PHYCOERYTHROCYANIN: ITS SPECTROSCOPIC BEHAVIOR AND PROPERTIES

Abstract— Phycoerythrocyanin is a biliprotein found in very few blue-green algae. Its properties have been examined under three conditions: in whole cells, in light-harvesting organelles (the phycobilisomes). and as an isolated protein. Absorption and fluorescence bands characteristic of the isolated protein are essentially the same as those in intact cells of the blue-green alga Anabaena variabilis. The same spectroscopic hallmarks are observed in purified phycobilisomes. Dissociation of these physobilisomes at low-phosphate concentrations resulted in increased phycoerythrocyanin fluorescence. This time-dependent increase in fluorescence demonstrates the function of this biliprotein in excitation-energy transfer to the other biliproteins when the organelles are intact. The relative stabilities of the various heteroprotein bonds within the phycobilisomes are shown to possess differing phosphate ion dependencies. Studies on the isolated protein from Mastigocladus laminosus include fluorescence measurements at both 23 and-196°C, as is generally observed with biliproteins, although phycoerythrocyanin has complex visible absorption and excitation spectra, only a single emission band is observed.     

SPECTRAL INHOMOGENEITY OF THE LIGHT-HARVESTING ANTENNA OF Rhodospirillum rubrum PROBED BY TRIPLET-MINUS-SINGLET SPECTROSCOPY AND SINGLET-TRIPLET ANNIHILATION AT LOW TEMPERATURES

–Triplet-minus-singlet (T-S) spectra and singlet-triplet annihilation was measured on Rhodospirillum rub-rum chromatophores at low temperatures (4–77K). The T-S spectrum of the LH-1 antenna was dependent on the wavelength of excitation and thus indicative of site-inhomogeneous broadening. The data cannot be understood adequately using the two pigment pool model (B880 + B896) but, in contrast, support a model for LH-1, in which the spectral properties are determined by site-inhomogeneous broadening. Consequently, the 4 K antenna domain for singlet-triplet annihilation in LH-1 is rather small. If the temperature is raised to 77 K the annihilation domain increases, and a fit of the fluorescence quenching and triplet yield data suggests that at 77 K the connectivity is between the extreme cases of the “lake” and the isolated “puddles” models. The implications of the observed antenna inhomogeneity on low temperature annihilation and fluorescence measurements are discussed.     

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.     

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.     

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.     

Ultra-low-temperature non-aqueous capillary electrophoretic separation--77 K fluorescence spectroscopic detection for the on-line identification of photo-converted analytes of trans-resveratrol

We demonstrate here, for the first time, that non-aqueous capillary electrophoresis (NACE) can be interfaced with any ultra-low-temperature (ULT) separation method and 77 K fluorescence spectroscopy (FS). This novel ULT-NACE-FS system consists of a modular CE system, a dry ice bath, and instrumentation for 77 K fluorescence detection. The ULT-NACE method serves to separate structurally similar molecules by a combination of a low electrophoresis current and a high voltage at approximately -70 degrees C. When the ULT-NACE-separated analytes move into the quartz Dewar flask and traverse into the capillary detection window, liquid nitrogen was added, thus freezing the separating analyte zones, allowing the collection of 77 K fluorescence spectra for on-line spectral fingerprint identification. The first application of the ULT-NACE-FS system is described for the analysis of photo-converted analytes of trans-resveratrol; prospects and future applications of ULT-NACE-FS are also briefly addressed.     

LOW-TEMPERATURE LUMINESCENCE SPECTRA AND FLUORESCENCE LIFETIMES OF POLYCYTIDYLIC ACID IN POLYALCOHOLIC GLASSES

Abstract— Corrected emission spectra and fluorescence lifetimes of polycytidylic acid in ethylene glycol: water glass at low temperatures are reported. Luminescence properties observed exhibit a strong dependence on pH and temperature. At neutral pH a vibronic structure of a blue part of the fluorescence spectrum is revealed when temperature is changed from 77 to 10 K, confirming that a monomer component of fluorescence is present. There is also a strong difference in decay of a red-shifted excimer fluorescence at 10 K at pH 7 and pH 3.9, reflecting a different protonation of cytosine residues and different conformations of polynucleotides in such conditions.     

SPECTROSCOPY OF POLYENES–III. ABSORPTION AND EMISSION SPECTRAL INVESTIGATION OF POLYENE SCHIFF BASES AND PROTONATED SCHIFF BASES RELATED TO VISUAL PIGMENTS

Abstract— A number of n -butylamine Schiff bases of polyenals related to retinals as homologues and analogues, and their protonated forms, have been studied for absorption and emission spectral properties. The polyene Schiff bases exhibit the same general features in their absorption spectra as those of the parallel polyenals except that the ^lB_u←¹A_g and π*← n singlet transitions are at substantially higher energy in the Schiff bases (the shift being larger for the π *← n transition). The Schiff bases with short polyene chainlength ( n = 2, 3 where n is the number of double bonds including C=N) do not fluoresce or phosphoresce in 3-methylpentane in the temperature range 298–77 K. The Schiff bases with intermediate chainlength ( n = 4, 5) show fluorescence at 77 K with intensity strongly dependent on the nature of solvent. The Schiff bases with relatively long chainlength ( n = 5–7) show strong or moderately strong fluorescence at 77 K and very weak fluorescence at 298 K ( n = 7) with intrinsic radiative lifetimes much longer than those estimated from the oscillator strength of the low-energy, strong absorption band (¹B_u←¹ A_g ). A discussion on the possible state order and nature of the fluorescing state of the various polyene Schiff base systems is presented.     

SPECTRA OF FLUORESCENCE LIFETIME AND INTENSITY OF Rhodopseudomonas sphaeroides AT ROOM AND LOW TEMPERATURE. COMPARISON BETWEEN THE WILD TYPE, THE C 71 REACTION CENTER-LESS MUTANT AND THE B800–850 PIGMENT-PROTEIN COMPLEX

Abstract— Spectra of the fluorescence lifetime and intensity of chromatophores from the wild type Rhodopseudomonas sphaeroides , from the C 71 reaction center-less mutant and of the B800–850 light harvesting pigment-protein complex have been studied by phase fluorimetry techniques at different light modulation frequencies at room and low temperature.
As already known, closed reaction centers (saturating light) are still quenchers of antenna fluorescence although with a lower efficiency than when they are opened. The fluorescence yields and lifetimes of both the C 71 mutant strain and the B800–850complex are found to increase by about 30% between room and low temperature.
The fluorescence lifetimes obtained for the C 71 strain (0.65 ns at 20C; 0.85 ns at 77 K) and for the B850 complex (1 ns at 20C; 1.3 ns at 77 K) indicate that the non-radiative deactivation pathways, in the antenna, remain important in the absence of the reaction centers even at low temperature. We suggest that these data arise from the presence of special antenna molecules which act as intrinsic quenchers of the B875 antenna fluorescence. Between room and low temperature, the fluorescence yield and lifetime of the wild type are found roughly constant. This result suggests that the energy trapping by the reaction centers is independent of the temperature. The mechanism governing the energy transfer from the antenna to the reaction centers may differ from the mechanism leading to the energy transfer within the antenna. We suggest that a partially irreversible trapping of the excitation energy, on its way to the reaction center, takes place in the vicinity of the reaction center.     

BILIPROTEINS FROM THE BUTTERFLY Pieris brassicae STUDIED BY TIME-RESOLVED FLUORESCENCE AND COHERENT ANTI-STOKES RAMAN SPECTROSCOPY

Abstract— The fluorescence decay time of the biliverdin IX7 chromophore present in biliproteins isolated from Pieris brassicae is determined to be 44 ± 3 ps. This value suggests a cyclic helical chromophore structure. The vibrational frequencies determined by CARS-spectroscopy are compared with those of model compounds. The data confirm that the chromophore in the protein-bound state adopts a cyclic-helical, flexible conformation.     

THE PRIMARY REACTION IN THE PHOTOREDUCTION OF PROTOCHLOROPHYLLIDE MONITORED BY NANOSECOND FLUORESCENCE MEASUREMENTS

Abstract— Time resolved fluorescence measurements, carried out on protochlorophyllide reductase enriched membranes from oat ( Avena sativa ), are described. A fast (1 ns at 293 K) decaying fluorescence component is assigned to the photoactive NADPH-protochlorophyllide-enzyme complex, while a slower (5 ns) component is ascribed to non-photoactive protochlorophyllide. The results are interpreted in terms of a new fast primary step in the light requiring step of chlorophyll synthesis. The temperature dependence of the rate of this reaction has been studied by measuring the decay time of the fast fluorescence component at various temperatures from 77 to 293 K. Complete spectra of the kinetic fluorescence components have been measured at 293, 160 and 77 K.     

POLARIZED PHOTOACOUSTIC SPECTRA OF PHYCOERYTHRIN and PHYCOCYANIN IN ANISOTROPIC POLYMER FILMS

Abstract— Photoacoustic spectra (PAS) of biliproteins, namely, R-phycoerythrin (PE) and C-phycocyanin (PC) and their mixture in anisotropic and isotropic polyvinylalcohol (PVA) films were measured under the illumination with polarized and natural light. Also samples in deuterated PVA were investigated. The yields of fluorescence of various chromophores of investigated biliproteins were obtained from PAS, absorption, fluorescence and fluorescence lifetime measurements. The deuteration of samples causes different changes in thermal deactivation of excitation of various chromophores. Ratios of PAS to absorption of the light polarized parallel and perpendicular to the direction of film stretching are different. The PAS amplitude of deuterated samples is higher than that for undeuterated.     

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.     

LUMINESCENCE STUDIES ON FORMYCIN, ITS AGLYCONE, AND THEIR N-METHYL DERIVATIVES: TAUTOMERISM, SITES OF PROTONATION AND PHOTOTAUTOMERISM

Abstract— A detailed study has been made of the luminescence spectra of 3-β-d -ribofuranosyl-7-amino-pyrazolo(4,3-d)pyrimidine (formycin A), 3-propyl-7-aminopyrazolo(4,3-d)pyrimidine (7APP), and their various N-methyl derivatives, at room temperature and in methanol-water glasses at 77 K. Comparisons of the foregoing, together with the observed dependence of the emission spectra of formycin and 7APP on excitation wavelength, demonstrated that these consist of two tautomeric species, N(1)H and N(2)H, both of which emit at 300 and 77 K. The two tautomers may be distinguished by the location of the emission maxima, especially for phosphorescence, and quantum yields for emission. Comparisons of the emission spectra of the protonated forms of 7APP and its N-methyl derivatives showed that the fluorescence of the cations of 7APP and its N,- and N₂-methyl derivatives originates from the forms protonated on N(4). By contrast, the forms protonated on N(6) contribute appreciably to the phosphorescence at 77 K. On the basis of the emission spectra at 77 K, it is concluded that the major tautomeric form of the formycin cation is N(1)H,N(4)H₊, but there is also some contribution by the form N(2)H,N(4)H₊. In acid medium at room temperature, there is photodissociation of a proton from the pyrazole ring of the formycin cation. This leads to formation in the state S! of the tautomeric species N(4)H, which does not exist in the ground state. This conclusion, similar to that previously reported for the analogous isomeric 4-aminopyrazolo(3,4-d)pyrimidines, is derived from a comparison of the fluorescence spectra of the cations of formycin and N₄-methylformycin, which exhibit two bands at 375 and 440 nm, the latter corresponding to the emission of the neutral form of N,i-methylformycin. The proposed mechanism of phototautomerization is supported by a study of solvent and salt effects.     

多变鱼腥藻藻胆蛋白共价复合物的合成及其分子内能量传递

利用双功能基偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀酰亚胺酯(SPDP)合成了两个藻胆蛋白复合物,藻红蓝蛋白-变藻蓝蛋白复合物PEC-APC和藻红蓝蛋白-藻蓝蛋白复合物PEC-PC.利用吸收光谱和荧光光谱证明了藻胆蛋白构型与构象在反应后得到保持。通过荧光光谱观察到能量传递现象。计算出复合物PEC-APC的分子内能量传递效率约为90%.复合物PEC-PC中藻红蓝蛋白PEC的荧光寿命比PEC本身的寿命大大缩短,证明存在分子内能量传递。二硫苏糖醇(DTT)还原二硫桥键后能量传递被阻断。这进一步证明复合物合成成功及分子内能量传递。     

On-line identification of trans-resveratrol in red wine using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy

The feasibility of combining the techniques of on-line concentration and capillary electrophoresis/low-temperature fluorescence spectroscopy (CE/LTFS) for the detection and identification of trans-resveratrol in red wine at 77 K is demonstrated for the first time. This technique, involving sweeping-micellar electrokinetic chromatography (sweeping-MEKC), was used for the initial on-line concentration and separation, after which a cryogenic molecular fluorescence experiment was performed at 77 K. In comparison with normal-MEKC mode, a approximately 1500-fold improvement in detection sensitivity could be obtained when the sweeping-MEKC was applied. The proposed method permits not only the separation and detection of trans-resveratrol from red wine extracts but also ensures that the on-line spectrum is readily distinguishable and can be unambiguously assigned at 77 K.     

IN VIVO CHLOROPHYLL a FLUORESCENCE TRANSIENTS AND THE CIRCADIAN RHYTHM OF PHOTOSYNTHESIS IN GONYAULAX POLYEDRA

Abstract— The intensity of chlorophyll a fluorescence during the early part of fluorescence induction at O , initial fluorescence, and P, peak fluorescence, was higher during the day phase of the circadian cycle than during the night phase in continuous light (LL) conditions and was positively correlated with the rate of oxygen evolution. The circadian rhythm in fluorescence in LL persisted in the presence of 10μM 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which blocks electron flow from photo-system (PS) II in photosynthesis. The rhythmic changes in fluorescence intensity are consistent with a lower rate constant for radiationless transitions during the day phase than during the night phase of the circadian rhythmicity. The circadian changes in the intensity of fluorescence were abolished at 77K, which may indicate the importance of structural changes in membranes in circadian oscillations.     

TWENTY-TWO CARBON HOMOLOGUE OF 11-CIS RETINAL. PHOTOPHYSICAL AND PHOTOCHEMICAL PROPERTIES

Abstract— Results concerning absorption-emission spectra and fluorescence quantum yields at 77 and 298K. triplet absorption spectra, and quantum yields of intersystem crossing and photoisomerization at 298 K, are presented for 11- cis β-apo-14'-carotenal (C₂₂-Ald), the immediate higher homologue of 11- cis retinal. The absorption spectra are characterized by two band-systems with maxima at 390–400 and 270–280 nm, respectively. Upon cooling from 298 to 77 K, the intensities of these two band-systems undergo changes in opposite directions indicating 12-s- cis: 12- s-trans conformational changes. No intermediate band-system analogous to the one located at 270–310 nm in cis retinals is observed for 11- cis C₂₂-Ald. In nonpolar hydrocarbon solvents (e. g. cyclohexane) at room temperature, quantum yields of fluorescence (0.01), intersystem crossing (0.6) and photoisomerization (0.4) are all quite pronounced. The photophysical and photochemical properties of 11- cis C₂₂-Ald are discussed in the light of similarities and dissimilarities with those of all- trans C₂₂-Ald and 11- cis retinal under comparable conditions.     

Determination of lysergic acid diethylamide (LSD) by application of online 77 K fluorescence spectroscopy and a sweeping technique in micellar electrokinetic chromatography

The principal advantage of the use of Shopl'skii effect (low temperature spectrum) is that spectral sharpening occurs both in absorption and emission. However, thus far using the technique of capillary electrophoresis/low temperature fluorescence spectroscopy (CE/LTFS) either at 77 or 4.2 K remains difficult to obtain an on-line spectrum, if the analyte is present at low concentration. This paper examines the feasibility of combining the techniques of online concentration and CE/LTFS to identify LSD and related compounds in urine at 77 K. To improve sensitivity, sweeping-micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used for on-line concentration which resulted in detection limits of approximately 20 approximately 60 ppt, respectively.     

Online identification of the fluorescent whitening agent 4,4-bis(2-sulfostyrol)biphenyl using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy

The feasibility of combining the techniques of online concentration and CE/low-temperature fluorescence spectroscopy in the detection and identification of E,E-4,4'-bis(2-sulfostyryl)biphenyl (DSBP) in synthetic detergents at 77 K is demonstrated. The technique involves the use of sweeping-MEKC, and was used for the initial online concentration and separation, after which a cryogenic molecular fluorescence experiment was performed at 77 K. The proposed method not only permits the separation and detection of E,E-DSBP in a synthetic detergent sample, but also ensures that the online spectrum is readily distinguishable and can be unambiguously assigned at 77 K. The photoconversion and isomer separation of DSBP are also described.