FLUORESCENCE RELAXATION KINETICS AND QUANTUM YIELD FROM THE PHYCOBILISOMES OF THE BLUE-GREEN ALGA NOSTOC SP. MEASURED AS A FUNCTION OF SINGLE PICOSECOND PULSE INTENSITY*

Abstract— A detailed experimental study of the effect of intensity of a 6 ps excitation pulse on the decay kinetics and yield from phycobilisomes (PBsomes) is presented. The fluorescence from the c-phycoerythrin (PE) emission from PBsomes was found to decay as a single exponential with a time of 31 ± 4ps for an excitation intensity <10¹⁴ photons/cm² per pulse. The risetime of the c-phycocyanin (PC) and allophycocyanin (APC) emission from PBsomes was found to be 34 ± 13 ps. Therefore, at low excitation intensities, the energy transfer time between the constituent phycobiliproteins, PE and PC, is measured to be 34 ± 13ps from the fluorescence decay time of PE and the fluorescence risetime of the PC and APC emission. The fluorescence yield from the PE emission component in PBsomes was found to be intensity dependent for excitation intensities >10¹⁴ photons/cm². The decrease in yield with increased intensity in this case occurred at a higher intensity than in the isolated phycobiliprotein PE. The fluorescence yield of the PC and APC emission component was also found to decrease markedly with increasing excitation intensity. This is in contrast to the case of the isolated phycobiliprotein APC which showed only a slight quenching of the fluorescence. The higher quenching observed for the APC emission in the PBsome evidences the higher effective absorption of APC via energy transfer from PE to PC and APC.     

EXCITON ANNIHILATION IN THE ISOLATED PHYCOBILIPROTEINS FROM THE BLUE-GREEN ALGA NOSTOC SP. USING PICOSECOND ABSORPTION SPECTROSCOPY

Abstract— Phycobilisomes from the blue-green alga Nostoc sp. contain the phycobiliproteins: c -phycoerythrin ( c -PE), c -phycocyanin ( c -PC) and allophycocyanin (APC). The depletion and the recovery of the ground states for the individual phycobiliproteins were measured using picosecond (ps) absorption spectroscopy. In all cases the depletion time was Glops. The recovery was found to be non-exponenzial which could be fitted to a single exponential ('fast' component) and a second component with a relaxation time of > 300ps. The recovery times of the fast component were found to be intensity dependent and for c -PE, c -PC and APC were measured to be 19, 27 and 35ps, respectively, at intensity ( I ) ∼ 7 × 10²⁰ photons/m² and increased to 54, 55 and 67 ps, respectively, at I ∼ 8 × 10¹⁹ photons/m2. The ps absorption data support the assignment of the'fast' component to singlet-singlet exciton annihilation.     

RECONSTITUTION OF ALLOPHYCOCYANIN FROM Mastigocladus laminosus WITH ISOLATED LINKER POLYPEPTIDE

The core linker polypeptide L_c^8.9 was isolated from Mastigocladus laminosus and purified on a preparative scale. A method for the reconstitution of allophycocyanin (AP)—linker complexes from isolated polypeptides was developed. The complex (α^AP(β^AP)₃ L_c^8.9 was reconstituted and compared to (α^APβ^AP) and (α^APβ^AP)₃ by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. Differences in the spectra of reconstituted and of directly isolated AP complexes are discussed.     

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.     

STRUCTURAL DIFFERENCES BETWEEN PHYCOCYANIN and ALLOPHYCOCYANIN FROM THEIR RESONANCE RAMAN SPECTRA*

Abstract— Resonance Raman spectra of the chromophores of the cyanobacterial light-harvesting proteins phycocyanin (CPC) and allophycocyanin (APC) were recorded using 364 nm excitation. The1500–1700 cm^-1 regions of these spectra were analyzed for the pH-induced structural changes accompanying the disruption of the native trimers into monomers as well as the progressive denaturation of these monomers. Computer-assisted decomposition of the 1642 cm^-1 marker bands of these spectra yielded up to four components (named I-IV), the frequencies of which were constant within 5 cm^-1 (CPC) and 10 cm^-1 (APC). The relative intensities of two of these components, namely I and III, were sensitive to chromophore conformations. The previously reported downshift of the 1642 cm^-1 band upon folding of the chromophores was shown to result from a weakening of component I and a concomitant enhancement of component III. Components I-IV had different relative intensity patterns in CPC and APC spectra. In particular, the higher relative intensity of component I at 1646 cm^-1 indicated more extended average conformations of the chromophores in trimeric APC than in trimeric CPC. This difference likely resulted from the extra β-155 chromophore present in CPC. Component III was sizably active in RR spectra of monomelic APC but was not observed either in those of monomelic CPC or in those of trimeric APC and CPC. This indicated that, in APC monomers, chromophore(s) did not assume the native conformations found in the trimer, while monomer formation did not sizably alter the structures of the CPC chromophores.     

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.     

FLUORESCENCE RELAXATION KINETICS AND QUANTUM YIELD FROM THE ISOLATED PHYCOBILIPROTEINS OF THE BLUE-GREEN ALGA NOSTOC SP. MEASURED AS A FUNCTION OF SINGLE PICOSECOND PULSE INTENSITY, I

Abstract— Phycobilisomes from the blue-green alga Nostoc sp. are known to contain the phycobiliproteins: c-phycoerythrin (c-PE), c-phycocyanin (c-PC) and four forms of allophycocyanin (APC I, II, III, and B). We have made a detailed study of the effects of the intensity of a single 6 ps excitation pulse on the decay kinetics and the yield of fluorescence in the individual isolated phycobiliproteins at pH 7 and 23°C. The risetime of the fluorescence of c-PE, c-PC and APC was > 12 ps. We found that the decay of the fluorescence was exponential at intensities of 10¹⁴ photons/cm² in all the phycobiliproteins; the lifetimes being 1552 ± 31ps for c-PE, 2111 ± 83ps for c-PC, 1932 ± 165ps for APC I, 1870 ± 90ps for APC II, 1816 ± 88ps for APC III, (1869 ± 62ps for the averaged APC's I, II, and III), and 2667 ± 233 ps for APC B. We also found that the fluorescence decay became non-exponential in c-PE at excitation intensities < 10¹⁴ photons/cm², but was exponential for all the other phycobiliproteins even at a pulse intensity of 10¹⁵ photons/cm². The relaxation times of c-PE and c-PC decreased with excitation intensity above 10¹⁴ photons/cm². For c-PE and c-PC the relative fluorescence vs excitation intensity was readily described by a relationship derived for a model in which exciton–exciton annihilation occurs. In APC the fluorescence yield and relaxation time were only slightly dependent on the excitation intensity. The results are interpreted to indicate the occurrence of singlet–singlet annihilation intramolecularly among the several phycobilin chromophores within the individual phycobiliprotein molecules in solution. The s to f transfer time is less than 12ps in c-PC.     

POLARIZED TIME-RESOLVED FLUORESCENCE SPECTRA OF PHYCOBILISOMES ISOLATED FROM Tolypothrix tenuis EMBEDDED IN POLY(VINYL ALCOHOL) FILM

–Time-resolved fluorescence spectra in the ps time range were measured on phycobilisome (PBS) embedded in poly(vinyl alcohol) films. The cyanobacterium Tolypothrix tenuis was used as a source of PBS because the pigment composition and the structure of the PBS are well defined. Isotropic PBS in the unstretched film and PBS uniaxially oriented in the stretched film were investigated. Diameters of PBS hemidiscs were oriented parallel to the film-stretching direction. The time-resolved fluorescence spectra of the unstretched sample and of the two polarized components in the stretched samples showed several differences in the rise and decay. The delay time, estimated from the time span between the maximum laser pulse and maximal intensity of the phycocyanin and allophycocyanin fluorescence, was much longer than that reported in the aqueous media. This suggests occurrence of a higher thermal deactivation of PBS in polymer film than in aqueous media. The excitation energy transfer from excited phycocrythrin to allophycocyanin was more efficient in the unstretched than in the stretched samples, and it was greater in the parallel polarized component of the stretched sample than in the perpendicular component. The present results are in agreement with a previous suggestion which states that there are two independent pathways of excitation energy transfer in PBS and that there is more than one final emitter of fluorescence. The molecules taking part in various pathways of energy transfer differ in their orientation within PBS.     

FLUORESCENCE QUENCHING OF ALLOPHYCOCYANIN AND PHYTOCHROME BY ESTRIOL*

Abstract— At 293 K the long-wavelength absorption and emission band of 1.4 μM allophycocyanin is decreased by estriol (Δ¹-³-⁵⁽¹⁰⁾-estratriene-3,16α, 17β-trio!) in the range 0.8-6.6 μM in the presence of 11% alcohol (vol/vol). The binding of estriol is shown to be of high affinity, 1:1 with allophycocyanin. The free energy of this binding process (ΔG^°) is -33.6 kJ mol' and single binding site dissociation constant (K_D) 1.0 ×10–6M. Estriol at 21 μM effectively quenches the fluorescence of 1.4 M large molecular weight phytochrome in its red absorbing form at 77 K while having little or no effect on the phototransformation difference spectrum at 293 K.     

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.     

PHOTOTRANSFORMATION OF AGGREGATED FORMS OF PROTOCHLOROPHYLLIDE IN ISOLATED ETIOPLAST INNER MEMBRANES

Abstract— Irradiation of an etioplast inner membrane fraction caused the transformation of two photoactive spectroscopically different protochlorophyllide forms into two chlorophyllide forms. A weak light flash, 6% of a saturating flash, preferentially caused the formation of a short wavelength chlorophyllide form absorbing at 672 nm and emitting at 676 nm. A saturating flash resulted in the formation of the 684 nm absorbing form of chlorophyllide with an emission maximum at 698 nm.
The circular dichroism (CD) signals of the newly formed chlorophyllide forms indicated that they are pigment aggregates of different sizes. These aggregates are probably connected to protochlorophyllide reductase and NADPH or NADP. In the absence of NADPH a decomposition of the pigment aggregates took place as revealed by a decrease in the CD-signal. A model is suggested which describes the structural changes of the pigment-protein aggregates after irradiation.     

SOLVENT-INDUCED PHOTOREVERSIBLE REACTIONS OF C-PHYCOCYANIN FROM SYNECHOCOCCUS SP.

Abstract— C-phycocyanin from Synechococcus sp. ( Anacystis nidulans ) shows photoreversible absorption changes when dissolved in buffer containing 75% ethylene glycol (vol/vol). Irradiation with red light (638 nm) causes a 7.5% decrease in absorbance around the absorption maximum (620 m), while the absorbance around 500 nm increases. Subsequent irradiation with green light (500 nm) partially reverses this change. Final photoreversibility at around 620 nm amounts to ca. 2.5% of the maximum absorbance. These reactions are ascribed to two interconvertible species PC_r and PC_g, the former with a higher absorbance in the red. the latter in the green. The rate of dark reversion from PC_g to PC_r is strongly enhanced by ferricyanide. It is proposed that with this reagent, dark reversion occurs via an oxidized form of PC_g. Furthermore, ferricyanide in the presence of ethylene glycol is capable of reversibly oxidizing part of the chromophores of C-phycocyanin, presumably to a radical. In the absence of ethylene glycol, however, ferricyanide causes total irreversible bleaching of the pigment in the dark. The induced photoreversibility of C-phycocyanin is ascribed to the perturbing action on the protein structure by ethylene glycol in high concentrations. This solvent proved the most suitable perturbant of several compounds tested.     

INCREASED ENERGY TRANSFER FROM PHYCOBILISOMES TO PHOTOSYSTEM II IN HIGH LIGHT ADAPTED Anabaena cylindrica

Excitation energy transfer from phycobilisomes to photosystem II in high-light adapted cells of Anabaena cylindrica was studied by fluorescence spectroscopy and compared to that of low-light adapted cells. Measurements were made on membrane fragments containing phycobilisomes, photosystem I and II, isolated in 0.75 M K-phosphate. Relative efficiency of 430 to 590 nm light in the excitation of F₆₈₀ chlorophyll fluorescence was compared in low and high light adapted cells, respectively. The values indicate that light energy absorbed by phycobilisomes is transferred to photosystem II antenna chlorophylls with higher efficiency in high-light adapted cells than in low-light adapted cells. Partial dissociation and uncoupling of energy transfer caused by low ion concentration were different in the membrane fragments isolated from the two kinds of cells and indicated a higher aggregation state of pigment-protein complexes of phycobilisomes in high-light adapted A. cylindrica cells.     

甲烷单加氧酶的催化机理

研究了来源于Ｍｅｔｈｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）催化机理，结果表明，用还原剂处理基于酶可得么不同还原态的羟基化酶，其中全还原态的羟基化酶具有ＭＭＯ催化活性，还原酶和调节蛋白单独存在的均没有ＭＭＯ活性，确定了羟基化酶组分是ＭＭＯ的催化活性中心，用紫外光谱法证实了ＮＡＤＨ和还原酶之间的相互作用，即还原酶接受ＮＡＤＨ的电子，使氧化态的ＦＡＤ转化为还原态的ＦＡＤ，ＭＭＯ的电子传     

TRYPTOPHAN EMISSION FROM HUMAN HEMOGLOBIN AND ITS ISOLATED SUBUNITS

Abstract— The emission spectra of human adult hemoglobin A₀ and its isolated α and ß subunits were obtained using a highly sensitive photon-counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A₀ and apohemoglobin. The fluorophore bis-ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.     

LIGHT SCATTERING SPECTROPHOTOMETRY FROM ISOLATED CONTROL AND TRIAZOLE-TREATED CHLOROPLASTS

Light scattering relaxation spectrophotometry has proven to be a useful technique to monitor rapid cytophysical changes in chloroplast suspensions brought about by flash illumination. This paper compares the Mg-ATP dependent light scattering behaviour of cholorplasts isolated from control and triazole-treated wheat seedlings. Our results suggest that triazole-treatment enhances Mg-ATP dependent activity. This same enhancement in control chloroplasts can be brought about by introducing potassium ion in the presence of valinomycin. Therefore, the potassium ion might account for part of the stress resistance conferred by triazole treatment.     

FLUORESCENCE PROPERTIES OF C-PHYCOCYANIN ISOLATED FROM A THERMOPHILIC CYANOBACTERIUM

Abstract The fluorescence lifetime of purified C-phycocyanin from the thermophilic cyanobacterium, Phormidium laminosum (strain OH-1-p. Cl 1), was measured as 1.48 ± 0.06 ns using the technique of time-correlated single-photon counting under very weak excitation pulses. The natural radiative lifetime (∼6.1 ns) of the pigment was calculated by integrating the absorption spectrum using the Strickler–Berg equation. From these two lifetimes we calculate a fluorescence quantum yield of ∼0.24 which is very close to the value ∼0.22 which we measure relative to the known value of cresyl violet in methanol. Both the fluorescence lifetime and the quantum yield of the pigment from this organism are lower than most previous values reported in the literature. We conclude that our lower values are not due to high light intensity, pH, buffer, concentration, instrumentation artifacts, aggregation effects or the thermophilic nature of the organism. Instead, we suggest that the photophysical properties of C-phycocyanin are species dependent, perhaps due to the specific molecular environment of the tetrapyrrole.     

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.     

STUDIES ON THE RED AND BLUE FORMS OF THE PIGMENT OF BLEPHARISMA*

Abstract— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.     

GLOW CURVES FROM PHOTOSYNTHETIC BACTERIA*

Abstract— Cells and chromatophores of the photosynthetic bacterium Rhodopseudomonas viridis were frozen in the dark and illuminated while frozen or were illuminated during freezing. Upon being heated they emitted light. The conditions under which glow curves could be produced and the kinetics of the light emission indicate that the glows resulted from the thermal release of electrons from secondary electron acceptors and their recombination with oxidized reaction center bacteriochlorophyll b. Glow curves could not be obtained with Rhodospirillum rubrum.