Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence.

To interpret accurately protein fluorescence and phosphorescence, it is essential to achieve a better understanding of the luminescence properties of tryptophan (Trp, or W) in peptides. In published literature data on luminescence of peptides of varied length are scarce. This article describes studies of fluorescence and phosphorescence properties of the eight Trp-containing synthetic peptides: WAK, AWK, SWA, KYLWE, AVSWK, WVSWAK, WAKLAWE, and AVSWAKLARE. The aim was to investigate which factors influence the fluorescence yield and phosphorescence-spectra and lifetimes. Absorption spectra, room temperature fluorescence emission and corresponding excitation spectra and time-resolved phosphorescence spectra (77K) have been recorded; the dependence of the fluorescence quantum yield on the specific peptide and its variation with the wavelength of excitation has been studied. The changes in fluorescence yield and shape of phosphorescence spectra are explained in terms of internal electron and proton transfer. The structured phosphorescence spectrum originates from proton transfer occurring upon excitation of Trp, while electron transfer gives rise to a non-structured luminescence spectrum. There is also electron transfer from higher vibronic S1 states. In the peptides there is higher probability of electron transfer than in Trp alone. The obtained data are interpreted in light of the peptides' sequence, length and conformation.

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution.

Under strong light conditions, long-lived chlorophyll triplets ((3)Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench (3)Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet-triplet energy transfer and quenching of (3)Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching (3)Chl.

Pulsed-source time-resolved phosphorimetry: comparison of a commercial gated photomultiplier with a specially wired ungated photomultiplier.

A common problem encountered in recording delayed light emission is that the signal of interest is preceded by a much more intense signal arising from prompt fluorescence. When a photomultiplier tube (PMT) is used as the photosensor in a pulsed-source phosphorimeter, two options are open to an experimenter who finds mechanical shutters inconvenient or impracticable and photon counting inappropriate: apply an electronic gate that suppresses the PMT gain for a brief period, or use a wiring scheme that enables the PMT to quickly regain normal operation after an intense burst of prompt emission. The performance of a squirrel-cage PMT that operates in the latter mode is compared with a new gateable PMT (Hamamatsu H11526 series) with a minimum gate time of 100 ns. The two detectors are found to provide practically the same temporal record of the delayed emission, but the ungated PMT is slightly superior in terms of recovery time and signal-to-noise ratio.

Model for conformational relaxation of flexible conjugated polymers: application to p-phenylenevinylene trimers in nonpolar solvents.

Photoexcitation of flexible conjugated polymers is invariably followed by a fast conformational/torsional relaxation towards a configuration favouring coplanarity of the conjugated segments. In general, the experimental relaxation rate constant (k(CR)) depends on the solvent viscosity (η) and temperature (T), and is not proportional to T/η. A theory capable of explaining the observed dependence of k(CR) on T and η over a wide range of these variables is not available. This gap is filled here by presenting a stochastic model that includes the participation of the oligomer side chain in storing and dissipating the stresses induced by photoexcitation. The model is able to account for the softening of solute-solvent interactions and its predictions are found to be in excellent agreement with the observed relaxation rate constants of a series of substituted p-phenylenevinylene trimers [ChemPhysChem 2009, 10, 448-454] on T, η and the size of the side-chains.

Trolox, a water-soluble analogue of α-tocopherol, photoprotects the surface-exposed regions of the photosystem II reaction center in vitro. Is this physiologically relevant?

Can Trolox, a water-soluble analogue of α-tocopherol and a scavenger of singlet oxygen ((1)O(2)), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of (1)O(2) in preparations of the five-chlorophyll PSII RC (RC5) containing only one ß-carotene molecule. The temporal profile of (1)O(2) emission at 1270 nm photogenerated by RC5 in D(2)O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 ± 1 µs and decay times of 54 ± 2 µs (without Trolox) and 38 ± 2 µs (in the presence of 25 µM Trolox). The ratio between the total (k(t)) and chemical (k(r)) bimolecular rate constants for the scavenging of (1)O(2) by Trolox in aqueous buffer was calculated to be ~1.3, with a k(t) of (2.4 ± 0.2) × 10(8) M(-1) s(-1) and a k(r) of (1.8 ± 0.2) × 10(8) M(-1) s(-1), indicating that most of the (1)O(2) photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RC5 and Trolox were mixed, revealed that Trolox was a better (1)O(2) scavenger than histidine and furfuryl alcohol at low concentrations (i.e., <1 mM). After its incorporation into detergent micelles in unbuffered solutions, Trolox was able to photoprotect the surface-exposed regions of the D1-D2 heterodimer, but not the RC5 pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by (1)O(2). These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a (1)O(2) scavenger in thylakoid membranes of photosynthetic organisms.

Comparison of the photochemical behaviors of α-tocopherol and its acetate in organic and aqueous micellar solutions.

Photoionization is known to take place when α-tocopherol (TOH) is excited to the S(1) state in a polar medium. It has been previously suggested that TO(•) is formed only as a result of proton release by TOH(•+), a process that is expected to occur, in a protic solvent, on the subnanosecond time scale. Recent redeterminations of the molar absorption coefficients of e(aq)(­) (Hare J. Phys. Chem. A 2010, 114, 1766) and of TOH(•+) and TO(•) (Naqvi J. Phys. Chem. A 2010, 114, 10795) have paved the way for testing the above suggestion, even if subnanosecond time resolution is not available, since it implies the equality of [e(aq)(­)](0) and [TO(•)](0), where [···](0) denotes the concentration of the enclosed species immediately after a nanosecond laser pulse. Nanosecond pump-probe spectroscopy of TOH in aqueous micellar solution (AMS) and two organic solvents with similar polarities (acetonitrile and methanol) has revealed that prompt formation of TO(•) through dissociation (TOH + hν → TO(•) + H(•)) is not negligible even in AMS. In acetonitrile, TOH(•+) and TO(•) are formed with comparable yields, and the former converts quantitatively into TO(•) within 15 µs. In methanol, TO(•) was observed, but no evidence was found for electron ejection from TOH. Only one photoproduct, namely TO(•), could be detected when α-tocopherol acetate (TOAc) was excited to the S(1) state in several polar and nonpolar solvents; TOAc has been found to be a more efficient energy degrader than TOH.

Kinetic studies of retinol addition radicals.

Retinol neutral radicals (RS-retinol˙), generated from the reaction of retinol with 4-pyridylthiyl and 2-pyridylthiyl radicals in argon-saturated methanol, undergo ß-elimination, which can be monitored via the slow secondary absorption rise at 380 nm attributed to the rearrangement of the unstable retinol neutral addition radicals to the more stable addition radicals. Rate constants for the ß-elimination reactions (k(ß)) of 4-PyrS-retinol˙ were measured at different temperatures and the Arrhenius equation for the reaction is described by log (k(ß)/s(-1)) = (12.7 ± 0.2) - (54.3 ± 1.3)/θ, where θ = 2.3RT kJ mol(-1). The reactivities of retinol addition radicals (RS-retinol˙), generated from the reaction of retinol with various thiyl radicals, towards oxygen have also been investigated in methanol. In the presence of oxygen, the decay of RS-retinol˙ fits to biexponential kinetics and both observed rate constants for the RS-retinol˙ decay are oxygen-concentration dependent. This suggests that at least two thiyl addition radicals, formed from the reaction of RS˙ with retinol, undergo oxygen addition reactions. In light of the estimated rate constants for oxygen addition to RS-retinol˙ and RS-CAR˙ (CAR: carotenoid), the antioxidant-prooxidant properties of retinol are discussed.

Spectroscopic characterization of neutral and cation radicals of α-tocopherol and related molecules: a satisfactory denouement.

Notwithstanding the facile occurrence of one-electron oxidation in α-tocopherol and its acetate (TOH and TOAc, respectively), and despite the remarkable stability, under appropriate conditions, of the oxidation products (TOH(•+), TO(•), and TOAc(•+)), their spectroscopic characterization is in an unsatisfactory state, calling for a fresh attempt to acquire reliable data. A new, model-free method is developed for analyzing time-resolved spectra showing the progress of the reaction TOH + R(•) → TO(•) + RH, where R(•) is a stable free radical. The resulting absorption coefficients of TO(•) in dichloromethane and hexane are in severe disagreement with some recent values derived from stopped-flow spectrophotometry. The discrepancy is traced to the imposition of boundary conditions that do not take proper account of the dead time of the apparatus; when multiplied by a factor of two, the stopped-flow data fall mostly in the range ε = (7.5 ± 0.5) × 10(3) M(-1) cm(-1), conforming with the results of this study and the values found by Boguth and Niemann in 1969. Absorption spectra of the radical cations produced (electro)chemically are found to be reliable only in the visible region. Incomplete conversion of the parent compound to the radical cation, an obstacle to the determination of absorption coefficients from electrochemical studies, is circumvented by combining EPR and optical spectroscopy. The absorption coefficients of TOH(•+) and TOAc(•+), determined in this manner, are found to be, respectively, 1.6 × 10(4) and 1.3 × 10(4) M(-1) cm(-1), in accord with the values found first through similar means.

Facile method for spectroscopic examination of radical ions of hydrophilic carotenoids.

Hydrophilic carotenoids, unusual members of an intrinsically hydrophobic family, and their radical ions are important reactants. An all-optical method for generating singly charged radical ions of a hydrophilic carotenoid (Car) is described. It relies on photolyzing an aqueous mixture of Car and a photoionizable auxiliary solute (A), and making conditions conducive to the capture, by Car, of the hydrated electron (e(aq)(-)) or the positive hole in A(*)(+) or both. When A is Trolox (TOH), only e(aq)(-) can be captured, since TOH (*)(+) deprotonates too rapidly to be a hole donor; when A is Trolox methyl ether (TOMe), both Car(*)(-) and Car(*)(+) are formed, since TOMe (+) lives long enough to transfer its positive hole to Car; formation of Car(*)(-) is prevented under aerobic conditions.

Light absorption by isolated chloroplasts and leaves: effects of scattering and 'packing'.

Light absorption was quantified in the following systems: isolated chloroplasts and leaves of spinach (Spinacea oleracea L.), a mutant of geranium (Pelargonium zonale L.) widely differing in pigment content, and coleus (Coleus blumei Benth.) at different stages of leaf ontogenesis. For these species and pea (Pisum sativum L.), scattering-compensated absorption spectra of chloroplast suspensions are presented. Comparison of leaf and chloroplast spectra showed considerable changes in the extent of the 'package' effect and the lengthening of the effective optical path in a leaf. The difference between leaf and isolated chloroplast absorption could be quantitatively described by adapting Duysens's treatment of flattening. It was found that the accumulation of chlorophyll in leaves is accompanied by a monotonous enhancement of the package effect. The results are discussed with special reference to the role of light scattering in leaf optics, light utilization in photosynthesis and wavelength-dependent light gradients in a leaf.

Light absorption by anthocyanins in juvenile, stressed, and senescing leaves.

The optical properties of leaves from five species, Norway maple (Acer platanoides L.), cotoneaster (Cotoneaster alaunica Golite), hazel (Corylus avellana L.), Siberian dogwood (Cornus alba L.), and Virginia creeper (Parthenocissus quinquefolia (L.) Planch.), differing in pigment composition and at different stages of ontogenesis, were studied. Anthocyanin absorption maxima in vivo, as estimated with spectrophotometry of intact anthocyanic versus acyanic leaves and microspectrophotometry of vacuoles in the leaf cross-sections, were found between 537 nm and 542 nm, showing a red shift of 5-20 nm compared with the corresponding maxima in acidic water-methanol extracts. In non-senescent leaves, strong anthocyanin absorption was found between 500 nm and 600 nm (with a 70-80 nm apparent bandwidth). By and large, absorption by anthocyanin in leaves followed a modified form of the Lambert-Beer law, showing a linear trend up to a content of nearly 50 nmol cm(-2), and permitting thereby a non-invasive determination of anthocyanin content. The apparent specific absorption coefficients of anthocyanins at 550 nm showed no substantial dependence on the species. Anthocyanin contribution to total light absorption at 550 nm was followed in maple leaves in the course of autumn senescence. Photoprotection by vacuolar anthocyanins is discussed with special regard to their distribution within a leaf; radiation screening by anthocyanins predominantly localized in the epidermal cells in A. platanoides and C. avellana leaves was also evaluated.

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection.

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.

Reaction center of photosystem II with no peripheral pigments in D2 allows secondary electron transfer in D1.

A pigment-deficient reaction center of photosystem II (PSII)-with all the core pigments (two molecules of chlorophyll a and one of pheophytin a in each D protein) but with only one molecule each of peripheral chlorophyll a (Chlz) and beta-carotene (Car)-has been investigated by pump-probe spectroscopy. The data imply that Car and Chlz are both bound to D1. The absence of Car and Chlz in D2 allows the unprecedented observation of secondary electron transfer in D1 of PSII reaction centers at room temperature. The absorption band of the Car cation in D1 (Car(D1)(+*)) peaks around 910 nm (as against 990 nm for Car(D2)(+*)), and its positive hole is shared by ChlzD1, whereas Car(D2)(+*) can disappear by capturing an electron from ChlzD2.

Formation and geminate quenching of singlet oxygen in purple bacterial reaction center.

The phosphorescence of singlet oxygen ((1)X( *)) photosensitized by the carotenoidless reaction center (RC) of Rhodobacter sphaeroides R26.1 has been investigated, using H(2)O and D(2)O as the suspending media. To enhance (under neutral conditions) the triplet quantum yield of the special pair P(870) (P) by the radical pair mechanism, the quinone acceptor Q(A) was removed by means of a chemical treatment. The phosphorescence signal fits the functional form P(0)[exp (-t/tau)-exp(-t/zeta)], regardless of whether (1)X( *) is sensitized by P(dagger) or M(dagger) (where the dagger denotes triplet excitation and M is a water-soluble molecule). The time constant zeta was identified with the decay time of (1)X( *); when P(dagger) is the sensitizer, one finds zeta(P)((1))=3.3+/-0.3 micros, and zeta(P)((2))=34+/-3 micros, where the superscripts 1 and 2 refer to H(2)O and D(2)O, respectively; the corresponding values for sensitization by M(dagger) (in the absence of RC) are zeta(M)((1))=3.7+/-0.4 micros, and zeta(M)((2))=75+/-5 micros. The addition of RC's to the solution of M in D(2)O reveals that the RC is a quencher of (1)X( *); however, for equal concentrations of the RC, zeta(P)((2))

Estimation of diffusive boundary layer thickness in studies involving diffusive gradients in thin films (DGT).

Recent laboratory experiments and field investigations involving diffusive gradients in thin films (DGT) have shown that the thickness (delta) of the diffusive boundary layer (DBL), which can affect the accuracy of the technique, is generally not negligible. Accordingly, the determination of delta has become a matter of considerable practical importance. Though the problem has been addressed in the recent literature, there is room for some improvement. An expression for estimation of delta is presented here, and a practical procedure for determining delta and the concentration of DGT-labile species from sparse experimental data is proposed and illustrated by analyzing data from four experiments with DGT samplers of different diffusive gel thicknesses.