is about 26 ns. Time-resolved emission spectroscopy in the nanosecond scale demonstrates that, despite the change in the orientation of the drug, the electronic structure of the PF-DNA complex is not substantially altered during the lifetime of the excited singlet electronic state of the drug.     

LASER PHOTOLYSIS STUDY ON POLY(N-VINYLCARBAZOLE) ADSORBED ON CELLULOSE SUBSTRATE

Abstract Triplet state and photoinduced charge separation dynamics under adsorption was Confirmed for the first time for the polymer without or with coadsorbed electron acceptor, respectively. The absorption spectrum of the triplet polymer is similar to the T_n← T₁ band of N -ethylcarbazole, showing no appreciable interchromophoric interaction. Absorption spectra of the coadsorbed systems comprise the polymer cation and electron acceptor anion. The ion radicals on the cellulose substrate undergo mutual recombination, and simple uni- and bimolecular decay kinetics do not hold. This mechanism was compared with that for poly( N -vinylcarbazole) film.     

INFLUENCE OF THE LOCATION OF TRYPTOPHANYL RESIDUES IN PROTEINS ON THEIR PHOTOSENSITIVITY

The environmental effect on Trp residues photolysis was investigated on four proteins containing a single Trp residue in environments of various polarities: glucagon (exposed residue), nuclease (partially buried residue), RNase T₁ (fully buried residue) and melittin (exposed or partially buried residue depending on the salt concentration). Direct photolysis was performed in neutral N₂-saturated phosphate solution at 20°C using 302 nm monochromatic light. Tryptophan loss was monitored by both absorption and fluorescence spectroscopy and by amino acid analysis. The results suggest that tryptophan photodegradation depends on the location of the residue in the protein, with regard to the exposure to the aqueous medium and to the neighbouring amino acids in the primary amino acid sequence and in the three dimensional structure. Photochemical products were not analysed but fluorescence spectra indicate that they vary with protein.     

THERMAL DENATURATION OF MONOMERIC AND TRIMERIC PHYCOCYANINS STUDIED BY STATIC AND POLARIZED TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

C. Phycocyanin (PC) and allophycocyanin (APC). as well as the α-subunit of PC. have been isolated from the blue-green alga (cyanobacterium). Spirulina platensis . The effects of partial thermal denaturation of PC and of its state of aggregation have been studied by ps time-resolved, polarized fluorescence spectroscopy. All measurements have been performed under low photon fluxes(10¹³ photons/pulse cm²) to minimize singlet-singlet annihilation processes. A complex decay is obtained under most conditions, which can be fitted satisfactorily with a bi-exponential (T₁=70–400 ps, T₂ -1000–3000 ps) for both the isotropic and the polarized part, but with different intensities and time constants for the two decay curves. The data are interpreted in the framework of the model first developed by Teale and Dale [ Biochem. J. 116, 161 (1970)], which divides the spectroscopically different chromophores in (predominantly) sensitizing ( s ) and fluorescing ( f ), ones. If one assumes temperature dependent losses in the energy transfer from the s to the f and between f chromophores. both the biexponential nature of the isotropic fluorescence decay and the polarization data can be rationalized. In the isotropic emission (corresponding to the population of excited states) the short lifetime is related to the sf transfer. the longer one to the "free" decay of the final acceptor( s ) (= f ). The polarized part is dominated by an extremely short decay time. which is related to sf transfer, as well as to resonance transfer between the f -chromophores.