Evidence that pyrene excimer formation in membranes is not diffusion-controlled

Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

A time-resolved fluorescence study of human copper-zinc superoxide dismutase

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.     

Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid fluorescence.

The fluorescent polyunsaturated parinaric acid incorporated in LDL particles is highly sensitive to the concentration of peroxyl radicals in the aqueous medium, undergoing rapidly oxidative degradation, as detected by a quenching of fluorescence, without delay after radical generation in solution. Ascorbate, cysteine, and urate suppress the parinaric acid fluorescence decay promoted by peroxyl radicals generated at a constant rate (thermal decomposition of 2,2'-azo-bis(2-amidino-propane hydrochloride)) in a concentration-dependent manner. The chain-breaking efficiencies of these antioxidants are evaluated from the time interval (inhibition period) of parinaric acid protection from oxidative degradation. The results correlate with the inhibition periods of LDL oxidation as monitored by O2 consumption. Therefore, the sensitive and simple parinaric acid assay can be used as a semiquantitative screening test for the detection of potentially important water-soluble chain-breaking antioxidants. Conversely to O2 consumption, the absence of any initial lag phase of probe degradation attests to the sensitivity of the assay. An improved methodology based on second-derivative spectroscopy to follow the formation of conjugated diene isomers directly in the preparation without the need for lipid extraction also confirms the sensitivity of this assay. To assess the usefulness of parinaric acid assay, strong chain-breaking activities of caffeic and chlorogenic acids are reported.     

Similarity of fluorescence lifetime distributions for single tryptophan proteins in the random coil state.

The picosecond time-resolved fluorescence decay data of nine single-tryptophan (trp) proteins and two multi-trp proteins in their native and denatured states were analyzed by the maximum entropy method (MEM). In the denatured state (6 M guanidine hydrochloride) a majority of the single-trp proteins show bimodal (at 25 degrees C) and trimodal (at 85 degrees C) distributions with similar patterns and similar values for average lifetimes. In the native state of the proteins the lifetime distributions were bimodal or trimodal. These results (multimodal distributions) are contradictory to the unimodal Lorentzian distribution of lifetimes reported for some proteins in the native and denatured states. MEM analysis gives a unimodal distribution of lifetimes only when the signal-to-noise ratio is poor in the time-resolved fluorescence decay data. The unimodal distribution model is therefore not realistic for proteins in the native and denatured states. The fluorescence decay components of the bi- or trimodal distribution are associated with the rotamer structures of the indole moiety when the protein is in the random coil state.     

Spectral characteristics of fluorophores formed via interaction between all-trans-retinal with rhodopsin and lipids in photoreceptor membrane of retina rod outer segments

It is shown that all-trans-retinal under model conditions of its excessive accumulation in photoreceptor membranes interacts with amino groups of rhodopsin and lipids, forming at least three distinct fluorophores with fluorescence quantum yield 20–40 times higher than that of free all-trans-retinal. These retinal derivatives are likely precursors of photo- and cytotoxic fluorophores of lipofuscin and in particular of A2E. Spectral characteristics of fluorophores have been described. Picosecond time-resolved laser fluorescence spectroscopy was used to study kinetics of fluorescence decay of both free and bound all-trans-retinal; fluorophores were determined and their lifetimes have been measured. Based on calculations it is shown that the decay kinetics of all-trans-retinal derivatives consists of three components with lifetimes equal to 48, 208, and 900 ps; kinetics of free all-trans-retinal is monoexponential with lifetime of 31 ps. The chemical nature of fluorophores with the lifetimes obtained is discussed.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Tryptophan photolysis is responsible for gramicidin-channel inactivation by ultraviolet light

The decay of gramicidin fluorescence resulting from ultraviolet exposure was compared to the decay of conductance from gramicidin-containing planar bilayer membranes under the same conditions of illumination. The decay rate was the same for both processes. The fluorescence decay was identical whether gramicidin was dissolved in methanol or incorporated into lipid vesicles, indicating that the peptide conformation does not affect the sensitivity of gramicidin to photolysis. The correlation of fluorescence decay and conductance decay imply that conductance loss from gramicidin-doped membranes illuminated with ultraviolet light is due to photochemical modifications of the channel tryptophans rather than simply to disturbance of the conformation of gramicidin channels.     

Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid.

A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37 degrees C, with 5 microM for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37 degrees C while being examined in an Epics Elite flow cytometer equipped with a 325 nm HeCd laser, and parinaric acid fluorescence at 405 nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant alpha-tocopherol, 200 microM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of the stretched exponential function to fluorescence lifetime imaging

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.     

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.     

Solvent dependent photocyclization and photophysics of some 2-ethynylbiphenyls.

The spectroscopic properties and photochemical behavior of molecules having 2-ethynylbiphenyl or 2-phenyldiphenylacetylene structures are reported. These molecules undergo photocyclization reactions to yield phenanthrene and dihydrophenanthrene products via putative isophenanthrene (cyclic allene) intermediates. Phenanthrene formation from the isophenanthrene intermediates does not occur via a unimolecular sigmatropic hydrogen shift, but rather by protonation or hydrogen abstraction mechanisms involving the solvent. Cyclization efficiencies are much lower than is the case for previously-investigated 2-vinylbiphenyl systems. The 2-phenyldiphenylacetylenes have high fluorescence quantum yields and long singlet lifetimes when compared to diphenylacetylene. The 2-ethynylbiphenyls decay via a combination of fluorescence and intersystem crossing.     

Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.

The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3-4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.     

Energy transfer and fluorescence mechanisms in photosynthetic membranes

Energy transfer in photosynthetic membranes involves the migration of excitons from light‐harvesting antenna chlorophyll‐protein complexes to the reaction center complexes. Recent efforts have focused on determining the time of arrival of excitons (trapping times) at the reaction centers following excitation with a single picosecond laser pulse. Three different approaches have been utilized: (1) determination of appearance of separated charges within the reaction centers by differential absorbtion spectroscopy, (2) determination of appearance of separated charges by fast photoemf measurements, and (3) kinetics of decay of fluorescence. The first two methods provide more direct information on exciton trapping by reaction centers than fluorescence methods, but are experimentally difficult to realize. Therefore, much activity has centered around the accurate measurement and analysis of fluorescence‐decay profiles by single‐photon counting methods. In green plants, about three different components with lifetimes of about 100 psec, 200 to 500 psec, and >1 nsec, have been reported. The first two components are believed to be related to trapping rates by reaction centers, while the third component is attributed to a charge recombination (Klimov) mechanism. Results from photoemf and exciton‐exciton annihilation experiments are consistent with the interpretation that the first decay component reflects exciton‐trapping rates. A critical analysis and discussion of these fast energy‐transfer phenomena in photosynthetic membranes of green plants are offered in this review.     

Temperature dependence of tryptophan fluorescence lifetime in aqueous glycerol and trehalose solutions

The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5–417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from –60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wave-length B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.     

Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.     

Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study.

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence of apoferritin and its subunits

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.     

Membrane dynamic alterations associated with viral transformation and reversion. Decay of fluorescence emission and anisotropy studies of 3T3 cells.

Fluorescence lifetimes, anisotropies and rotational correlation time values of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of normal, transformed, and revertant 3T3 cells were determined by nanosecond (nsec), photon counting spectrofluorimetry. No change in lifetime values with transformation or reversion is observed. Fluorescence anisotropy decay curves show at least two components; an initial relatively fast decay and a non-zero “plateau” level component. The observed changes in the average anisotropy values, which qualitatively follow steady-state fluorescence polarization values, is due primarily to changes in the non-zero “plateau” level component. The anisotropy decay curves suggest that the rotational motion of the probe is restricted to a limited angular range. The present results are compared with model membrane systems.