Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach.

A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.     

Fluorescence lifetime quenching and anisotropy studies of ribonuclease T1

The time-resolved fluorescence of the lone tryptophanyl residue of ribonuclease T1 was investigated by using a mode-locked, frequency-doubled picosecond dye laser. The fluorescence decay could be characterized by a single exponential function with a lifetime of 3.9 ns. The fluorescence was readily quenched by uncharged solutes but was unaffected by iodide ion. These observations are interpreted in terms of the electrostatic properties of the amino acid residues at the active site of the protein, which would appear to restrict the access of solute species to the tryptophanyl residue. The temperature dependence of the fluorescence lifetime and anisotropy decay time could be rationalized in terms of a model which postulates a significant ordering of the solvent layer immediately surrounding the surface of the protein.     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

A method for deciding whether two experimental fluorescence anisotropy decay curves are significantly different

We describe a method to compare two fluorescence anisotropy decay curves. After numerical deconvolution of both decays by a non a priori method [1], their difference, D, is considered. The variance is computed for each point of D. A confidence interval is defined which allows a decision to be made as to the significance of D. Information on the time range in which the changes of the fluorescence anisotropy decay occur is directly available. This analysis is particularly well suited for following the perturbations induced by an effector. It has been tested on 3-phosphoglycerate kinase in the presence and in the absence of ATP and 3-phosphoglycerate. We consider that this method leads to a significant improvement in the application of time resolved depolarization experiments.     

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.     

Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier.

In many proteins fluorescence from single tryptophan exhibits a nonexponential decay function. To elucidate the origin of this nonexponential decay, we have examined the fluorescence decay function and time-resolved fluorescence anisotropy of a fluorophore covalently bound to a macromolecule by solving a rotational analogue of the Smoluchowski equation. An angular-dependent quenching constant and potential energy for the fluorophore undergoing internal rotation were introduced into the equation of motion for fluorophore. Results of numerical calculations using the equations thus obtained predict that both the fluorescence decay function and time-resolved anisotropy are dependent on rotational diffusion coefficients of fluorophore and potential energy for the internal rotation. The method was applied to the observed fluorescence decay curve of the single tryptophan in apocytochrome c from horse heart. The calculated decay curves fit the observed ones well.     

Fluorescence correlation spectroscopy and Brownian rotational diffusion

A theroy relating rotational Brownian motion to the time autocorrelation function of the intensity of radiation from a fluorescent system composed of spherical rotors is presented. The calculation shows three relaxation times, two associated with the rotational diffusion, and the third associated with the natural decay of the fluorescence. The correlation function contains terms that relax independently of the fluorescence decay time, thus arbitrarily extending the time range over which rotational diffusion can be studied by fluorescence.     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Time-resolving luminescence techniques for possible detection of forest decline

Needles from spruces at different environmental and physiological conditions were analyzed by picosecond fluorescence spectroscopy using a novel laser diode and single photon counting detection. The decay curves of chlorophyll fluorescence showed a superposition of three exponentially decaying components with time constants of T1 = 100-200 ps, T2 = 300-500 ps and T3 = 2.0-3.5 ns. A high relative intensity of the long-lived component was found in damaged spruces as well as in trees showing first symptoms of yellowing, needle loss or parasite infection, although all measurements were carried out with green needles which appeared visually intact. Therefore, fluorescence spectroscopy with subnanosecond time resolution seems to be a valuable attempt for an early detection of forest decline.     

Tryptophan interactions of gramicidin A′ channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.     

Tryptophan interactions of gramicidin A' channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.     

Simultaneous analysis of multiple fluorescence decay curves by Laplace transforms. Deconvolution with reference or excitation profiles

The properties and potentials of the noniterative Laplace deconvolution (LAP2) (M. Ameloot and H. Hendrickx, Biophys. J. 44 (1983) 27) are further investigated. It is shown that LAP2 is exact and that no extrapolations have to be calculated or assumed for the data measured in the actual time window if the impulse response function of the investigated system can be described by a sum of exponentials. The formulas for the LAP2 deconvolution against the measured decay of a reference compound instead of the recorded excitation profile are derived. The procedure for the simultaneous analysis of multiple fluorescence decay curves by LAP2 is described in detail. This global analysis allows one to link any decay parameter, is fast and compares favorably with the nonlinear least-squares iterative reconvolution methods. Because of its short computation time the global analysis by LAP2 provides an efficient way to analyze the fluorescence decay surface in terms of decay associated spectra.     

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.     

Effects of Growth Irradiance and Nitrogen Limitation on Photosynthetic Energy Conversion in Photosystem II

Photosynthetic energy conversion was investigated in five species of marine unicellular algae, (Dunaliella tertiolecta, Thalassiosira pseudonana, T. weisflogii, Skeletorema costatum, Isochrysis galbana) representing three phylogenetic classes, which were grown under steady state conditions with either light or inorganic nitrogen as a limiting factor. Using a pump and probe fluorescence technique we measured the maximum change in variable fluorescence yields, the flash intensity saturation curves for the change in fluorescence yields and the kinetics of the decay in fluorescence yields. Under all growth irradiance levels nutrient replete cells exhibited approximately the same changes in fluorescence yields and similar fluorescence decay kinetics. The apparent relative absorption cross-section of photosystem II, calculated from the slope of the flash intensity saturation curves, generally increased as cells shade adapted. The decay kinetics of the fluorescence yield following a saturating pump flash can be expressed as the sum of three exponential components, with half-times of 160 and 600 microseconds and 30 to 300 milliseconds. The relative contribution of each component did not change significantly with growth irradiance. As cells became more nitrogen limited, however, the maximum change in fluorescence yield decreased, and was accompanied by a decrease in the proportion of a 160 microsecond fluorescence decay component, which corresponds to the transfer of electrons from Q_a⁻ to Q_b. Changes in fluorescence yields were also accompanied by changes in the levels of D1, a protein which is integral in reaction center II, and CP47, a chlorophyll protein forming part of the core of photosystem II. These results are consistent with a loss of functional photosystem II reaction centers. Moreover, in spite of losses of total cellular chlorophyll, which invariably accompanied nitrogen limitation, the apparent absorption cross-sections of photosystem II increased. Our results suggest that nitrogen limitation leads to substantial decreases in photosynthetic energy conversion efficiency.     

Simultaneous analysis of single-photon timing data for the one-step determination of activation energies, frequency factors and quenching rate constants. Application to tryptophan photophysics

A general global analysis of single-photon timing data is presented in which each fluorescence decay curve can be described by a different decay law. The model parameters can be held in common within one curve and/or between related curves. Any or all parameters can be kept fixed, or they may be variable to seek optimum values. This general analysis allows the determination of activation energies, frequency factors and quenching rate constants in one step. The construction of the global mapping table which relates parameters in one experiment to those in another is explained in detail. The use and performance of this general simultaneous analysis are examined using tryptophan fluorescence decays at pH 6.0 obtained at various emission wavelengths as a function of temperature and added solute quencher. The results show that tryptophan at pH 6.0 decays as a biexponential with decay times which are independent of the analysis wavelength. The decay component with the short lifetime has a deactivation rate constant of 1.4 x 10(9) s-1 independent of temperature. The decay component with the long lifetime has an activation energy of 28 kJ/mol and a frequency factor of 3 x 10(13) s-1; its temperature-independent decay rate constant equals 1 x 10(8) s-1. Recursion formulas for a computer program to estimate activation energies, frequency factors, and decay rate constants are provided.     

Diphenylhexatriene as a Fluorescent Probe for Monitoring Low Density Lipoprotein Peroxidation

The use of the fluorescent probe diphenylhexatriene (DPH) for monitoring low density lipoprotein (LDL) peroxidation has been investigated. The DPH incorporation into LDL results in a high fluorescence signal which decreases with time after addition of cupric ions. A strong correlation was found between the decay of the DPH fluorescence signal and the appearance of the thiobarbituric reactive substances (TBARS). HPLC and spectrofluorometric analyses demonstrated that DPH is destroyed during the time course of the copper-induced LDL peroxidation. The decrease in DPH fluorescent signal is prevented by addition of EDTA, vitamin E and drugs which protect LDL against peroxidation such as probucol or calcium antagonists. The high fluorescence of DPH allows the use of very small quantities of LDL (less than 5 μg/ml LDL protein). We thus suggest that DPH could be of use for continuous monitoring of LDL autooxidation, especially for the in vitro testing of the protective effect of antioxidant compounds.     

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b.

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the Fo and Fm levels. It was found that: (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (less than 2 x 10(14) photons . cm(-2). (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps (Fo) or 1300 ps (Fm) in the presence of Mg2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps (Fo) and 424 ps (Fm). (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres (Fm/Fo) or on the addition of MgCl2 (+Mg2+/-Mg2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.     

Interpretation of fluorescence decays using a power-like model

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl-L-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.     

A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.