Origin of Tryptophan Fluorescence Lifetimes Part 1. Fluorescence Lifetimes Origin of Tryptophan Free in Solution

Fluorescence intensity decays of L-tryptophan free in polar, hydrophobic and mixture of polar-hydrophobic solvents were recorded along the emission spectrum (310–410 nm). Analysis of the data show that emission of tryptophan occurs with two lifetimes in 100 % polar and hydrophobic environments. The values of the two lifetimes are not the same in both environments while their populations (pre-exponentials values) are identical. Fluorescence lifetimes and pre-exponentials values do not change with the excitation wavelength and thus are independent of excitation energy. Our results indicate that tryptophan emission occurs from two specific sub-structures existing in the excited state. These sub-structures differ from those present in the ground states and characterize an internal property and/or organization of the tryptophan structure in the excited state. By sub-substructure, we mean here tryptophan backbone and its electronic cloud. In ethanol, three fluorescence lifetimes were measured; two lifetimes are very close to those observed in water (0.4–0.5 ns and 2–4 ns). Presence of a third lifetime for tryptophan in ethanol results from the interaction of both hydrophobic and hydrophilic dipoles or chemical functions of ethanol with the fluorophore.     

New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Origin of Fluorescence Lifetimes in Human Serum Albumin. Studies on Native and Denatured Protein

Human serum albumin consists of a single polypeptide of 585 amino acid residues with 1 Trp residue. In the present work, we measured fluorescence lifetimes of the protein in both native and denatured states. The results indicate that Trp emission occurs with three lifetimes in both states. Lifetimes values and contribution to the global emission decay differ between the two states. Data are interpreted as the results of an emission occurring from three substructures of the tryptophan formed in the excited state. Two of these substructures are already present for the tryptophan free in solution. The third lifetime is the result of the interaction between the tryptophan residue and surrounding microenvironment. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the fluorophore microenvironment and on the global protein structure.     

Fluorescence Origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) Bound to Proteins

6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.     

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme’s absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.     

Sub-Structures Formed in the Excited State are Responsible for Tryptophan Residues Fluorescence in β-Lactoglobulin

Origin of tryptophan residues fluorescence in β-lactoglobulin is analyzed. Fluorescence lifetimes and spectra of β-lactoglobulin solution are measured at pH going from 2 to 12 and in 6 M guanidine. Tryptophan residues emit with three lifetimes at all conditions. Two lifetimes (0.4–0.5 ns and 2–4 ns) are in the same range of those measured for tryptophan free in solution. Lifetimes in the denatured states are lower than those measured in the native state. Pre-exponential values are modified with the protein structure. Data are identical to those already obtained for other proteins. Fluorescence lifetimes characterize internal states of the tryptophan residues (Tryptophan sub-structures) independently of the tryptophan environments, the third lifetime results from the interaction that is occurring between the Trp residues and its environment. Pre-exponential values characterize substructures populations. In conclusion, tryptophan mission occurs from substates generated in the excited state. This is in good agreement with the theory we described in recent works.     

Effect of 1-Aminoanthracene (1-AMA) Binding on the Structure of Three Lipocalin Proteins,the Dimeric β Lactoglobulin,the Dimeric Odorant Binding Protein and the Monomeric α<Subscript>1</Subscript>-Acid Glycoprotein. Fluorescence Spectra and Lifetimes Studies

We studied effect of 1-aminoanthracene (1-AMA) binding on the structures of dimeric β lactoglobulin, dimeric odorant binding protein (OBP) and monomeric α₁-acid glycoprotein (lipocalin family proteins) by monitoring fluorescence excitation spectra and measuring fluorescence lifetimes of the tryptophan residues of the proteins. Results show that binding of 1-AMA to β lactoglobulin and OBP modifies their conformation even at low probe concentration compared to that of the proteins. Structural modification induces a red shift of the fluorescence excitation spectra maximum of tryptophan residues accompanied with an increase of the third fluorescence lifetime and a decrease of its pre-exponential factor. These effects were not observed for α₁-acid glycoprotein, probably as the result of carbohydrate presence. These data raise doubts concerning use of 1-AMA as a probe to study biological properties of β lactoglobulin and OBP.     

Nonlinear Excitation of Tryptophan Emission Enhanced by Silver Nanoparticles

We measured and analyzed the behavior of the fluorescence of tryptophan water solutions with and without silver nanoparticles, excited by one, two and three photon processes. Two different colloids with silver nanoparticles with distinct diameters (0.65 nm and 9 nm) were used in the experiments. Fluorescence quenching was observed with one and two photon excitation. However, upon three-photon excitation, significant fluorescence enhancement was observed in the colloid. In this case excitation of the amino acid is assisted by the nonlinear absorption of infrared light by the silver nanoparticles. In this paper we are proposing a new way to explore metallic nanoparticles to enhance autofluorescence of biomolecules.     

Fluorescence Lifetimes of Tryptophan: Structural Origin and Relation with So → 1Lb and So → 1La Transitions

We measured fluorescence lifetimes of L-Tryptophan dissolved in de-ionized water and in ethanol in the absence and the presence of high progesterone concentrations. The hormone absorbs between 220 and 280 with a peak around 250 nm, while its absorption is equal to zero beyond 280 nm. Tryptophan excitation spectrum recorded in presence of progesterone shows that the S_o → ¹L_a transition is completely abolished while the S_o → ¹L_b transition is not affected. Emission of L-tryptophan in water occurs with two fluorescence lifetimes, 0.40 and 2.8 ns. In ethanol, three fluorescence lifetimes equal to around 0.2, 1.8 and 4.8 ns were observed. Addition of progesterone to the medium does not affect any of the fluorescence lifetimes indicating clearly that both transitions could induce tryptophan excitation and that recorded fluorescence lifetimes could be assigned to sub-structures generated in the excited state.     

Dipolar Relaxation Times of Tryptophan and Tyrosine in Glycerol and in Proteins: A Direct Evaluation from Their Fluorescence Decays

The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, as well as in several single tryptophan-containing proteins (staphylococcal nuclease, ribonuclease T1, melittin and albumin), has been studied by dynamic fluorescence measurements. A new theoretical model has been developed, including the relaxation dynamics directly in the fluorescence decay function. The phase shift and demodulation data have been fitted with this new algorithm which allows to resolve the different relaxation times influencing the fluorophore excited state. These parameters are in a good agreement with those measured with the traditional time-resolved emission spectroscopy. The results indicate that indeed a correlation exists between the radiative rate change obtained with the new model and the temporal spectral shift reported in the literature. Finally, this new approach has also been extended to the case of superoxide dismutase and phosphofructokinase, allowing to measure the relaxation time even in proteins lacking a temporal spectral shift during the fluorphore's lifetime.     

Fluorescence decay time measurements in tetracene crystals excited with synchrotron radiation

Fluorescence decay times from tetracene single crystals excited at room temperature with synchrotron radiation have been recorded as a function of the excitation wavelength (in the 400–500 nm range). A non-exponential decay with two decay rates is observed. The analysis of our data shows that the first singlet exciton level of tetracene (single crystal) decays radiatively mainly through, as we call it, channel 1, with a lifetime of 0.200 ± 0.020 ns. About 10% of the emitted fluorescence transits through channel 2 with a lifetime of 1.7 ± 0.2 ns. These results do not agree with previously published decay data obtained when tetracene is excited by means of powerful lasers. Thus there is experimental evidence to believe that the decay properties of condensed materials can be very dependent on the excitation density. Because synchrotron radiation compared to lasers is a very weak source, and therefore secondary effects are minimized in our experimental conditions, the decay values reported in the present work are the true lifetimes of the tetracene single crystal.     

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6 nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.     

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.     

Red-Edge Excitation Study of Nonexponential Fluorescence Decay of Indole in Solution and in a Protein

Proteins are known to be heterogeneous systems with a hierarchy of internal motions. However, those properties are often ignored when the complex fluorescence decay of tryptophan residues is compared to model studies with indole derivatives in solution. Here two simple models are presented, which illustrate different aspects of protein organization: (1) Trp zwitterion in buffer exemplifies ground-state heterogeneity and (2) indole in water/glycerol mixture exemplifies excited-state reconfiguration of solvate. Both systems are known to produce nonexponential fluorescence decay, attributed to the existence of multiple species (rotamers) or to the effects of slow dipolar relaxation, for (1) and (2), respectively. In the latter case a substantial dependence of decay on the excitation wavelength is expected. Indeed such dependence is observed for indole in water/glycerol mixture but not for Trp zwitterion in buffer. Therefore, excitational dependence can be used as a criterion to distinguish effects of multiple conformations in the ground state from effects of excited state reactions on tryptophan decays in proteins. The example of the bee venom peptide melittin indicates that both phenomena are important for interpretation of heterogeneity of decay, and therefore, caution should be exercised when assigning individual decay components to conformational subspecies in proteins.     

Quenchers Induce Wavelength Dependence on Protein Fluorescence Lifetimes

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein’s aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher’s extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.     

Reduced Lifetimes are Directly Correlated with Excitation Irradiance in Metal-Enhanced Fluorescence (MEF)

We describe a fundamental observation in Metal-Enhanced Fluorescence (MEF), which has become a leading technology in the life sciences today, namely, how the lifetime of fluorophores near-to metallic plasmon-supporting silver islands/nanoparticles, modulates as a function of excitation power irradiance. This finding is in stark contrast to that observed in classical far-field fluorescence spectroscopy, where excitation power does not influence fluorophore radiative decay/lifetime.     

Uranyl Soil Extraction and Fluorescence Enhancement by Nanoporous Silica Gel: pH effects

Nanoporous silica gel was employed to extract uranyl from contaminated soil and to enhance the fluorescence intensity and lifetime. The fluorescence lifetime and intensity of uranyl ions absorbed within nanoporous silica gel was measured from pH 1?C13. The results show that the uranyl fluorescence intensity can be enhanced by approximately two orders of magnitude by the silica nanoporous matrix from pH 4?C12 with the greatest enhancement occurring from pH 4?C7. The enhanced fluorescence lifetime can be used in time-gated measurements to help minimize the influence of background environmental fluorophores.     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Rhodamine B,Coumarin 6 and Lucifer Yellow

Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine which wavelengths we should use for the excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10^?2– 10^?6 M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-photon excitation and obtaining lifetime data in the time-domain using time-correlated single-photon counting.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

Fluorescence Dynamics of Three UV-B Sunscreens

Polarity of the surrounding medium affects the excited states of UV-B sunscreens. Therefore understanding excited state processes in a mixed polarity model system similar to skin is essential. We report the excited state lifetimes, quantum yields, radiative and non-radiative rates of three sunscreens. Among the three UV-B sunscreens studied, octyl salicylate emits from a single excited state, while padimate O and octyl methoxy cinnamate show multiple states. The radiative rates of salicylate and cinnamate are approximately constant, while that of padimate O depends strongly on solvent. The non-radiative rates of all sunscreens vary with solvent polarity. Compared to salicylate and cinnamate, padimate O is complex to analyze because of its two emission peaks and one peak’s strong dependence on the dielectric constant. High absorbance, broad absorption peak with small fluorescence quantum yield, and low radiative rate make octyl methoxy cinnamate a superior UV-B sunscreen ingredient. The complexity in excited-state analysis shows that the lifetimes of the sunscreens are critical parameters, in addition to absorbance and quantum yield. Fluorescence lifetime substantiates the use of polystyrene nanospheres as a model host to study the photo-physical properties of sunscreen in a heterogeneous environment.