Long-lived Fluorescence Research Articles

B Side Electron Transfer in a Rhodobacter sphaeroides Reaction Center Mutant in Which the B Side Monomer Bacteriochlorophyll Is Replaced with Bacteriopheophytin: Low-Temperature Study and Energetics of Charge-Separated States

The mutation, HL(M182), in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll (BChl) on the inactive side (B side) of the RC with a bacteriopheophytin (BPheo; the new cofactor is referred to as φB). In φB-containing RCs, the first excited state of the primary donor (P*) decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature as compared to 3.1 ± 0.2 ps in wild-type (WT) RCs. At low temperatures, P* decay is essentially the same in the HL(M182) mutant and WT RCs: 1.4 ± 0.1 ps at 77 K and 1.1 ± 0.1 ps at 9 K. The yield of the charge-separated P+φB- state decreases from 35% at room temperature to 12% at 77 and 9 K. The decreased P+φB- yield is apparently due to the fact that the rate of the charge separation along the A side of the RC at low temperature increases, while the rate along the B side remains essentially unchanged. From measurements of the long-lived fluorescence decay at room temperature, the standard free energy of the P+φB- state is estimated to be about 0.16 ± 0.04 eV below P*. Given a difference between the midpoint potentials of BChl and BPheo of 0.26 ± 0.03 V, the standard free energy of the P+BB- state in WT RC is estimated to be 0.1 ± 0.07 eV above P*.

A joint structural, kinetic, and thermodynamic investigation of substituent effects on host-guest complexation of bicyclic azoalkanes by beta-cyclodextrin.

Derivatives of the azoalkane 2,3-diazabicyclo[2,2,2]oct-2-ene (1a) with bridgehead 1,4-dialkyl (1b), 1,4-dichloro (1c), 1-hydroxymethyl (1d), 1-aminomethyl (1e), and 1-ammoniummethyl (1f) substituents form host-guest inclusion complexes with beta-cyclodextrin. They were employed as probes to assess substituent effects on the kinetics and thermodynamics of this complexation by using time-resolved and steady-state fluorimetry, UV spectrophotometry, induced circular dichroism (ICD) measurements, and (1)H NMR spectroscopy. The kinetic analysis based on quenching of the long-lived fluorescence of the azoalkanes by addition of host provided excited-state association rate constants between 2.6 x 10(8) and 7.0 x 10(8) M(-)(1) s(-)(1). The binding constants for 1a (1100 M(-1)), 1b (900 M(-1)), 1c (1900 M(-1)), 1d (180 M(-1)), 1e (250 M(-1)), and 1f (ca. 20 M(-1)) were obtained by UV, NMR, and ICD titrations. A positive ICD signal of the azo absorption around 370 nm was observed for the beta-cyclodextrin complexes of 1a, 1d, and 1f with the intensity order 1a >> 1d approximately 1f, and a negative signal was measured for those of 1b, 1c, and 1e with the intensity order 1c < 1b approximately 1e. The ICD was employed for the assignment of the solution structures of the complexes, in particular the relative orientation of the guest in the host (co-conformation).

Homogeneous Time-Resolved Fluorescence DNA Hybridization Assay by DNA-Mediated Formation of an EDTA–Eu(III)–β-Diketonate Ternary Complex

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA–Eu3+–β-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA–Eu3+ complex on the 5′-terminus and the other, labeled with a bidentate β-diketone on the 3′-terminus. After hybridization of two DNA probes with target DNA, EDTA–Eu3+ and β-diketone come close to each other, and an EDTA–Eu3+–β-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate β-diketonate–Eu3+ complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Time-resolved fluorescence study of isolated and aggregated permethyldecasilanes

Time-resolved fluorescence spectra were measured for two kinds of permethyldecasilanes, Me(SiMe 2) 10Me and Ph(SiMe 2) 10Ph, in fluid solutions at room temperature, in frozen solutions and in vacuum-deposited thin films. Time-dependent red-shifts were clearly observed in fluid solutions but not in dilute frozen solutions, which was discussed in terms of conformational relaxation in the excited states. In concentrated frozen solutions, significantly red-shifted, long-lived fluorescence bands appeared, indicating the formation of molecular aggregates. Temperature dependence of fluorescence lifetimes as well as peak wavelengths for films were discussed with respect to exciton migration followed by trapping in localized sites.

A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.

Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.

Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants.

Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.

A Photoactivable Fluorophore Based on Thiadiazolidinedione as Caging Group

Photoactivable (“caged”) fluorescent dyes and probes are crucial for temporally and spatially resolved tracer experiments, e.g., in cell biology or fluid physics. The thiadiazolidinedione 1 represents a new class of caged fluorophore. Upon UV-irradiation it releases in a rapid photoreaction with high quantum yield the azoalkane 2. Longer wavelength excitation of 2 to the singlet excited state results in strong and long-lived fluorescence with maximum intensity at 425 nm. It has been demonstrated that one single uncaging laser pulse suffices for time-resolved or steady-state detection of the fluorescence.

Red GFP and endogenous porphyrins

The finding that green fluorescent protein (GFP) can ‘turn red’ on photoactivation [1,2] has stirred considerable interest. The fluorescent properties of a chromophore, however, are rigidly determined by its molecular structure [3] and the mechanism of a GFP switch to different excitation and emission wavelengths [1,2], and from short-lived to long-lived fluorescence [1] remains to be clarified.

Fluorescence from molecules and aggregates in polycrystalline thin films of α-oligothiophenes

We report stationary fluorescence, fluorescence excitation spectra, site-selective fluorescence spectra, and femtosecond time-resolved fluorescence spectra of vacuum-evaporated α- quaterthiophene (T4) and α-quinquethiophene (T5) polycrystalline thin films at various temperatures. It is found that the fluorescence spectrum of each compound studied is a superposition of emission spectra originated from energetically and spatially separated fluorescent species or sites, which are suggested to be disordered-molecules, pre-aggregates, aggregates, and physical defects. The disordered molecules lying at grain boundaries display a solutionlike fluorescence decay feature. Multiple fluorescence spectra with identical vibrational developments from pre-aggregate species are observed in the T5 film. A long-lived fluorescence decay component observed in the T4 film is attributed to the dipole-allowed transition in the aggregates. A slow rise observed in the time-resolved fluorescence of the T4 film is interpreted in terms of energy transfer from the energetically high-lying species to the physical defects in crystal.

A Fluorescent Probe for Antioxidants

The n,π* singlet-excited azoalkane 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO) is efficiently quenched by the natural antioxidants α-tocopherol (kq = 5.3 × 109 M-1 s-1), uric acid (3.4 × 109), ascorbic acid (2.05 × 109), and glutathione (0.82 × 109). This trend in reactivity is the same as that observed for alternative probes for antioxidants, e.g., triplet-excited benzophenone and the trichloromethylperoxyl radical. Since the quenching of singlet-excited DBO can be readily quantified by means of its long-lived (325 ns in aerated water) and strong fluorescence (λmax ca. 425 nm), this azoalkane serves as the first fluorescent probe for the reactivity of antioxidants. The effects of pH and deuterium substitution and kinetic solvent effects were also examined. The data suggest the involvement of hydrogen atom transfer in the fluorescence quenching of DBO by antioxidants. The reaction efficiency for radical formation in the quenching of singlet-excited DBO by α-tocopherol was found to be one order of magnitude lower (only ca. 10%) than for triplet-excited benzophenone. This contrast is attributed to the variation in spin multiplicity, since reactions of singlet-excited states encounter additional deactivation channels. The potential of the fluorescent probe DBO in providing direct information on antioxidant activity in biological systems is evaluated.

Second-Order Nonlinear Optical Properties of the Four Tetranitrotetrapropoxycalix[4]arene Conformers

This paper reports a systematic experimental and theoretical study of the second-order nonlinear optical properties of multichromophoric molecules that range from dipolar symmetry to three-dimensional octupolar symmetry. The four possible conformers of tetranitrotetrapropoxycalix[4]arene (cone, pace, 1,2-air, 1,3-alt) were studied by nanosecond hyper-Rayleigh scattering and a newly developed time-resolved femtosecond hyper-Rayleigh scattering technique. The latter enables to correct for long-lived fluorescence contributions to the second harmonic scattering intensity. The depolarization ratios D-x/z prove the (partial) octupolar symmetry of the 1,2-alt and 1,3- alt conformers, and thus explain why their hyperpolarizabilities beta(FHRS) are of the same order of magnitude as those of the dipolar cone and pace conformers. The corresponding theoretical second-order nonlinear optical properties (both beta(FHRS) and D-x/z) were calculated using the conformations obtained from single-crystal X-ray diffraction, molecular mechanics (MM), and molecular dynamics (MD) calculations. In contrast with sum-over-state calculations presented in the literature, our theoretical method takes also into account octupolar contributions by linearly adding the NLO-properties of the separate chromophoric groups and using Bersohn's theory. The agreement between experimental and theoretical results is good both for the conformers having dipolar symmetry and for the conformers having (partly) three- dimensional octupolar symmetry. The 1,2-alt and 1,3-alt conformers of the tetranitrotetrapropoxycalix[4]arene represent the first examples of multichromophoric molecules that have high hyperpolarizabilities beta due to 3D octupolar symmetry.

Liposome immunoassay by long-lived fluorescence detection.

Immunoassay by fluorescence energy transfer from a europium chelate in liposome to allophycocyanin (APC) was demonstrated. Streptavidin or antibody to biotin was bonded to the liposome containing the europium chelate of 2-naphthoyltrifluoroacetone in the bilayer. When the biotin bonded to APC (APC-BT) was added to the prepared liposomes and the long-lived fluorescence (lambda ex 336 nm, lambda em 665 nm, delay time 0.05 ms, gate time 3.9 ms) was measured by a flow system, the fluorescence energy of the europium chelate was found to be transferred to APC-BT and the long-lived fluorescence intensity to increase linearly as the concentration of APC-BT (1-10 micrograms ml-1) increased. Further, the intensity decreased competitively with the concentration of biotin (0.1-100 microM) when biotin was added to the liposome solution containing a constant concentration of APC-BT.

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC4 bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values ( ) obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above Tc as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid “melt” profiles are broad and shifted to higher midpoint EA pressure values (P1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported “fluid–gel” lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.

Photocycloaddition Reactions of Pyrazinopsoralen with Simple Olefins

Direct photolyses of pyrazinopsoralen (PzPs) with excess olefins such as dimethyl fumarate (DMFu), dimethyl maleate (DMMa) and dimethyl ethylidenemalonate (DMEM) gave C4-photocycloadducts. The photoproducts were determined to be 1:1 C4-cycloadducts formed through the addition of 4′,5′-furan double bond of the excited PzPs to the olefins. The fluorescence of PzPs was quenched by olefins with rate constants on the order of 109–1010M-1/s. The appearance of the long-lived fluorescence component implies a singlet exciplex mechanism for the photocycloaddition reaction of PzPs with excess olefins.

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O2-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680+ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced QA(QA -). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated QA (QAH2). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680*QA → P680+QA -), is nearly the same in O2-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the QAH2-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of QA leading to QAH2 formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.

Unimolecular decomposition of NO3: The NO+O2 threshold regime

The unimolecular decomposition of expansion-cooled NO3 has been investigated in the threshold regime of the NO+O2 channel. Photoexcitation in the region 16 780–17 090 cm−1 (596–585 nm) prepares ensembles of molecular eigenstates, each of which is a mixture of the B 2E′ bright state and lower electronic states. The X 2A2′ ground state is believed to be the probable terminus of 2E′ radiationless decay, though participation of A 2E″ is also possible. For these photon energies, unimolecular decomposition occurs exclusively via the NO+O2 channel, and NO yield spectra and state distributions have been obtained. The yield spectra are independent of the rotational state monitored, as expected for a large reverse barrier. The state distributions are insensitive to the photolysis photon energy and can be rationalized in terms of dynamical bias. The NO yield goes to zero rapidly above the O+NO2 threshold (17 090±20 cm−1). Because of tunneling, the NO+O2 channel does not have a precise threshold; the value 16 780 cm−1 is the smallest photon energy that yielded signals under the present conditions. Very small decomposition rates were obtained via time-domain measurements in which reactive quenching of long-lived NO3 fluorescence was observed. The rates varied from 1×104 at 16 780 cm−1 to 6×107 s−1 at 16 880 cm−1, and their collision free nature was confirmed experimentally. These data were fitted by using a one-dimensional tunneling model for motion along the reaction coordinate combined with the threshold Rice–Ramsperger–Kassel–Marcus (RRKM) rate. The top of the NO+O2 barrier is estimated to lie at 16 900±15 cm−1. Translational energy measurements of specific NO (X 2ΠΩ,v,J) levels showed that O2 is highly excited, with a population inversion extending to energies above the a 1Δg threshold, in agreement with previous work. It is possible that the main O2 product is X 3∑g−, though some participation of a 1Δg cannot be ruled out. Within the experimental uncertainty, b 1∑+g is not produced.

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.

Excimer fluorescence from phenanthrene-9-carboxylate derivatives

Phenanthrene and most of its known derivatives do not form stable, fluorescent excimers. Moderately intense, long-lived excimer fluorescence has been observed for lactone and lactam derivatives of phenanthrene. The solvatochromic shift of this excimer fluorescence is similar to that previously reported for the pyrene excimer. Stabilization of the excimer is attributed to the increased polarizability of phenanthrene by a coplanar carbonyl substituent. Excimer formation is not observed for a coplanar cyano substituent or an orthogonal carbonyl substituent.

Spectroscopic properties of aromatic dicarboximides part 3: Substituent effect on the photophysical properties of N-phenyl-2,3-napthalimides

In the present article we describe the effect of the substitution on the photolysis properties of a series of N-phenyl-2,3-napthalimides. It is found that the decrease in the electron-donating character of the substituent on the N-phenyl ring changes the fluorescence emission from a weak, broad and short-lived long-wavelength (LW) emission into a structured and long-lived fluorescence localized at the short wavelengths (SW) which behaves like that of the unsubstituted 2,3-napthalimide. Both SW and LW emissions can be observed in some cases. The different results can be explained on the basis of a three-level scheme where the vibrationally relaxed Franck-Condon state populates two different excited states, the one (SW) emitting at short wavelengths, and the other (LW) emitting at long wavelengths. The substitution on the phenyl ring influences mainly the energy of the LW excited state. The nature of the substituent on the N-phenyl ring has a determining influence also on the internal conversion process by virtue of the solvent- and rotation-induced pseudo-Jahn-Teller coupling of the two excited states.