Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

Oxygen fluorescence quenching studies with single tryptophan-containing proteins

The work of Lakowicz and Weber [Biochemistry 12, 4161 (1973)] demonstrated that molecular oxygen is a powerful quencher of tryptophan fluorescence in proteins. Here we report studies of the oxygen quenching of several proteins that have a single, internal tryptophan residue. Among these are apoazurin (Pseudomonas aeruginosa), asparaginase (Escherichia coli), ribonuclease T₁ (Aspergillus oryzae), and cod parvalbumin. Both fluorescence intensity and phase lifetime quenching data are reported. By comparison of these data we find that there is a significant degree of apparent static quenching in these proteins. The dynamic quenching rate constants,k _q, that we find are low compared to those for tryptophan residues in other proteins. For example, for apoazurin we find an apparentk _q of 0.59×10⁹ M ^–1 s^–1 at 25°C. This value is the lowest that has been reported for the oxygen quenching of tryptophan fluorescence.     

Distance-dependent fluorescence quenching ofN-acetyl-l-tryptophanamide by acrylamide

We examined the time-dependent intensity decays ofN-acetyl-l-tryptophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol over a range of temperatures. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature, and quenching of NATA by acrylamide was observed even in vitrified propylene glycol, where translational diffusion cannot occur during the lifetime of the excited state. These frequencydomain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide, and the results are consistent with a rate constant for quenching that depends exponentially on the fluorophore-quencher separation distance. The exponential distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, and the steady-state data aid in determining the interaction distance between NATA and acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of acrylamide quenching of proteins.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.     

Perturbation of Conformational Dynamics of ASCUT-1 from Ascaris lumbricoides by Temperature and Sodium Dodecyl Sulfate

ASCUT-1 is a protein found in cuticlin, the insoluble residue of the cuticles of the nematode Ascaris lumbricoides. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The monomeric form of ASCUT-1 contains a single tryptophan residue. An understanding of the structure-function relationship of the protein under different chemical-physical conditions is of fundamental importance for an understanding of its structure and function in cuticles. In this paper we report the effect of the temperature and sodium dodecyl sulfate on the structural stability of this protein. The structure of the protein was studied in the temperature range 25–85°C in the absence and in the presence of sodium dodecyl sulfate by frequency-domain measurements of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that the tryptophanyl emission decays were well described by a bimodal lifetime distribution, and that the temperature increases resulted in the sharpening and in the shortening of the tryptophanyl lifetime distribution. In the presence of SDS an unimodal fluorescence lifetime distribution as well as a marked decrease in the anisotropy decay values were observed.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Study on the interaction between theasinesin and human serum albumin by fluorescence spectroscopy

The binding properties on theasinesin to human serum albumin (HSA) have been studied for the first time using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results showed that theasinesin strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and non-radiation energy transfer happened within molecules. The number of binding site was 1, and the efficiency of Förster energy transfer provided a distance of 4.64 nm between tryptophan and theasinesin binding site. At 298, 310 and 323 K, the quenching constants of HSA–theasinesin system were 2.55×10³, 2.16×10³ and 1.75×10³ mol L⁻¹. ΔH^θ, ΔS^θ and ΔG^θ were obtained based on the quenching constants and thermodynamic theory (ΔH^θ<0, ΔS^θ>0 and ΔG^θ<0). These results indicated that hydrophobic and electrostatic interactions are the mainly binding forces in the theasinesin–HSA system. In addition, the results obtained from synchronous fluorescence spectra showed that the binding of theasinesin with HSA could induce conformational changes in HSA.     

Determination of the Constants of Binding of Acridine Dyes with Micelles of Sodium Dodecyl Sulfate Using the Quenching of Delayed Fluorescence by Heavy Atoms

The constants of binding dye molecules with the micelles of sodium dodecyl sulfate are determined using quenching of delayed fluorescence of acridine dyes by sodium iodide in aqueous–micellar solutions. Kinetic equations have been composed that describe the processes of deactivation of the excited states of dyes. By solving these equations at the concentration of the quencher sodium iodide corresponding to the minimum lifetime of triplet states and at the concentration of micelles corresponding to the least value of the delayed fluorescence quenching rate constants, we obtained the constants of binding dyes with micelles equal to 1.3·10⁷, 2.9·10⁷, and 3.1·10⁷ M^–1 for trypaflavine, acridine orange, and acridine yellow, respectively. We calculated the rate constants of quenching of the triplet states of the molecules of dyes by iodide ions (I ^–) that decreased in transition from trypaflavine to acridine orange and acridine yellow.     

The tyrosyl fluorescence of angiotensin II in alcoholic solvents

Angiotensin II is an octapeptide hormone and contains a single tyrosyl residue and no tryptophyl residues. Intramolecular interactions of the tyrosyl residue with, for example, ionizable side chains or hydrogen bond acceptors can potentially perturb its fluorescence properties. The intrinsic fluorescence of angiotensin II was used to determine if the interactions of the tyrosyl residue were altered, as a consequence of conformational changes induced by certain alcoholic solvents. Steady state and time-resolved fluorescence data for angiotensin II in neutral aqueous buffer, isopropanol and 1,2=propanediol, provided no evidence for specific conformations, which facilitated intramolecular association of the tyrosyl residue with other moieties. Multiexponential decay kinetics in which the decay times were <5 ns were observed in all cases. No fluorescence which could be attributed to tyrosinate anion was detected in the solvent systems studied.Issued as NRCC publication No. 34266.     

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Nitrogen atom detection in low-pressure flames by two-photon laser-excited fluorescence

Nitrogen atoms have been detected in stoichiometric flat premixed H₂/O₂/N₂ flames at 33 and 96 mbar doped with small amounts of NH₃, HCN, and (CN)₂ using two-photon laser excitation at 211 nm and fluorescence detection around 870 nm. The shape of the fluorescence intensity profiles versus height above the burner surface is markedly different for the different additives. Using measured quenching rate coefficients and calibrating with the aid of known N-atom concentrations in a discharge flow reactor, peak N-atom concentrations in these flames are estimated to be on the order of 10¹²–5×10¹³ cm^–3; the detection limit is about 1×10¹¹ cm^–3.     

Aggregation ofNaja nigricollis cardiotoxin: Characterization and quantitative estimate by time-resolved polarized fluorescence

After purification to homogeneity by Bio-Rex 70 ion exchange chromatography, micromolar solutions ofNaja nigricollis cardiotoxin were found to contain significant amounts of aggregates, as detected by time-resolved polarized fluorescence of its single tryptophan residue. The level of cardiotoxin aggregation depends strongly and reversibly on the protein concentration and pH. However, supplementary reverse-phase HPLC completely suppresses this aggregation, resulting in all cases in fluorescence anisotropy decays characteristic of the pure cardiotoxin monomer. The self-association properties of cardiotoxin, in the presence of a possible cofactor eliminated by the HPLC step, may be functionally relevant, and would deserve further investigation. The physical heterogeneity of the cardiotoxin samples required an appropriate model for the analysis of fluorescence depolarization, which was iteratively improved by comparison with experimental results. In this way, an approximate molar fraction of 10–15% aggregated cardiotoxin at a 90M total protein concentration, pH 7, was determined. The fluorescence of the partly aggregated samples is significantly perturbed as compared to the HPLC-treated monomer, indicating that the cardiotoxin aggregate must have an increased average fluorescence lifetime and a strongly decreased initial anisotropy. The decrease in initial anisotropy suggests either an increased mobility of the tryptophan residue upon aggregation or fast energy transfers between residues of different cardiotoxin molecules brought within a short distance in the aggregate. This study illustrates the high sensitivity of the time-resolved fluorescence technique, through both total fluorescence and anisotropy parameters, to low levels of physical or chemical heterogeneity in a protein sample.     

Near-field time-resolved fluorescence studies of poly(phenylmethyl silane) with subwavelength resolution

We present an example of the first time-correlated single-photon counting (TCSPC) near-field optical measurement. The aperture size of our prepared aluminun-coated fiber-optic probe was approximately 50 nm, which represents a spatial resolution of _ex/7 for our UV measurements. Near-field fluorescence decays of poly(phenylmethyl silane) in solid thin films excited in the range 325–360 nm were obtained and the steady-state excitation spectra compared with the excitation spectral information obtained in the far field. Fluorescence decays showed single exponential lifetimes ranging from 45 to 277 ps, which was dependent on the excitation wavelength and the selected near-field tip. The proximity of the metal-coated tip to the sample may be the reason for the modulation in fluorescence lifetime.     

A study on the fluorescence quenching of 9-Aminoacridine by certain antioxidants

The fluorescence quenching of 9-Aminoacridine by certain estrogens and flavonoids in water was studied using absorption, steady state and time-resolved measurements. The bimolecular quenching rate constants for the chosen estrogens and flavonoids were found to be in the range of 3.2-9.2×10¹¹ and 0.36-14.46×10¹¹ M⁻¹s⁻¹, respectively. From lifetime measurement we observed that the quenching was mainly due to static mechanism through ground state complex formation. The binding constant (K) and the number of binding sites (n) were calculated based on the fluorescence quenching data. The free energy change (ΔG_et) for electron transfer process was calculated by Rehm-Weller equation.     

Role of the Carbohydrate Moiety and of α-L-Fucose in the Stabilization and the Dynamics of the Lens culinaris Agglutinin–Glycoprotein Complex. A Fluorescence Study

Interaction between the fluorescent Lens culinaris agglutinin–fluorescein complex (LCA-FITC) and two glycoproteins, lactotransferrin (LTF) and serotransferrin (STF), was studied. The two glycoproteins have the same glycan structures, with one difference: the lactotransferrin glycans contain a fucose residue -1,6-linked to the N-acetylglucosamine residue involved in the N-glycosylamine linkage. Fluorescence intensity quenching of the LCA-FITC complex shows that affinity between LCA and lactotransferrin is 50 times higher than that between LCA and serotransferrin, the fucose playing a major role in this high affinity (K _a is equal to 9.66 and 0.188 M ^–1 for the LCA–LTF complex and LCA–STF complex, respectively). Time-resolved anisotropy decay indicates that the rotational correlation time of LCA (20 ns) does not change to a large extent whether the glycoproteins are bound to LCA or not. This suggests that there is no extended physical contact between LCA and the glycoproteins. The interaction between LCA and the glycoproteins occurs likely only via the carbohydrate chains, the STF and the LTF rotating almost-freely in the vicinity of LCA, with the glycans as an anchor.     

Characterization of ultra-weak fluorescence using picosecond non-collinear optical parametric amplifier

We report a technique for characterization of ultra-weak fluorescence based on a 355-nm pumped picosecond non-collinear optical parametric amplifier (OPA). In the experiment, we effectively reduced the strong super-fluorescence background by using a series of methods. With the picosecond OPA as the pre-amplifier and the gating pulse, the decay of the fluorescence of Rhodamine 6G dye in ethanol was measured and the fluorescence lifetime was found to be about 941 ps. The gain factor of this parametric fluorescence amplifier was measured to be ∼4.2 × 10⁶, while the energy detection limit was ∼160 aJ per pulse within a 15-ps gating pulse.     

On the mechanism of rhodamine 6G fluorescence quenching

The fluorescence of the widely used TPF dye rhodamine 6G is quenched by photons of the ruby as well as the Nd-glass laser. Taking into consideration the polarisation of the ground- and excited-state absorptions of rhodamine 6G a simple model of quenching results. The main process is an S₁ absorption with subsequent leave of the ordinary rhodamine 6G singlet system. For Nd-laser photons the cross section of the concerned transition following from our quenching experiments is σS₁→S₂ = 1.2×10^-16 cm².     

Pump–probe analysis of polaron decay in InAs/GaAs self-assembled quantum dots

We report on polaron decay in InAs/GaAs self-assembled quantum dots. The polarons are probed by pump–probe spectroscopy through their optical intersublevel absorption around 62 meV (20 μm wavelength). A T₁ polaron lifetime of the order of tens of picosecond is deduced from the low-temperature pump–probe measurements. We show that a long-lived component can be additionally observed on the pump–probe measurements. The spectral dependence of this long-lived component is, however, not correlated to the polaron absorption. It is thus not a signature of polaron relaxation quenching. The origin of this long-lived component is attributed to the two-phonon absorption of the bulk GaAs substrate.     

Distributions of fluorescence decay times for synthetic melittin in water-methanol mixtures and complexed with calmodulin,troponin C,and phospholipids

Frequency-domain measurements of the intensity decays of melittin were used to recover the distribution of decay times displayed by its single tryptophan residue. Melittin was examined in the monomeric random coil state (water), in the monomeric -helical state (water-methanol), in the tetrameric state, and with 6M guanidine hydrochloride. In the presence of denaturant, where melittin is expected to be devoid of secondary structure, we observed a narrow distribution of lifetimes, similar to a double-exponential decay. In water the intensity decay of melittin was found to be described better by the distribution of decay times, which became progressively wider as the amount of -helix was increased by the methanol cosolvent or upon formation of the -helical tetrameric state. We also examined the intensity decays of melittin when complexed with calmodulin, troponin C, or lipid vesicles of 1-palmitoyl-2-oleyl-l--phosphatidylcholine (POPC). The lifetime distributions of the complexes with lipid were comparable to those observed in methanol-water, suggesting a similarity of the structure and/or dynamics of the environment surrounding the tryptophan residue. A broad lifetime distribution was observed for the melittin-calmodulin complex, suggesting a rigid structure and/or heterogeneity in the form of the complex. The lifetime distribution of the melittin-troponin C complex was more narrow, suggesting a more uniform structure, at least in the region surrounding the tryptophan residue. These results demonstrate that the lifetime distributions of a single tryptophan protein can be a sensitive indicator of the conformational heterogeneity and dynamics of proteins.