Picosecond Time-resolved Fluorescence Studies Research Articles

Excited state dynamics of 9,9′-bianthryl in room temperature ionic liquids as revealed by picosecond time-resolved fluorescence study

Picosecond time-resolved fluorescence measurements have been carried out on 9,9′-bianthryl in three imidazolium ionic liquids to probe the excited state dynamics. In the early time-scale, the fluorescence spectra of bianthryl have been found to consist of emission from both locally excited (LE) and charge transfer (CT) states. The LE → CT relaxation time, as estimated from the decay of the fluorescence intensity of the LE emission, is found to vary between 230 and 390 ps, while the average solvent relaxation time, as estimated from the analysis of time-dependent fluorescence Stokes shift, is found to vary between 620 ps and 1840 ps, depending on the viscosity of the ionic liquids. The results confirm that while in conventional less viscous solvents the CT formation kinetics of bianthryl occurs simultaneously with the solvation dynamics, in ionic liquids the two processes mostly occur in different time scales.

Picosecond time-resolved fluorescence study of poly vinyl methyl ether aqueous solution

Picosecond time-resolved fluorescence spectroscopy was carried out for the aqueous solution of poly vinyl methyl ether (PVME) at two different temperatures by using a fluorescent probe molecule. The dehydration of coiled PVME, which is the initial process of coil-to-globule phase transition, was observed below the low critical solution temperature (LCST), whereas two hydrophobic environments were found inside and outside PVME fine particles above LCST.

Site-Selective Binding and Dual Mode Recognition of Serum Albumin by a Squaraine Dye

With the objective of developing small molecule based probes for proteins, interactions of polyhydroxyl-substituted squaraine dye (SQ) with bovine serum albumin (BSA) have been investigated by absorption, steady-state and time-resolved fluorescence, circular dichroism (CD), cyclic voltammetry (CV), 1H NMR, scanning electron, and tapping mode atomic force microscopic techniques. Increase in addition of BSA resulted in increase in absorbance and fluorescence quantum yields (80-fold) of SQ, along with significant bathochromic shifts in the absorption and fluorescence maxima. Half-reciprocal analysis of the absorption data gave a 1:1 stoichiometry for the complex between BSA and SQ with high association (Kass) constant of (1.4 +/- 0.1) x 106 M-1 and change in free energy of -35 kJ/mol. The complex formation was further confirmed by observation of induced CD signal corresponding to the SQ chromophore at 610 nm, upfield shift (about Deltadelta 0.1 ppm) of aromatic protons of SQ in 1H NMR spectra, and decrease in current intensity (CV) of SQ when bound to BSA. The picosecond time-resolved fluorescence studies indicated that the BSA-SQ complex exhibits biexponential decay with significantly enhanced lifetimes of 0.5 and 1.5 ns when compared to the lifetime of SQ (tau = 121 ps) in the absence of BSA. Employing displacement cum fluorimetry using site-specific binding ligands, such as dansylproline and dansylamide, indicated that SQ binds with protein selectively at site II involving hydrophobic, hydrogen bonding, and electrostatic interactions. The uniqueness of this molecular system is that it interacts with BSA selectively at site II and signals the binding event through dual mode recognition of "visual color" change and "turn on" fluorescence mechanism.

Photophysical study of two alternating polyfluorene copolymers exhibiting dual fluorescence

We present a photophysical study of two fluorene alternating coporymeis in dilute solutions. The UV-Vis absorption spectra of the copolymers show two absorption bands in the 300–600 nm wavelength range. In spite of the alternating nature of the copolymers the fluorescence spectrum, recorded by excitation within the higher energy absorption band, is also composed of two bands. The excitation spectra show that the lower energy emission band has contributions from both absorption bands, while the picosecond time-resolved fluorescence studies do not provide evidence for any energy transfer process, meaning that, if it occurs, is faster than the time resolution of our set up (≈5 ps).

Dual probe solubilisation in two distinct regions of pure and mixed micelles: a pico-second time resolved fluorescence study

Photophysical parameters of a newly synthesised fluorescence probe, 2-[3-( N-methyl- N-phenyl amino)-2-propenylidene] indanone, have been studied in three different micellar media formed by the surfactants sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium bromide (CTAB) and triton X-100 (TX100) and their binary mixtures (SDS–TX100, CTAB–TX100). Our study indicates that the probe molecules are distributed between the core and Stern/ palisade layer of the micelle. The extent of distribution depends on the nature of the micelle. The radiative decay constant ( k r) for the excited S 1 state has been found to be independent of the nature of micellar media. However, the non-radiative decay constant ( k nr) exhibits significant medium dependence in the order SDS > CTAB ≈ TX100. Non-linear variation of k nr with component surfactant composition in the mixed micelle has been observed and has been explained in terms of synergistic interaction between the surfactant molecules.

Conformational states of HRPA1 induced by thermal unfolding: effect of low molecular weight solutes.

Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1. The fraction of heme-quenched fluorophore decreased to 0.4 after unfolding as shown by acrylamide quenching. A new unfolding pathway for HRPA1 was proposed and the effect of the low molecular weight solutes trehalose, sorbitol, and melezitose on this pathway was analyzed. Native HRPA1 unfolds with an intermediate between the native and the unfolded conformation. The unfolded conformation can refold to the native state or to a native-like conformation with no calcium ions upon cooling or can give an irreversible denatured state. The refolded conformation with no calcium ions was clearly identified in a second thermal scan in the presence of EDTA and shows secondary and tertiary structures, heme reincorporation in the cavity, and at least 59% of activity. Melezitose stabilized the refolded Ca2+-depleted protein and induced a more complex mechanism for heme disruption. The effect of sorbitol and trehalose were mainly characterized by an increase in the temperature of unfolding.

Spectroscopic studies on human serum albumin and methemalbumin: optical, steady-state, and picosecond time-resolved fluorescence studies, and kinetics of substrate oxidation by methemalbumin.

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity ( K(M)=433 microM, molar catalytic activity=0.33 s(-1)), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by approximately 80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (approximately 20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of approximately 3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the approximately 80% populated conformer of methemalbumin allows the orientation factor, kappa(2), to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 A was deduced.

Steady-State and Picosecond Time-Resolved Fluorescence Studies on Native and Apo Seed Coat Soybean Peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase super family. The protein has a very similar 3-dimensional structure with that of horseradish peroxidase (HRP-C). The fluorescence characteristics of the single tryptophan (Trp117) present in SBP and apo-SBP have been studied by steady-state and pico-second time-resolved fluorescence spectroscopy. Fluorescence decay curve of SBP was best described by a four exponential model that gave the lifetimes, 0.035 ns (97.0%), 0.30 ns (2.0%), 2.0 ns (0.8%), and 6.3 ns (0.2%). These lifetime values agreed very well with the values obtained by the model independent maximum entropy method (MEM). The three longer lifetimes that constituted 3% of the fluorophore population in the SBP sample are attributed to the presence of trace quantities of apo-SBP. The pico-second lifetime value of SBP is indicative of efficient energy transfer from Trp117 to heme. From fluorescence resonance energy transfer (FRET) calculations, the energy-transfer efficiency in SBP is found to be relatively higher as compared to HRP-C and is attributed mainly to the higher value of orientation factor, κ2 for SBP. Decay-associated spectra of SBP indicated that the tryptophan of SBP is relatively more solvent exposed as compared to HRP-C and is attributed to the various structural features of SBP. Linear Stern–Volmer plots obtained from the quenching measurements using acrylamide gave kq = 5.4 × 108 M−1 s−1 for SBP and 7.2 × 108 M−1 s−1 for apo-SBP and indicated that on removal of heme in SBP, Trp117 is more solvent exposed.

Excited-State Dynamics in the Enhanced Green Fluorescent Protein Mutant Probed by Picosecond Time-Resolved Single Photon Counting Spectroscopy

The complex photophysics of the wild-type green fluorescent protein (GFP), one of the most popular fluorescent probes in biology, has been extensively documented in literature. The excited-state dynamics of GFP was explained by means of a model implying excited-state proton transfer (ESPT) and three forms of the chromophore, a protonated A form absorbing at 400 nm and two deprotonated I and B forms absorbing at around 475 nm. We report here a systematic picosecond time-resolved fluorescence study of the enhanced green fluorescent protein (EGFP) variant, carrying the Ser65-Thr and Phe64-Leu mutations. By means of multiple excitation wavelength time-resolved experiments, we were able to distinguish between the fluorescence decay times of the deprotonated I* and B* states (3.4 and 2.7 ns). Spectrally, we found the I form being red shifted in comparison with the B form, both in absorption and in emission. Evidence for an excited-state reaction, namely, proton transfer, is also reported. An additional protonated species is proposed in the photophysical scheme in order to explain the excited-state dynamics of EGFP on the basis of our results as well as previous reported data. Two alternative models are presented, both of them applicable also to the data reported in relation with wild-type GFP.

Excited-state intramolecular proton transfer coupled-charge transfer of p- N, N-dimethylaminosalicylic acid in aqueous β-cyclodextrin solutions

The single emission at 400 nm originated from mostly excited-state intermolecular hydrogen bonding has been observed for 0.05 mM p- N, N-dimethyaminosalicylic acid (DMAS) in aqueous solutions (pH=1.7). Upon addition of β-CD, however, the 400 nm emission band is diminished, accompanied by a broadening of the bandwidth along with an appearance of a new band at 350 nm. According to the band analysis, the broad emission was found to be composed of two emission bands arising from the excited-state intramolecular proton transfer (ESIPT) and the excited-state intramolecular charge transfer (ICT), respectively. Being consistent with these steady-state spectroscopic results, the picosecond time-resolved fluorescence study unraveled three decay components corresponding to two different proton transfer processes and the excited ICT state with ca. 5.6 ns, 160 ps and 1.5 ns, respectively. It is noteworthy that the relative amplitude of 1.5 ns component attributable to the ICT state is enhanced in parallel with an increase of 160 ps ESIPT component. These results are interpreted in terms of the proton transfer coupled-charge transfer processes.

Picosecond time-resolved fluorescence studies on excitation energy transfer in a histidine 117 mutant of the D2 protein of photosystem II in Synechocystis 6803.

The role of the peripheral reaction center chlorophyll a molecule associated with His117 of the D2 polypeptide in photosystem II was investigated in Synechocystis sp. PCC 6803 using a combination of steady state, pump-probe, and picosecond time-resolved fluorescence spectroscopy. Data were obtained from intact cells and isolated thylakoid membranes of a control mutant and a D2-H117T mutant, both of which lacked photosystem I. Excitation energy transfer and trapping were investigated by analyzing the data with a kinetic model that used an exact numerical solution of the Pauli master equation, taking into account available photosystem II spectral and structural information. The results of our kinetic analysis revealed the observed difference in excited-state dynamics between the H117T mutant and the control to be consistent with a retardation of the rate of excitation energy transfer from the peripheral chlorophyll of D2 (Chl at His117) to the electron-transfer pigments and an increase of the rate constant for charge recombination in the H117T mutant. The kinetic model was able to account for the experimentally observed changes in absorption cross section and fluorescence decay kinetics between the control and mutant by invoking changes in only these two rate constants. The results rule out quenching of excitation by a chlorophyll cation radical as a mechanism responsible for the lower efficiency of excitation energy utilization in the H117T mutant. Our work also demonstrates the importance of the chlorophyll associated with His117 of the D2 protein for excitation energy transfer to the PSII electron-transfer pigments and for the effective stabilization of the primary radical pair.

Steady state and picosecond time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase

Steady state and time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase (XO) have been carried out to investigate the conformational changes associated with the replacement of the molybdenum double bonded sulphur by oxygen and the removal of the flavin adenine dinucleotide (FAD). The steady state quenching experiments of the intrinsic tryptophan residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and deflavo enzymes. However, the number of the tryptophan residues accessible to the ionic quenchers, potassium iodide and cesium chloride, increases upon removal of the FAD centre from the enzyme. This indicates that two tryptophan residues move out from the core of the enzyme to the solvent upon the removal of the FAD. The time-resolved fluorescence studies were carried out on the native, desulpho and deflavo XO by means of the time-correlated single photon counting technique, and the data were analysed by discrete exponential and maximum entropy methods. The results show that the fluorescence decay curve fitted best to a three-exponential model with lifetimes τ 1=0.4, τ 2=1.4 and τ 3=3.0 ns for the native and desulpho XO, and τ 1=0.7, τ 2=1.7 and τ 3=4.8 ns for the deflavo XO. The replacement of the molybdenum double bonded sulphur by oxygen in the desulpho enzyme does not cause any significant change of the lifetime components. However, removal of the FAD centre causes a significant change in the shortest and longest lifetime components indicating a conformational change in the deflavo XO possibly in the flavin domain. Decay-associated emission spectra at various emission wavelengths have been used to determine the origin of the lifetimes. The results show that τ 1 and τ 3 of the native and desulpho XO originate from the tryptophan residues which are completely or partially accessible to the solvent but τ 2 corresponds to those residues which are buried in the core of the enzyme and not exposed to the solvent. For deflavo enzyme, τ 2 is red shifted compared to the native enzyme indicating the movement of tryptophan residues from the core of the enzyme to the solvents.

Binding of camphor to Pseudomonas putida cytochrome P450 cam: steady-state and picosecond time-resolved fluorescence studies

The binding of camphor to cytochrome P450 cam has been investigated by steady-state and time-resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time-resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site-directed mutagenesis studies.

Redox-linked conformational changes in bovine heart cytochrome c oxidase: picosecond time-resolved fluorescence studies of cyanide complex.

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.

Unfolding pathway of cytochromec oxidase induced by ionic surfactants: Circular dichroism and picosecond time-resolved fluorescence studies

The unfolding of the membrane protein, cytochromec oxidase (CcO) induced by ionic surfactants have been studied by using circular dichroism, optical absorbance and time resolved tryptophan fluorescence spectroscopic methods. Ionic surfactant cetyltrimethyl ammonium bromide (CTAB) was found to cause denaturation of this membrane protein leading to release of both, the hemea residues from CcO indicated by both CD and optical titration. Upon dissociation of the hemes from the protein matrix; the tryptophan fluorescence intensity of CcO increased drastically and the fluorescence lifetimes became much longer compared to the short lifetimes observed in the native protein. The shortest lifetime of 70 ps observed in the native protein due to strong quenching (energy transfer) of the heme groups, increased ∼10-fold in the CTAB-unfolded protein indicating complete removal of the heme groups from the protein matrix. Remarkable differences were observed between the mode of actions of ionic surfactants and the commonly used denaturant guanidine hydrochloride. Improved data analysis of maximum entropy method showed that the lifetime distribution pattern in the two cases of unfolding were very different. The lifetimes in guanidine hydrochloride unfolded CcO were much shorter and more widely distributed indicating that the hemes are probably not separated away from the protein matrix and that the unfolded state is highly heterogeneous. Our results further showed that the lauryl maltoside inhibits denaturation of CcO by the ionic surfactant and the initial step of the denaturation possibly involves quantitative replacement of the lauryl maltoside by the ionic surfactant at the surface of the enzyme.

Cu(II)-inhibitory effect on photosystem II from higher plants. A picosecond time-resolved fluorescence study.

The influence of Cu(II) inhibition on the primary reactions of photosystem II (PSII) electron transport was studied by picosecond time-resolved fluorescence on isolated PSII membranes. The fluorescence decay from Cu(II)-inhibited PSII centers showed a dominant amplitude of a fast phase (100-300 ps) similar to PSII centers in the uninhibited "open state" and minor contributions of components around 600 ps and 2.6 ns. These data indicate efficient primary charge separation in PSII membranes incubated with Cu(II). The quantum yield of primary reactions in the inhibited PSII centers was similar to that of "open" PSII centers. Kinetic analysis of the decay curves in the framework of the exciton/radical pair equilibrium model showed no significant changes in the rate constants associated with the charge separation/recombination equilibrium. However, in closed centers (QA reduced), a decrease in the rate constant K23, associated with the back-reaction of a relaxed radical pair, by a factor of 4 was calculated. The free energy losses upon primary charge separation (delta G1) and during subsequent radical pair relaxation (delta G2) were also determined in Cu(II)-inhibited centers and were compared with uninhibited centers. No changes in the delta G1 values and a significant decrease in the delta G2 values were found as compared with those of control PSII centers in the "closed" state. These data indicate that Cu(II) does not affect primary radical pair formation, but strongly affects the formation of a relaxed radical pair, by neutralizing the negative charge on QA- and eliminating the repulsive interaction between Pheo- and QA- and/or by modifying the general dielectric properties of the protein region, surrounding these cofactors. Moreover, a close attractive interaction between Pheo-, QA-, and Cu2+ can be proposed. Our results are in good agreement with very recent EPR results indicating an additional effect of Cu2+ on the acceptor side [Jegerschöld et al. (1995) Biochemistry 34, 12747-12758].

Studies on the Dissociation of Cryptomonad Biliproteins

The spectroscopic properties of two biliproteins, phycocyanin 645 and phycoerythrin 566, have been studied by treating the proteins with two different agents, NaSCN at pH 6.0, or pH 4.0 without NaSCN. For phycoerythrin 566, treatment with NaSCN revealed that the visible CD spectrum of its chromophores was separated into a pair of different spectra, and each of these spectra was observed as a negative and one or more positive bands. For phycocyanin 645, two negative CD bands have been observed previously, together with two or more positive bands, in the dimer (alpha 2 beta 2) state, and NaSCN treatment caused elimination of one of these negative bands. The dimer was stable at pH 6.0, but at pH 4.0 the spectra of phycocyanin 645 had one less negative band than those at pH 6.0. Chromatography demonstrated that phycocyanin 645 was a monomer (alpha beta) at pH 4.0. Monomers of cryptomonad biliproteins have never been observed before. Excitation at 514 nm, in picosecond time-resolved fluorescence studies, produced lifetimes of 11.0 and 45.2 ps for dimers and monomers, respectively. Excitation at 566 nm yielded times of 1.38 and 1.24 ps, for dimers and monomers, respectively. CD in the far UV showed that monomers and dimers had very similar secondary structures. These results have been used to test an hypothesis that proposed two types of exciton splitting among the chromophores of phycocyanin 645, and perhaps phycoerythrin 566 could also have this chromophore organization.

Time-resolved fluorescence study of the single tryptophan in thiocyanate and azide derivatives of horseradish peroxidase: Implication for apH-induced conformational change in the heme cavity

Detailed pH-dependent steady state and picosecond time-resolved tryptophan fluorescence studies on thiocyanate and azide complexes of horseradish peroxidase have been carried out. The fluorescence decay of the single tryptophan in these species was fitted to a discrete three exponential model. Maximum entropy method analysis also gave three distinct regions of lifetime distributions. The fast subnanosecond lifetime component was found to have > 97% amplitude contribution while other two longer lifetime components have small contributions. Small contributions from the nanosecond lifetime components possibly arise from apoprotein impurity or some small amount of disordered heme conformer of the protein. pH dependence of the fast picosecond lifetime components was found to show a systematic behavior which has been interpreted in the light of obligatory conformation change associated with activation of the enzyme at low pH.

Initial spectroscopic characterization of the ciliate photoreceptor stentorin

Stentorin serves as the primary photosensor in the single cell ciliate, Stentor coeruleus, for its photophobic and phototactic responses to light of visible wavelengths. We separated two subunits, stentorin-2A and -2B, from the previous stentorin complex (‘stentorin-2’) of greater than half a million molecular mass isolated from the photoreceptor organelle (pigment granule). Stentorin-213 bears the chromophore covalently linked to an approx. 50 kDa apoprotein, as determined by SDS-urea-PAGE. Partial amino acid sequences were obtained from this 50 kDa subunit. Its visible and CD spectra were found to be similar to those of stentorin-2. The steady-state and time-resolved fluorescence spectra of stentorin-2B, in H 2O and D 2O buffers, were also similar to those of stentorin-2. This suggests that the 50 kDa subunit retains the spectral integrity and primary photoreactivity of the stentorin-complex. The picosecond time-resolved fluorescence study revealed that the short picosecond emission component ( τ F ≅ 8–10 PS) was the predominant emitting species in stentorin-2B and -2, followed by longer decaying species. No deuterium solvent effect was seen in this fast-decaying species. The possible mechanism for the primary photoreaction appears to involve electron transfer coupled with proton transfer.

Picosecond time-resolved fluorescence study of all-trans retinal. The existence of two fluorescent singlet excited states

Picosecond time-resolved fluorescence spectra of all-trans retinal in hexane have been measured with 8 ps time-resolution with use of a highly sensitive streak camera. The observed fluorescence consists of a fast ( τ<1 ps, λ max≈470 nm) and a slow ( τ=33–34 ps, λ max≈540 nm) component. The lifetime of the slow component agrees well with the reported rise time of the triplet-triplet absorption. It is concluded that the photoexcitation first produces the optically allowed S 2 state and it relaxes to the optically forbidden S 1 state within 1 ps. The fast and slow fluorescence components are assigned to the S 2 and S 1 states, respectively.