Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-stale protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (I) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (φ) larger than 90° or by measurement of ratios of m/cos φ > 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overtap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene(PRODAN) and N-acetyl-l-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos φ measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.     

Phase-sensitive fluorescence spectroscopy: A new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores

A novel phase fluorometric method is described which permits direct recording of individual emission spectra from a mixture of two flourescent compounds. Additionally, the lifetimes of each component may be determined by examination of the phase-sensitive fluorescence spectra. The method utilizes phase-sensitive detection of the sinusoidally modulated emission from a phase fluorometer. Resolution of the individual emission spectra in the mixture requires different fluorescence lifetimes for each components. Determination of the individual lifetime requires knowledge of the steady-state emission spectra of the components. Use of low-frequency (≈ 10 Hz) cross-correlated signals eliminates the need for high-frequency frequency (≈10⁶ Hz) phase-sensitive detection. A mixture of 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) and 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) was used to demonstrate the possibility of phase resolution of fluorophore mixture and to confirm theoretical predictions. A mixture of dibenzo[a,h]anthracene and dibenzo[c,g]carbazole was used to demonstrate that phase resolution is possible for spectra which overlap strongly and which are highly structured. In addition, the possibility of using phase-sensitive emission spectra for the resolution of excited-state reactions was demonstrated with anthracene and its diethylaniline exciplex. From a sample whose steady-state emission displayed both components we directly recorded the emission spectrum of anthracene monomer and the exciplex. For all these samples the dependence of the individual intensities on the phase angle of the detector agreed precisely with that expected on the basis of the individual fluorescence lifetimes. The detector phase angles chosen for suppression of each component in the mixture also agreed with the measured lifetimes. Thus, phase-sensitive fluorescence spectra can reveal individual spectral distributions or lifetimes. This method will be useful in the analysis fluorescence emissions which frequently occur from proteins, membranes and other biological samples.     

Proof of nanosecond timescale relaxation in apomyoglobin by phase fluorometry

Apomyoglobin was labeled with the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). Apparent phase shift and demodulation lifetimes of bound TNS were measured at various emission wavelengths. The lifetimes increased with increasing wavelength. Similar results were obtained for TNS in the viscous solvent glycerol at 10°C but not for TNS in vitrified or fluid solvent. The wavelength-dependent lifetimes suggest apomyoglobin is relaxing around the TNS molecule during its fluorescent lifetime. Importantly, the apparent phase lifetimes exceeded the apparent modulation lifetimes on the long wavelength side of the emission for TNS in apomyoglobin at 3°C and for TNS in glycerol at 10°C. This result proves the increasing lifetimes are a result of an excited state reaction during the lifetime of the excited state and are not a result of heterogeneity in the fluorescence emission. From the lifetimes on the short wavelength side of the emission the relaxation time of apomyoglobin was estimated to be 18 nsec.     

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.     

Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence.

The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.     

Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity. This procedure was used to resolve the emission spectra of the initially excited and solvent relaxed states of a tryptophan derivative in viscous solution. Specifically, we examined N-acetyl-l-tryptophanamide (AcTrpNH₂) in viscous and nonviscous solutions of propylene glycol. Time-resolved decays of fluorescence intensity were collected at wavelengths across the emission spectra. Instead of the usual procedure of deconvolving these data with the time profile of the exciting pulse, we deconvolved these data using the response observed on the short-wavelength side of the emission. If one assumes that this emission results only from the initially excited state (F), then the nonzero decay time calculated using deconvolution is that of the solvent relaxed state (R). For our specific case of AcTrpNH₂ the emission spectra of the F and R states overlap at most wavelengths longer than the short-wavelength side of the emission (310 nm). As a result, differential-wavelength deconvolution yields two lifetimes and amplitudes, one pair representing the relaxed state and the other the initially excited state. The latter appears as a zero-decay-time component whose amplitude can be readily quantified. The wavelength-dependent amplitude of this zero-lifetime component can be used to calculate the emission spectrum of the F state and. by difference, the emission spectrum of the relaxed state. For AcTrpNH₂ in propylene glycol at ?20°C the emission maxima of the F and R states are near 320 and 350 nm, respectively, and the relative proportion of the emission from each state was near 50%. At lower temperatures the emission from the F state becomes dominant and at high temperatures the emission from the R state dominates. We note that this resolution of states is somewhat arbitrary because we assumed a two-state model and the absence of solvent relaxed emission at 310 nm. Nonetheless, differential-wavelength deconvolution simplifies and facilitates the analysis of time-resolved fluorescence data from samples which undergo excited state reactions. Moreover, this deconvolution procedure considerably simplifies the determination of the kinetic constants for reversible excited state reactions. The application of differential-wavelength deconvolution does not increase the time reqaired for data acquisition. This differential analysis procedure should enhance the usefulness and precision of pulse fluorometric methods in studies of nanosecond time scale processes in proteins and membranes.     

Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry

We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational correlation times of TNS bound to apomyoglobin are consistent with those expected for this probe rigidly bound to the protein. TNS in propylene glycol also displayed a single exponential decay of anisotropy. These results, in conjunction with the previous successful resolution of multiexponential decays of fluorescence intensity (Lakowicz, J. R., Gratton, E., Laczko, G., Cherek, H., and Limkeman, M. (1984) Biophys. J., in press; Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J., in press) demonstrate that frequency-domain measurements provide information which is, at a minimum, equivalent to that obtainable from time-domain measurements.     

Nanosecond dynamics of a mimicked membrane-water interface observed by time-resolved stokes shift of LAURDAN

We studied the dipolar relaxation of the surfactant-water interface in reverse micelles of AOT-water in isooctane in the nanosecond and subnanosecond time ranges by incorporating the amphipathic solvatochromic fluorescent probes LAURDAN and TOE. A negative component was observed in the fluorescence decays in the red edge of the emission spectrum-the signature of an excited state reaction-with LAURDAN but not for TOE. The deconvolution of the transient reconstructed spectra of LAURDAN based on a model constructed by adding together three log-normal Gaussian equations made it possible to separate the specific dynamic solvent response from the intramolecular excited state reactions of the probe. The deconvoluted spectrum of lowest energy displayed the largest Stokes shift. This spectral shift was described by unimodal kinetics on the nanosecond timescale, whereas the relaxation kinetics of water-soluble probes have been reported to be biphasic (on the subnanosecond and nanosecond timescales) due to the heterogeneous distribution of these probes in the water pool. Most of this spectral shift probably resulted from water relaxation as it was highly sensitive to the water to surfactant molar ratio (w(0)) (60-65 nm at w(0) = 20-30). A small part of this spectral shift (9 nm at w(0) = 0) probably resulted from dipolar interaction with the AOT polar headgroup. The measured relaxation time values were in the range of the rotational motion of the AOT polar headgroup region as assessed by LAURDAN and TOE fluorescence anisotropy decays.     

Fluorescence of quinacrine mustard conjugated to proteins.

Proteins are readily labeled by quinacrine mustard to yield conjugates whose spectral properties are well-suited for fluorescence studies. Data on these conjugates and on the parent compound, quinacrine, are presented including lifetimes, quantum yields, and corrected excitation and emission spectra. Polarization studies using the Perrin-Weber equation show that rotational relaxation times can be obtained with quinacrine mustard conjugates. Such conjugates had lifetimes ranging from 4 to 13 ns and quantum yields from about 0.1 to 0.3. Quinacrine mustard is a useful reporter group, as shown by the changes in fluorescence parameters of conjugates undergoing conformational changes induced by denaturants. An excited state $\text{p}\text{K}{}_{\mathrm{a}}{}^1$ of 4.9 was identified for quinacrine, but the protonation was suppressed in the mustard conjugate of serum albumin until the N-F transition took place. The properties of the mustard conjugates are discussed in terms of potential uses and compared with properties of other types of fluorescent conjugates.     

Stark effect in P700, the primary electron donor of Photosystem I

Quadratic Stark effect in CP1 pigment-protein complex was examined at low temperatures in the red spectral region. The Stark spectra of samples containing P700 in reduced form exhibit a strong negative band at 704 nm, which disappears on chemical oxidation of P700. The change in permanent dipole moment, delta mu, of P700 on electronic excitation estimated from these spectra was found to be between 4.7 and 7.7 Debye units. It is suggested to reflect the charge-transfer contribution to the excited state of P700. For antenna chlorophyll, delta mu approximately equal to 1 D was obtained in accordance with the data for monomeric chlorophyll.     

Influence of vesicle curvature on fluorescence relaxation kinetics of fluorophores

The effect of membrane curvature on the fluorescence decay of 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter dm = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1-40 degrees C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times tau(r) than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5 degrees C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesicles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1-3 ns) are practically identical for small and large vesicles, the longer decay times (5-14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature.     

Dipolar solvent relaxation on a nanosecond time scale in ether phospholipid membranes as determined by multifrequency phase and modulation fluorometry

The present study reports on the observation of dipolar solvent relaxation in phospholipid membranes using multifrequency phase and modulation fluorometry. We measured the time-resolved emission spectra of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) in artificial bilayer membranes of chemically defined acyl-, alkyl-, and alkenyl-substituted phospholipids at 15 degrees C. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 3-O-hexadecyl-2-oleoyl-sn-glycero-1-phosphocholine, or 1-O-hexadec-1'-enyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmalogen) were used as matrix lipids. The chemical structures of these lipids differ only with respect to the type of linkage (carboxyl ester, ether, or enol ether bond) between glycerol and the hydrophobic chain linked to the primary hydroxyl of glycerol. At 15 degrees C, all the lipids are in the liquid crystalline state. PRODAN probably localizes at the hydrophobic-hydrophilic interface of the phospholipid bilayer [Chong, P. L. (1988) Biochemistry 27, 399-404]. We found faster solvent relaxation of PRODAN in membranes composed of the ether lipid compared to that in the ester lipid membranes. On the other hand, the fluorescence anisotropies of the label were very similar, showing that the motion of the label itself is similar in ether and carboxyl ester lipids. We conclude that the spectral differences observed for PRODAN in ether and ester lipids could be due to different dipolar relaxation of the immediate surroundings of the label, i.e., reorientation of lipid dipoles in the glycerol region and of water molecules residing therein.     

On the nanosecond mobility in proteins: Edge excitation fluorescence red shift of protein-bound 2-(p-toluidinylnaphthalene)-6-sulfonate

The fluorescence spectra of 2-(p-toluidinylnaphthalene)-6-sulfonate associated with β-lactoglobulin, β-casein. and bovine and human serum albumins are shown to depend on excitation wavelength. A long-wave shift of the spectra is observed at the long-wave edge excitation, reaching 10 nm and above. A similar phenomenon is found in glucose glass and in glycerol at + 1°C, i.e., in systems with delayed dipolar solvent relaxation, but not in liquid solutions. This phenomenon is proposed to be based on relaxation processes in the excited state. There exists a distribution of chromophore microstates with different interactions with surrounding groups which results in heterogeneous broadening of the electronic spectra and allows photoselection of a part of this distribution, being characterized by a low transition energy. The fast structural relaxation results in an altered distribution and, if this is the case, the effect of edge excitation of fluorescence spectra is not observed. If the structural relaxation during the excited state lifetime is absent, this effect is maximal. This interpretation is in agreement with results on the influence of red edge excitation on the low-temperature fluorescence spectra of dyes and with the data on time-resolved nanosecond fluorescence spectroscopy. The results of this work strongly support the significant dye fluorescence spectral shifts on protein binding, being determined not only by polarity changes in their environment, but also by relaxation properties of protein groups in this environment. These results also indicate that on the nanosecond time scale, the structural relaxation around the excited chromophore in proteins may be incomplete.     

Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

We describe the use of phase-sensitive detection of fluorescence to resolve the lifetimes and fractional intensities from multi-component fluorescence samples, using data obtained at a single modulation frequency. Phase-sensitive spectra of the mixture are recorded at arbitrarily chosen detector phase angles. The steady-state spectrum of each component must be known. The phase-sensitive spectra are fitted, using a nonlinear least-squares algorithm, to obtain the lifetimes and fractional intensities of each fluorophore in the mixture. Simulations for two- and three-component mixtures are presented to illustrate how the resolution is affected by spectral overlap and lifetime separation. Experimentally, we resolved two- and three-component mixtures of protein-like fluorophores (N-acetyl-L-tyrosinamide, N-acetyl- L-tryptophanamide, indole and 2,3-dimethylindole) using data collected at 30 MHz. These fluorophores have closely spaced lifetimes of 1.5, 2.9, 4.5 and 4.3 ns, respectively, and display extensive spectral overlap. These results demonstrate that phase-sensitive spectra, recorded at only one modulation frequency with a standard phase fluorometer, can be used to resolve multi-component emissions.     

Time-resolved fluorescence studies on protoporphyrin IX-apohorseradish peroxidase

The hemin moiety of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was removed and the apoprotein reconstituted with the fluorescent protoporphyrin IX. Steady-state and time-resolved fluorescence properties of the HRP(desFe) adduct were examined; the multifrequency phase and modulation method was utilized for lifetime and dynamic polarization studies. The emission spectrum of HRP(desFe) had maxima at 633 and 696 nm. The lifetime of this emission was characterized by a single exponential decay of 16.87 ns at 22 degrees C. Debye rotational relaxation times for HRP(desFe) were determined using both static (Perrin plot) and dynamic (differential phase and modulation fluorometry) methods; these two approaches gave values of 96 and 86 ns, respectively. A spherical protein of HRP's molecular weight and partial specific volume would be expected to have a Debye rotational relaxation time, at 22 degrees C, in the range of 50 to 60 ns, depending upon the extent of hydration. Hence our results indicate that HRP(desFe) is asymmetric; the global rotational relaxation times observed are consistent with those of a prolate ellipsoid with an axial ratio of 3:1.     

Nonequilibrium radiative properties in fluctuating plasmas1

A general kinetic model was developed to simulate the radiative properties of nonstationary fluctuating plasmas and characterize the relationship between the nonstationary fluctuation time and the atomic relaxation times. The developed theory is applied to the radiative line emission in the case of instabilities in tokamaks. It is shown by exact time dependent simulations that involve explicitly LSJ-split excited states that the radiation emission in fluctuating plasma can be larger than in the corresponding stationary limits. For regular fluctuations like the sawtooth activity, also the startup phase of sawtooth activity can lead to higher emission compared to the time dependent regular phase. It is demonstrated that the sawtooth crash can be almost exactly followed by resonance line emission like H-like Lyman-alpha and He-like Helium-alpha of, e.g., argon impurity ions, whereas the effective charge state distribution lags seriously behind.     

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.     

Zwitterionic dipoles as a dielectric probe for investigating head group mobility in phospholipid membranes

For phospholipid membranes with zwitterionic head groups, the dipole can be considered as a specific label for tracing the changes in the dynamic behaviour of this region of the bilayer in its various phases. Measurements of the dielectric properties of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the frequency range 1--50 MHz show a dispersion which is attributed to the motion of the phosphocholine dipoles in the plane of the bilayers. When the temperature is varied, both the permittivity and loss factor increase sharply at the pretransition (35 degrees C) and the main transition (42 degress C). The relaxation time and amplitude were also determined for this dispersion and these further reflect the structural changes occurring with temperature. The relaxation times varied between 4 ns at 30 degrees C and 2.3 ns at 50 degrees C. Due to steric hindrances a restriction in the angle of head group rotation occurs at lower temperatures but is greatly reduced above the main transition.     

Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the     

Intramolecular charge transfer processes in donor-acceptor substituted vinyltetrahydropyrenes.

Two novel donor-acceptor-substituted vinyltetrahydropyrene derivatives, 2-N,N-dimethylamino-7-(1-carbethoxyvinyl)-4,5,9,10-tetrahydropyrene, , and 2-N,N-dimethylamino-7-(1,1-dicyanovinyl)-4,5,9,10-tetrahydropyrene, , were synthesized and their photophysical properties investigated in solvents of different polarities. Our studies revealed the existence of intramolecular charge transfer excited states in these molecules. For both compounds the fluorescence maxima exhibited solvent polarity-dependent red shifts. These were quantitatively analysed by the Lippert-Mataga and Liptay equations to obtain the excited state dipole moments. Our results indicated that in the case of , emission takes place from a planar (1)CT state in all non-protic solvents. In the case of , the nature of the excited state depends on the solvent. A fast relaxation to a triplet state is proposed in cyclohexane. The emitting state in medium polar solvents is a planar (1)CT state. In highly polar solvents a twisted (1)CT state is invoked to explain the low fluorescence quantum yield. For both compounds CT nature of the emitting states were further confirmed by studies in acidic medium. The ground and excited state pK(a) values for and were determined using absorption and emission spectral changes observed in the presence of protic acids.