Electron Spin Echo Spectroscopy Research Articles

Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes.

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) from the group of phenylacetic acid derivatives, which has analgesic, anti-inflammatory and antipyretic properties. The interaction of non-steroidal anti-inflammatory drugs with cell membranes can affect their physicochemical properties, which, in turn, can cause a number of side effects in the use of these drugs. Electron paramagnetic resonance (EPR) spectroscopy could be used to study the interaction of diclofenac with a membrane, if its spin-labeled analogs existed. This paper describes the synthesis of spin-labeled diclofenac (diclofenac-SL), which consists of a simple sequence of transformations such as iodination, esterification, Sonogashira cross-coupling, oxidation and saponification. EPR spectra showed that diclofenac-SL binds to a lipid membrane composed of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). 2H electron spin echo spectroscopy (ESEEM) was used to determine the position of the diclofenac-SL relative to the membrane surface. It was established that its average depth of immersion corresponds to the 5th position of the carbon atom in the lipid chain.

Geminate recombination in organic photovoltaic blend PCDTBT/PC71BM studied by out-of-phase electron spin echo spectroscopy.

The key process in organic solar cell operation is charge separation under light illumination. Due to the low dielectric constant of organic materials, the Coulomb attraction energy within the interfacial charge-transfer state (CTS) is larger than the thermal energy. Understanding the mechanism of charge separation at the organic donor/acceptor interface still remains a challenge and requires knowledge of the CTS temporal evolution. To address this problem, the CTS in the benchmark photovoltaic blend PCDTBT/PC71BM was studied by the out-of-phase Electron Spin Echo (ESE). The protocol for determining the CTS geminate recombination rate for certain electron-hole distances was developed. Simulating the out-of-phase ESE trace for the CTS in the PCDTBT/PC71BM blend allows precise determination of the electron-hole distance distribution function and its evolution with the increase in the delay after the laser flash. Distances of charge separation up to 6 nm were detected upon thermalization at a temperature of 20 K. Assuming the exponential decay of the recombination rate, the attenuation factor β = 0.08 Å-1 is estimated for the PCDTBT/PC71BM blend. Such a low attenuation factor is probably caused by a high degree of hole delocalization along the PCDTBT chain.

Electron spin echo detection of stochastic molecular librations: Non-cooperative motions on solid surface.

In frozen biological media and molecular glasses only restricted motions exist; because of the weakness and disorder of intermolecular bonds these motions may have stochastic nature. Electron spin echo (ESE) spectroscopy of spin-labeled molecules allows detecting their restricted stochastic rotations (stochastic molecular librations). As in molecular disordered media motions may be highly cooperative, it would be desirable to investigate their spectroscopic manifestation also in the systems where cooperative effects would be certainly ruled out. In this work, ESE of spin-labeled molecules adsorbed on inorganic SiO2 surface was investigated in a wide temperature range. The rate of motion-induced spin relaxation was found to become measurable above 130 K, increasing with temperature and attaining then a saturating behavior with a well-defined maximum near 250 K. For two types of molecules differing remarkably in their size and polarity (a small highly-polar nitroxide radical and a large spin-labeled peptide), quite similar results were obtained. This saturating behavior was quantitatively reproduced in simulations within a simple model of jump between two close orientations. Comparison with experiment allowed estimate that at 250 K the correlation time of the motion τc is of the order of several tens of nanoseconds and the angle α between two orientations is around 0.02 rad. As the found saturating behavior is a property of individual motions, for any other molecular system an excess of the spin relaxation rate above the maximum found here for adsorbed molecules may be ascribed to cooperative motions. Comparison with literature data on molecular systems of different origin has shown that effects of cooperativity indeed are present and, moreover, may be very essential.

Spin-dependent recombination of the charge-transfer state in photovoltaic polymer/fullerene blends

ABSTRACTQ-band electron spin echo (ESE) spectroscopy was applied for studying the spin-dependent recombination of charge transfer (CT) states in the benchmark organic photovoltaics (OPV) blend of poly(3-hexylthiophene-2,5-diyl) and [6,6]-phenyl C61 butyric acid methyl ester (P3HT/PC60BM). Selective microwave excitation and a special protocol for ESE data treatment allowed to suppress the ESE signal of thermalised polarons and weakly coupled CT states and to address CT states with a relatively short distance between positive and negative polarons (1.5 nm < r < 2.5 nm). Inversion of the in-phase ESE signal with increase of the delay after laser flash was observed for the regioregular P3HT•+/PC60BM•− CT state at a temperature of 40 K. This effect is very similar to the inversion of the time resolved (TR) EPR spectrum of the same system obtained previously. Both effects can be explained by spin-dependent recombination of the CT state, with the recombination via the triplet channel proceeding much slower than via the singlet channel. For the regiorandom P3HT•+/PC60BM•− CT state no ESE sign inversion was observed in an analogous experiment. The result suggests the importance of CT state formation via a triplet exciton, a process which was not considered previously for the P3HT/PC60BM blend.

The Power of EPR Techniques in Investigating Functionalization and Penetration into Fibers of Cotton-Bound Antimicrobial Peptides

The development of protective and safe textiles is of fundamental importance for defending the human body from bacterial infections. To this aim, garments are often functionalized with antibacterial agents. We recently started a program aimed at covalently linking antimicrobial peptides to cotton tissues. To optimize the process of binding, it is necessary to know the degree of functionalization and how deeply peptides penetrate into the cotton fiber. Here, we present a spin-label electron paramagnetic resonance (EPR) approach for obtaining data on the peptide incorporation into the fibers. The approach is based on the line broadening in conventional EPR and on the signal decays in electron spin echo spectroscopy that is a pulsed version of EPR.

A THz spectrometer combining the free electron laser FLARE with 33 T magnetic fields

The free electron laser Free electron Laser for Advanced spectroscopy and high Resolution Experiments (FLARE) at the FELIX Laboratory generates powerful radiation in the frequency range of 0.3–3 THz. This light, in combination with 33 T Bitter magnets at the High Field Magnet Laboratory, provides the unique opportunity to perform THz magneto spectroscopy with light intensities many orders of magnitude higher than provided by conventional sources. The performance of the THz spectrometer is measured via high-field electron spin resonance (ESR) in the paramagnetic benchmark system 2,2-diphenyl-1-picrylhydrazyl (DPPH). The narrow ESR linewidth of DPPH allows us to resolve a fine structure with 3 GHz spacing, demonstrating a considerable coherence of the individual THz micropulses of FLARE. The spectral resolution Δν/ν is better than 0.1%, which is an order of magnitude higher than typical values for a rf-linac based free electron laser. The observed coherence of the high power THz micropulses is a prerequisite for resonant control of matter, such as THz electron spin echo spectroscopy.

Light-induced charge separation in a P3HT/PC70BM composite as studied by out-of-phase electron spin echo spectroscopy.

A composite material of semiconducting polymer P3HT and fullerene derivative PC70BM was studied by means of electron spin echo (ESE) spectroscopy. The out-of-phase ESE signal was observed under laser irradiation of the composite at low temperature. We assume that during the charge separation process firstly the spin-correlated radical pairs in the singlet-polarized spin state are formed, and then the net polarization of radical pairs arises due to spin evolution. Both types of polarizations contribute to the out-of-phase ESE signal in the case of non-ideal microwave pulses. Analytical calculation of the echo shape for both types of initial polarization revealed that the contribution of the net polarization becomes zero after averaging over the whole EPR spectrum of the radical pair. This behavior was experimentally confirmed; thus the analysis of the out-of-phase ESE signal was simplified. Interspin distance distributions in the charge transfer state were obtained by modeling the out-of-phase ESE envelope modulation measured at different delays after laser flash TDAF from 300 ns to 3.3 μs at a temperature of 65 K. Due to geminate recombination and diffusion of the radicals from the interface the distribution becomes significantly broader with larger distances prevailing at longer TDAF values. The average distance between charges increases from 3.5 nm to 5.6 nm with an increase in TDAF.

Heterogeneities in Cholesterol-Containing Model Membranes Observed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

Biological membranes are supposed to have heterogeneous structure containing lipid rafts-lateral micro- and nanodomains enriched in cholesterol (chol) and sphingolipids. In this work, lipid bilayers containing a small amount of the spin-labeled chol analogue 3β-doxyl-5α-cholestane (chlstn) were studied using electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR). Bilayers were prepared from an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with chol added at different concentrations. The ESE decays recorded at 77 K become faster with increase of chlstn concentration. The chlstn-dependent contribution to ESE decay is remarkably nonexponential; however, the logarithm of this contribution can be rescaled for different chlstn concentrations to a universal function with the rescaling factor approximately proportional to concentration. This result shows that the chlstn-dependent contribution to the ESE decay can be employed to estimate the local (at the nanometer scale of distances) chlstn concentration. Analogous rescaling behavior is also observed for the bilayers with different chol concentrations, with the rescaling factor increasing with increase of the chol concentration. This result is evidence that chlstn molecules are distributed heterogeneously in the chol-containing bilayer and form clusters with enhanced chlstn (and probably chol) local concentration. The local concentration of chlstn molecules for large chol content (∼30 mol %) was enhanced by at least ∼70% versus chol-free bilayers. The suggested approach appears to be useful for exploring heterogeneities in lipid composition of biological membranes of different types.

Out-of-Phase Electron Spin Echo Studies of Light-Induced Charge-Transfer States in P3HT/PCBM Composite.

The light-induced charge-transfer (CT) state in the composite of the conductive polymer poly(3-hexylthiophene) (P3HT) and the fullerene derivative [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) has been studied by electron spin echo (ESE) spectroscopy. The out-of-phase ESE signal corresponding to the spin-correlated radical pair P3HT(+)/PCBM(-) has been observed in this composite material. The time-domain ESE shape for different delays between the laser flash and the microwave pulse sequence has been analyzed. In order to explain the evolution of the out-of-phase ESE signal as a function of the delay between the microwave pulses, a model of the CT state is proposed. The hole is assumed to be delocalized on the P3HT chain over several thiophene subunits, while the point-dipole approximation is used to describe the interaction with the electron on PCBM. The distribution of distances between the positive and negative charges in the CT state has been evaluated.

Communication: Orientational self-ordering of spin-labeled cholesterol analogs in lipid bilayers in diluted conditions.

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3β-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of "instantaneous diffusion" in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.

EPR, ESE, and pulsed ENDOR study of the nitrogen donors in 15R SiC grown under carbon‐rich conditions

X‐band field‐sweep electron spin echo and pulsed electron nuclear double resonance (ENDOR) spectroscopy were used to study n‐type 15R SiC wafers grown under carbon (C)‐rich conditions with the aim to verify the recently proposed concept that nitrogen (N) donors substitute both carbon (C) and silicon (Si) sites and may occupy nonequivalent positions at Si sites. It was found that besides the 14N ENDOR spectra of the C substituting quasicubic “k1”, “k2”, and “k3” positions five doublet lines due to 14N nuclei at other lattice positions were observed in the ENDOR spectrum of highly compensated C‐rich n‐type 15R SiC. Three of them with the hyperfine interaction (HFI) constants 34.66, 32.20, and 29.77 MHz were attributed to the N donors substituting “k1”, “k2”, and “k3” positions at Si sites. Two other additional ENDOR spectra were explained by the presence of the C antisite (CSi) defects in C‐rich 15R SiC, which leads to the formation of CSiNC complexes with donor levels close to the conduction‐band minimum and isotropic 14N HFI constants of 21.58 and 25.94 MHz. The energy levels of N donors have been evaluated by comparing the number of ENDOR spectra observed in C‐rich 15R SiC with different degrees of compensation.

Low-temperature molecular motions in lipid bilayers in the presence of sugars: insights into cryoprotective mechanisms.

Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose, and sorbitol at concentrations of 0.2 and 1 M and containing incorporated spin-labeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR). The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and the stearic acids were labeled with nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th carbon positions. The ratio of the echo time traces for the two field positions in the EPR spectrum possessing the largest and smallest anisotropies gave the anisotropic contribution to the echo decay, which obeys exponential time dependence with good accuracy. At low temperatures, the anisotropic contribution is induced by stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations), with the exponential decay rate Wanis proportional to <α(2)>τc, where <α(2)> is the mean angular amplitude of the motion and τc is the correlation time. In all cases, it was found that Wanis begins to increase sharply above 170-200 K, which was ascribed to the dynamical transition known for biological systems at these temperatures. For hydration by the sucrose and trehalose solutions, Wanis was found to increase noticeably also above ∼120 K, which was explained by bilayer expansion due to direct bonding of sugar molecules to the bilayer surface. The Wanis temperature dependencies were found to be close to those obtained for the simple systems of the nitroxide spin probe TEMPONE in aqueous sorbitol and sugar 1 M solutions. This correlation suggests a possible mechanism of cryoprotective action of sorbitol and sugars due to the similarity of low-temperature motions in the membrane and in the cryoprotectant-containing surrounding liquid.

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

Hydrogen bonding to the nitroxide of protein bound spin labels

Deuterium electron spin-echo envelope modulation spectroscopy (2H-ESEEM) (9.3 GHz, 0.33T) and continuous wave high-field (95 GHz, 3.4T) electron paramagnetic resonance (EPR) spectroscopy were applied to disentangle the sources of protons which are hydrogen bonded to nitroxides of protein bound spin labels. For a set of 12 colicin A mutants with nitroxide spin labels site-specifically introduced at residue positions 26, 33, 42, 62, 91, 105, 115, 154, 166, 169, 181 and 192 of the channel forming domain we analysed the heterogeneity in the gxx region of EPR spectra detected at 95 GHz and quantified the fractions of nitroxide populations engaged in H-bonding to water and protein protons. The amplitudes of the narrow and broad components of 2H-ESEEM spectra arising from non-bonded and bonded water (D2O) molecules or deuterated protein components reflect the local H-bonding equilibria. The combination of both methods allowed estimating the fraction of nitroxide populations hydrogen bonded to non-exchangeable protein protons. Hydrogen bonds are known to play a governing role in protein folding and function, and the presented approach extends the information obtainable from site-directed spin labelling in the investigation of complex biomolecular systems and their conformational changes.

Multifrequency EPR and DENR of polyacetylene composite

The organo-inorganic composite MoCl1.9 ± 0.1(C30 ± 1H30 ± 1), a product of interaction of MoCl5 with C2H2, has been studied by X- and W-band EPR, double electron nuclear resonance, and W-band electron spin echo spectroscopy. The composite consists of nanosized organometallic molybdenum clusters in the polyacetylene matrix. It has been shown that the composite contains three types of magnetic centers: the first is related to the existence of paramagnetic molybdenum atoms in the polyacetylene matrix, and the other two are paramagnetic defects of the matrix.

Molecular Motion in Frozen Phospholipid Bilayers in the Presence of Sucrose and Sorbitol Studied by the Spin-Echo EPR of Spin Labels

Electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), was applied to spin-labeled stearic acids in phospholipid bilayers hydrated in the presence of sucrose and sorbitol, which are known for their cryoprotective action on biological membranes. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Stearic acids were labeled by nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th specific carbon positions. ESE detects fast stochastic small-angle restricted molecular rotations (stochastic molecular librations) with correlation times on the nanosecond timescale. These motions are believed to have the same nature as the anharmonic motions of hydrogen atoms in biological substances detected by neutron scattering and Mossbauer spectroscopy, which become active above 200 K. To ensure that the echo decays indeed originate from fast stochastic molecular librations, a three-pulse stimulated spin echo was employed. It was found that the presence of sucrose or sorbitol suppresses the observed molecular motions. The observed effect was nearly the same for both label positions, indicating that the motions are similarly suppressed near the bilayer surface and in the bilayer interior. This finding suggests non-specific interactions of sugars with bilayer surface, which are likely to influence only the bulk physical properties of hydrated membranes. The results obtained show the usefulness of spin-echo EPR of spin labels when applied to investigate the molecular mechanisms of action of cryoprotective agents on biological systems.

EPR and potentiometric studies of copper(II) binding to nicotinamide adenine dinucleotide (NAD+) in water solution

Coordination of Cu(II) by nicotinamide adenine dinucleotide (NAD+) molecule has been studied in water solutions of various pH by potentiometry and electron paramagnetic resonance (EPR) and electron spin echo (ESE) spectroscopy. Potentiometric results indicate Cu(II) coordination by protonated NAD+ at low pH and by deprotonated NAD+ at high pH. At medium pH value (around pH=7) NAD+ is not able to coordinate Cu(II) ions effectively and mainly the Cu(H2O)6 complexes exist in the studied solution. This has been confirmed by EPR results. Electronic structure of Cu(II)–NAD complex and coordination sites is determined from EPR and ESE measurements in frozen solutions (at 77K and 6K). EPR spectra exclude coordination with nitrogen atoms. Detailed analysis of EPR parameters (g||=2.420, g⊥=2.080, A||=–131×10−4cm−1 and A⊥=8×10−4cm−1) performed in terms of molecular orbital (MO) theory shows that Cu(II)NAD complex has elongated axial octahedral symmetry with a relatively strong delocalization of unpaired electron density on in-plane and axial ligands. The distortion of octahedron is analyzed using A|| vs. g|| diagram for various CuOx complexes. Electron spin echo decay modulation excludes the coordination by oxygen atoms of phosphate groups. We postulate a coordination of Cu(II) by two hydroxyl oxygen atoms of two ribose moieties of the NAD molecules and four solvated water molecules both at low and high pH values with larger elongation of the octahedron at higher pH.

Silver atoms in the structural channels of beryl

A silver atom in synthetic beryl is investigated by the EPR and electron spin echo (ESE) spectroscopy. It is established that silver ions were first introduced into the structural channels of beryl by thermodiffusion at 800υC. The Ag+ ions are then converted to the Agυ state by the X-ray irradiation of samples at room temperature. Charge changes in manganese and chromium impurities located at the aluminum positions are observed at the same conditions. Four different Agυ centers with isotropic hyperfine interactions (HFI) with 107 Ag and 109 Ag nuclei and hyperfine constants less than those for the free Ag atom are revealed by the EPR method. ESE investigations enable us to confirm the positions of silver atoms that are stable up to 230υC.

Small-Amplitude Backbone Motions of the Spin-Labeled Lipopeptide Trichogin GA IV in a Lipid Membrane As Revealed by Electron Spin Echo

Trichogin GA IV is a lipopeptide antibiotic of fungal origin, which is known to be able to modify the membrane permeability. TOAC nitroxide spin-labeled analogues of this membrane active peptide were investigated in hydrated bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by electron spin echo (ESE) spectroscopy. Because the TOAC nitroxide spin label is rigidly attached to the peptide backbone, it may report on the backbone orientational dynamics. The ESE signal in this system is observed below ∼150 K. Previously, three-pulse stimulated ESE was found to be sensitive to two types of orientational motion of spin-labeled POPC lipid bilayers at these temperatures. The first type is fast stochastic librations, with a correlation time on the nanosecond scale (which also manifests itself in a two-pulse primary ESE experiment). The second type is slow millisecond inertial rotations. In the present work, we find that at low molar peptide to lipid ratio (1:200), where the individual peptide molecules are randomly distributed at the membrane surface, the spin labels show only a fast type of motion. At the high molar peptide to lipid ratio (1:20), a slow motion is also observed. Because at this high concentration trichogin GA IV is known to change its orientation from the in-plane topology to the transmembrane disposition, the observed onset of a slow motion may be safely attributed to the dynamics of peptides, which are elongated along the lipid molecules of the membrane. The possible interrelation between this backbone rotational motion of the peptide antibiotic and the membrane leakage is discussed.

ChemInform Abstract: Electronic Structure of Retinal and Related Polyenals in the Lowest Triplet State: An Electron Spin Echo Study.

The authors have observed transitions between the spin sublevels of the lowest triplet state T{sub 0} of all-trans isomers of retinal and methyl-substituted polyenals by means of electron spin echo spectroscopy in a magnetic field and in zero field. This triplet state appears to be {pi}{pi}* in nature. the frequencies of the zero-field transitions have been determined. For retinal, the authors have observed the T{sub z}-T{sub x} transition at 2370 MHz and the T{sub z}-T{sub y} transitions at 1990 MHz. The upper spin sublevel T{sub z} dominates the populating of T{sub 0} and decays fastest, but the selectivity in the decay is clearly less than in the populating. The lifetimes of the individual sublevels of the lowest triplet state or retinal are 71 {mu}s for T{sub x}, 17 {mu}s for T{sub y}, and 12 {mu}s for T{sub z} at 1.2 K. The data for retinal correspond to those for a linear polyenal with six double bonds. This shows that the carbon-carbon double bond in the cyclohexene ring of potential is also involved in the excitation, which suggests that the chromophoric part of retinal is approximately planar in the lowest triplet state. 22 refs., 6 figs., 4 tabs.