Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: Lifetime of the Excited State In Vivo: I. Chlorophyll a in Algae, at Room and at Liquid Nitrogen Temperatures; Rate Constants of Radiationless Deactivation and Trapping

Using a mode-locked laser (λ, 632.8 nm), fluorescence decay of chlorophyll (Chl) a in the green alga Chlorella pyrenoidosa, the red alga Porphyridium cruentum, and the blue-green alga Anacystis nidulans was measured by the phase-shift method under conditions when photosynthesis was not operative (3-(3,4-dichlorophenyl)-1,1-dimethylurea [DCMU] poisoning, or cooling to 77°K). In the presence of 10^-5 M DCMU, the lifetime of Chl a fluorescence (τ) at room temperature is about 1.7 nsec in Chlorella, 1.0 nsec in Porphyridium, and 0.7 nsec in Anacystis. At 77°K, τ is 1.4 nsec (for fluorescence at about 685 nm, F-685) and 2.3 nsec (for F-730) in Chlorella, 0.9 nsec (F-685) and 1.2 nsec (F-730) in Porphyridium, and 0.8 nsec (F-685 and F-730) in Anacystis. From the above measurement, and the assumption that τ₀ (the intrinsic fluorescence lifetime) for Chl a in all three algae is 15.2 nsec, we have calculated the rate constants of radiationless transition (that includes energy transfer to weakly fluorescent system I) processes competing with fluorescence at room temperature to be about 5 × 10⁸ sec^-1 in Chlorella, 9 × 10⁸ sec^-1 in Porphyridium, and 13 × 10⁸ sec^-1 in Anacystis. At 77°K, this rate constant for Chl a that fluoresces at 685 nm remains, in the first approximation, the same as at room temperature. From the τ data, the rate constant for the trapping of excitation energy is calculated to be about 1.2 × 10⁹ sec^-1 for Chlorella, 2 × 10⁹ sec^-1 for Porphyridium, and 2 × 10⁹ sec^-1 for Anacystis. The efficiency of trapping is calculated to be about 66% (Chlorella), 68% (Porphyridium), and 60% (Anacystis). (It is recognized that variations in the above values are to be expected if algae grown under different conditions are used for experimentation.) The maximum quantum yield of Chl a fluorescence for system II (λ, 632.8 nm), calculated from τ measurements, is about 10% in Chlorella, 6-7% in Porhyridium, and 5% in Anacystis under conditions when photosynthesis is not operative; the values at 77°K appear to be very close to those with DCMU added at room temperature. ø for F-730 at 77°K, however, is somewhat higher than for F-685. The predicted quantum yields of fluorescence for Chl a in intact cells (both systems I and II) at low intensities of 632.8 nm light are about 2-3, 1-2, and 1% for Chlorella, Porphyridium, and Anacystis, respectively.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.     

Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm² is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Picosecond decay kinetics and quantum yield of fluorescence of the photoactive yellow protein from the halophilic purple phototrophic bacterium, Ectothiorhodospira halophila

The photoactive yellow protein (PYP) has been previously shown to be partially bleached and red shifted (in less than 10 ns) by a pulse of laser excitation at the wavelength maximum (445 nm), to further bleach (k = 7.5 × 10³ s^-1), and then to slowly recover in the dark (k = 2.6 s^-1) (Meyer, T. E., G. Tollin, J. H. Hazzard, and M. A. Cusanovich. 1989. Biophys. J. 56:559-564). The quantum yield for the formation of the fully bleached form was found to be 0.64. We have now shown that the yellow protein is weakly fluorescent with an emission maximum at 495 nm (which mirrors excitation at 445 nm) and a fluorescence quantum yield of 1.4 × 10^-3. Measurement of the picosecond kinetics of the fluorescence decay shows that ~90% of the emission occurs with a lifetime of 12 ps. This is in good agreement with the quantum yield determination, which suggests that a single quenching process (presumably the photochemical event) is primarily responsible for the excited state decay. The lifetime of the excited state of PYP is remarkably similar to that for the rise of the first photochemical intermediate of bacteriorhodopsin, and underscores the fundamental similarity in their photocycles despite a lack of structural relationship.     

Spectral properties of stabilized D1/D2/cytochrome b-559 photosystem II reaction center complex Effects of Triton X-100, the redox state of pheophytin, and β-carotene

Absorption, fluorescence, and CD spectral properties of the isolated D1/D2/cytochrome b-559 photosystem II reaction center complex were examined in stabilized reaction center material at 77 K. Spectral properties were dependent on the presence or absence of 0.05% Triton X-100 in the RC suspension medium, on the redox state of pheophytin, and on the state of inactivation of the complex. The specific spectral properties of the PS II RC complex in the red suggest that the primary donor is not a bacterial-type special pair and could be a monomer. Furthermore, the spectral properties in the PS II RC may be the result of excitonic interactions among all the porphyrin molecules in the complex. Interactions between β-carotene and porphyrins indicate a significant role for β-carotene in the PS II RC.     

Mass spectrometry and spectroscopic characterization of a tetrameric photosystem I supercomplex from Leptolyngbya ohadii,a desiccation-tolerant cyanobacterium

Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77 K revealed two emission bands at ～690 nm and 725 nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725 nm emission band is associated with low energy Chls a with absorption band clearly resolved at ～710 nm at 77 K. In summary, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.     

Kinetic and Energetic Model for the Primary Processes in Photosystem II

A detailed model for the kinetics and energetics of the exciton trapping, charge separation, charge recombination, and charge stabilization processes in photosystem (PS) II is presented. The rate constants describing these processes in open and closed reaction centers (RC) are calculated on the basis of picosecond data (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1987. Proc. Natl. Acad. Sci. USA. 84:8414-8418) obtained for oxygen-evolving PS II particles from Synechococcus sp. with ~80 chlorophylls/P₆₈₀. The analysis gives the following results. (a) The PS II reaction center donor chlorophyll P₆₈₀ constitutes a shallow trap, and charge separation is overall trap limited. (b) The rate constant of charge separation drops by a factor of ~6 when going from open (Q-oxidized) to closed (Q-reduced) reaction centers. Thus the redox state of Q controls the yield of radical pair formation and the exciton lifetime in the Chl antenna. (c) The intrinsic rate constant of charge separation in open PS II reaction centers is calculated to be ~2.7 ps^-1. (d) In particles with open RC the charge separation step is exergonic with a decrease in standard free energy of ~38 meV. (e) In particles with closed RC the radical pair formation is endergonic by ~12 meV. We conclude on the basis of these results that the long-lived (nanoseconds) fluorescence generally observed with closed PS II reaction centers is prompt fluorescence and that the amount of primary radical pair formation is decreased significantly upon closing of the RC.     

Acclimation of Arabidopsis thaliana to the light environment: changes in photosynthetic function

Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was grown under light regimes of differing spectral qualities, which results in differences in the stoichiometries of the two photosynthetic reaction centres. The acclimative value of these changes was investigated by assessing photosynthetic function in these plants when exposed to two spectrally distinct actinic lights. Plants grown in an environment enriched in far-red light were better able to make efficient use of non-saturating levels of actinic light enriched in long-wavelength red light. Simultaneous measurements of chlorophyll fluorescence and absorption changes at 820 nm indicated that differences between plants grown under alternative light regimes can be ascribed to imbalances in excitation of photosystems I and II (PSI, PSII). Measurements of chlorophyll fluorescence emission and excitation spectra at 77 K provided strong evidence that there was little or no difference in the composition or function of PSI or PSII between the two sets of plants, implying that changes in photosynthetic stoichiometry are primarily responsible for the observed differences in photosynthetic function.Abbreviations Chl chlorophyll - FR far-red light - HF highirradiance FR-enriched light (400 mol·m^–2·s^–1, RFR = 0.72) - HW high-irradiance white light (400 mol·m^–2 1·1 s^–1RFR = 1.40) - LHCI, LHCII light-harvesting complex of PSI, PSII - qO quenching of dark-level chlorophyll fluorescence - qN non-photochemical quenching of variable chlorophyll fluorescence - qP photochemical quenching of variable chlorophyll fluorescence - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Dr. Sasha Ruban for assistance with the 77 K fluorescence measurements and for helpful discussions. This work was supported by Natural Environment Research Council Grant GR3/7571A.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Excited state properties of chlorophyll f in organic solvents at ambient and cryogenic temperatures

Chlorophyll f is a photosynthetic pigment that was discovered in 2010. In this study, we present investigations on spectral and dynamic characteristics of singlet-excited and triplet states of Chl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The pigment was studied at room temperature in two organic solvents: pyridine and diethyl ether that have different characters of coordination of the chlorophyll magnesium (Mg) atom (hexa- and penta-coordination, respectively). Cryogenic measurements (77 K) were performed in 2-methyltetrahydrofuran (hexa-coordination). The singlet-excited state lifetime was measured to be 5.6 ns at room temperature regardless of Mg coordination and 8.1 ns at 77 K. The fluorescence quantum yield of Chl f was also determined in pyridine to be 0.16. The triplet state lifetime was studied in detail in pyridine at room temperature, and the inherent lifetime was estimated to ~150 μs. Selective measurements at 77 K demonstrated that the metastability of the triplet state greatly enhances, and its lifetime increases by a factor of more than three.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of Photosystem II

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides in the 29.5–21 kDa region. The Chl a forms at 668, 676, and 690 nm that belong to LHC and antenna part of PS I disappear completely after treatment. The intensity of the Chl b form at 648 nm is sharply decreased in treated seedlings grown under 30 or 100 lx light intensity. The bands of carotenoid absorption at 421, 448 (Chl a), 452, 480, 492, 496 (β-carotene), and 508 nm also disappear. The band shift from 740 to 720 nm and decrease in its intensity relative to the 687 nm emission peak in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chla form at 710–712 nm.     

Resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at 77 and 295 K through fluorescence excitation anisotropy

Fluorescence excitation spectra of highly anisotropic emission from Photosystem I (PS I) were measured at 295 and 77 K on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). When PS I was excited with light at wavelengths greater than 715 nm, fluorescence observed at 745 nm was highly polarized with anisotropies of 0.32 and 0.20 at 77 and 295 K, respectively. Upon excitation at shorter wavelengths, the 745-nm fluorescence had low anisotropy. The highly anisotropic emission observed at both 77 and 295 K is interpreted as evidence for low-energy chlorophylls (Chls) in cyanobacteria at room temperature. This indicates that low-energy Chls, defined as Chls with first excited singlet-state energy levels below or near that of the reaction center, P700, are not artifacts of low-temperature measurements.If the low-energy Chls are a distinct subset of Chls and a simple two-pool model describes the excitation transfer network adequately, one can take advantage of the low-energy Chls' high anisotropy to approximate their fluorescence excitation spectra. Maxima at 703 and 708 nm were calculated from 295 and 77 K data, respectively. Upper limits for the number of low-energy Chls per P700 in PS I from S. 6803 were calculated to be 8 (295 K) and 11 (77 K).Abbreviations Chl - chlorophyll - BChl - bacteriochlorophyll - LHC - light-harvesting chlorophyll - PS - Photosystem - RC - reaction center - S. 6803 - Synechocystis sp. PCC 6803     

A large field CCD system for quantitative imaging of microarrays

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities 1 molecule/µm². The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation ~±4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from ~ 450 to ~750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K × 2K pixel, cooled CCD chip with a well depth of ~10⁵ electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 µm, sufficient for obtaining quantitative multicolor images from >30 000 array elements in an 18 mm × 18 mm square.     

Discrete bathochromic shifts exhibited by fluorescein ligand bound to rabbit polyclonal anti-fluorescein Fab fragments

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by 14 to 15 nm and/or energies of 1.5 kcal mol^–1 relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.     

Gaussian band analysis of absorption,fluorescence and photobleaching difference spectra of D1/D2/cytb-559 complex

A study of the absorption and fluorescence characteristics of the D1/D2/cytb-559 reaction centre complex of Photosystem II has been carried out by gaussian decomposition of absorption spectra both at room temperature and 72 K and of the room temperature fluorescence spectrum. A five component fit was found in which the absorption and fluorescence sub-bands could be connected by the Stepanov relation. The photobleaching and light-activated degradation in the dark of long wavelength pigments permitted a further characterisation of the absorption bands. The absorption (fluorescence) maxima of the five bands at room temperature are 660 nm (670 nm), 669 nm (675 nm), 675 nm (681 nm), 680 nm (683 nm), 681 nm (689 nm). A novel feature of this analysis is the presence of two approximately isoenergetic absorption bands near 680 nm at room temperature. The narrower one (FWHM=12.5 nm) is attributed to pheophytin while the broader band (FWHM=23 nm) is thought to be P680. The P680 band width is discussed in terms of homogeneous and site inhomogeous band broadening. The P680 fluorescence has a large Stokes shift (9 nm) and most fluorescence in the 690–700 nm range is associated with this chromophore.The three accessory pigment bands are broad (FWHM=17–24 nm) and the 660 nm gaussian is largely temperature insensitive thus indicating significant site inhomogeneous broadening.The very slight narrowing of the D1/D2/cytb-559 Q_y absorption at crytogenic temperatures is discussed in terms of the coarse spectral inhomogeneity associated with the spectral forms and the apparently large site inhomogeneous broadening of short wavelength accessory pigments.     

Fluorescence characteristics and photophysical parameters of light-harvesting chlorophyll <Emphasis Type="Italic">a/b</Emphasis> complex aggregates

Fluorescence characteristics and molecular photophysical parameters of light-harvesting chlorophyll a/b complexes isolated from pea were studied in relation to their aggregation state. The aggregate size was varied by changing the Triton X-100 concentration from 0 to 0.23 mM at a chlorophyll concentration of 2.45 μg/ml. Molecular photophysical parameters were determined with laser fluorimetry. Dispersion of large aggregates into smaller ones drastically decreased the intensity of low-temperature (77 K) fluorescence at 700 nm, reduced the singlet-singlet annihilation rate by more than two orders of magnitude, and prolonged the fluorescence lifetime from 0.16 to 3.2 ns.     

4-amino-1H-benzo[g]quinazoline-2-one: a fluorescent analog of cytosine to probe protonation sites in triplex forming oligonucleotides

We developed a new fluorescent analog of cytosine, the 4-amino-1H-benzo[g]quinazoline-2-one, which constitute a probe sensitive to pH. The 2′-O-Me ribonucleoside derivative of this heterocycle was synthesized and exhibited a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of Φ = 0.62 at pH 7.1. The fluorescence emission maximum shifted from 456 to 492 nm when pH was decreased from 7.1 to 2.1. The pK_a (4) was close to that of cytosine (4.17). When introduced in triplex forming oligonucleotides this new nucleoside can be used to reveal the protonation state of triplets in triple-stranded structures. Complex formation was detected by a significant quenching of fluorescence emission (~88%) and the N-3 protonation of the quinazoline ring by a shift of the emission maximum from 485 to 465 nm. Using this probe we unambiguously showed that triplex formation of the pyrimidine motif does not require the protonation of all 4-amino-2-one pyrimidine rings.     

Interaction of Triton X-100 with the pigment-protein complexes of photosynthetic membranes.

The interaction of the non-ionic detergent Triton X-100 with photosynthetic membrane components of Pisum sativum (pea) is described. The detergent affected both the wavelength and the intensity of the 77K fluorescence-emission peaks of both Photosystem I and Photosystem II preparations, in addition to the effects on whole thylakoids recently described by Murphy & Woodrow [(1984) Biochem. J. 224, 989-993]. Below its critical micellar concentration, Triton X-100 had no effect on 77K fluorescence emissions even after prolonged incubations of up to 30 min. Above the critical micellar concentration of about 0.16 mg X ml-1, Triton X-100 caused a dramatic increase in the intensity of the 680 nm emission. The intensity of the 680 nm fluorescence emission continued to increase as more Triton X-100 was added, until limiting concentrations of detergent were reached. These limiting concentrations were proportional to the amount of membrane present and generally occurred at Triton X-100/chlorophyll (w/w) ratios of 100-200:1. In all cases the detergent effect was seen within 10 min, and is often considerably faster, with longer detergent treatments causing no further effects. The data are discussed in terms of a three-stage mechanism for detergent solubilization of membrane components.