TRYPTOPHAN FLUORESCENCE LIFETIMES AS A FUNCTION OF EXCITATION WAVELENGTH*

Abstract— –Fluorescence decay times of aqueous dilute solutions (?20 µM) of L-tryptophan have been determined using the phase shift technique as well as single photon-counting coupled with synchrotron radiation (ACO at Orsay and SPEAR at Stanford). Decay times were obtained as a function of the excitation wavelength (in the spectral region 220–320 nm) monitoring emission of λ> 320 nm (in certain specified cases, λ> 360 nm). We have found that, at neutral pH and 20°C. fluorescence decays are single exponentials and independent of the excitation wavelength; under these conditions we find τ= 3.1 ± 0.1 ns.     

FLUORESCENCE OF MELANIN-DEPENDENCE UPON EXCITATION WAVELENGTH AND CONCENTRATION

Abstract— An introduction to the fundamental characteristics of synthetic melanin fluorescence is presented. The particular difficulties associated with the detection and reduction of the relatively weak signal are discussed and a technique is described for correcting the fluorescence spectra for attenuation of the excitation and emission beams. Spectra are reported for the excitation wavelength range 340–400 nm and an emission range of 360–560 nm. The concentration dependence of the corrected fluorescence signal is examined and is shown to be linear. The variation of the fluorescence spectra with excitation wavelength suggests a two-component fluorescence, for the wavelength range studied. The presence of an isosbestic point in the spectra is used to identify the fluorophores as components of a reaction equilibrium. The possible relationship of this equilibrium to that associated with the melanin photo ESR is discussed     

DISTANCE-DEPENDENT FLUORESCENCE QUENCHING OF TRYPTOPHAN BY ACRYLAMIDE

Abstract We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N -acetyl -L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20°C. 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. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NAT A by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.     

FLUORESCENCE OF TYROSINE AND TRYPTOPHAN IN PROTEINS USING ONE- AND TWO-PHOTON EXCITATION

Abstract— We examined the emission spectra of tyrosine- and tryptophan-containing proteins using one-photon (270–310 nm) and two-photon (565–610 nm) excitation. Emission spectra for two-photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one-photon excitation below 290 nm. We examined the one-photon and two-photon excitation spectra of tyrosine-tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short-wavelength shift of the tyrosine two-photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett . 208 , 276–282.     

TUMORIGENESIS BY A LONG WAVELENGTH UV-A SOURCE

Albino hairless mice (Skh-hr1) were exposed daily to radiation from a high-power long wavelength UV-A source (wavelengths longer than 340 nm). The irradiations lasted 2 h per day. The daily dose was 220 kJ/m2. Heavy scratching marks were observed in 13 out of 48 animals. However during the experiment 31 of the animals developed tumors of 1 mm or larger before any scratching was observed. The median induction time was 265 days for 1 mm tumors.     

QUENCHING OF PYRENE FLUORESCENCE BY TRYPTOPHAN IN MICELLAR SOLUTIONS

Deactivation of excited pyrene incorporated to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecyl sulfate) and neutral (Triton X-100) micelles by tryptophan has been investigated over a wide pH range. Data obtained allow an estimation of the tryptophan association to the micelles, of the tryptophan apparent p K in the micellar solutions, and of the dynamics of tryptophan incorporation to the micellar pseudophase. In particular, the data obtained at pH 7 allow an estimation of the effect of the micellar charge upon the binding capacity of the tryptophan zwitterion.     

FLUORESCENCE OF TRYPTOPHAN IN KERATIN

The excitation and emission spectra have been determined for the fluorescence from trypto-phan residues in dry keratin. The fluorescence decay was also measured and shown to be a single exponential with a rather long lifetime of 6.9 ns. It is suggested that the emission takes place from a state formed by interaction between the ¹L_a state of the tryptophan residues and neighbouring polar or polarizable groups in the protein. The fluorescence excitation spectrum displays a peak at 290 nm, and its appearance at this position rather than at 280 nm, which is the absorption maximum of tryptophan, is believed to arise from inner filtering by the tyrosine residues in keratin.     

INFLUENCE OF THE ENVIRONMENT ON THE EXCITATION WAVELENGTH DEPENDENCE OF THE FLUORESCENCE QUANTUM YIELD OF INDOLE

Abstract— The influence of excitation wavelength, pH, oxygen and solvents, upon fluorescence quantum yields, were measured for insole Indole in neutral aqueous solution exhibits the same wavelength dependence as tryptophan, which indicates that COO- absorption is not responsible for the effect. Parameters such as pH and oxygen influence only the absolute fluorescence quantum yields but not their relative variation with wavelength, indicating competition with fluorescence emission for S1 deactivation. without any influence upon deactivation of higher excited states. In contrast, solvents exhibit a specific influence upon the wavelength dependence; for indole, the decrease of fluorescence yield excited around 215 nm, compared with the yield in the first absorption band is about 40% in water, 10% in acetonitrile, 70% in n-hexane and cyclohexane, whereas no appreciable decrease occurs in methanol, ethanol or n-butanol. These observations, together with the Stokes shifts of the emission spectra, may be well correlated with Kosower's Z-values, expressing microscopic solvent-solute interactions.     

DIFFERENT CHROMATOGRAPHIC BEHAVIOR OF A THYMINE-CONTAINING NEW ULTRAVIOLET PHOTOPRODUCT AND SPORE PHOTOPRODUCT

Abstract— One- and two-dimensional cochromatography in five different solvents of a near UV photo-product induced by 365 nm irradiation of DNA in aqueous solution and the 'spore photoproduct', 5-thyminyl-5,6-dihydrothymine, produced by 254 nm irradiation of dry DNA, indicate that the two photoproducts seem to have related structures but are different.     

A COMPARISON OF FLUORESCENCE QUENCHING AND FLUORESCENCE DECAY STUDIES WITH TRYPTOPHAN

Abstract— The quenching of the fluorescence of aqueous tryptophan solutions has been studied as a function of emission wavelength using acrylamide as a collisional quencher. Our quenching studies are consistent with recent observations of the heterogeneity of tryptophan fluorescence, but they show a slight discrepancy when compared to certain analyses of the decay of tryptophan fluorescence in terms of two components.     

NEAR-UV-INDUCED BREAKS IN PHAGE DNA: SENSITIZATION BY HYDROGEN PEROXIDE (A TRYPTOPHAN PHOTOPRODUCT)

Abstract— Near-UV irradiation of l -tryptophan yields a large number of photoproducts. When this mixture is added to recombinationless ( rec ) mutants of bacteria, the cells are killed. The most toxic component of tryptophan photoproducts has been identified as hydrogen peroxide (H₂O₂). We now report that both tryptophan photoproducts and H₂O₂ sensitize phage DNA to near-UV radiation resulting in enhanced killing as well as enhanced DNA breakages. We conclude that the in situ production of H₂O₂ via tryptophan photolysis may be an important biological event.     

DIRECT RECORDING OF THE INITIALLY EXCITED AND THE SOLVENT RELAXED FLUORESCENCE EMISSION SPECTRA OF TRYPTOPHAN BY PHASE SENSITIVE DETECTION OF FLUORESCENCE

Abstract Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent-relaxed emission spectra of N-acetyl-L-tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation.
The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.     

FLUORESCENCE OF BACTERIORHODOPSIN UNDER SUBPICOSECOND LIGHT EXCITATION

Abstract— A streak camera detection technique has been used to record the fluorescence of bacteriorhodopsin at room temperature induced by single subpicosecond light pulses. The fluorescence lifetime of bacteriorhodopsin has been found to be less than 2 ps.     

ORIENTATION OF SHORT WAVELENGTH AND LONG WAVELENGTH PROTOCHLOROPHYLL SPECIES IN GREENING CHLOROPLASTS

Abstract— The orientation of protochlorophyll and Chi species with respect to the plane of thylakoid membranes was studied by measuring the fluorescence polarization ratio in magnetically oriented chloroplasts isolated from greening maize leaves and cucumber cotyledons. With viewing direction parallel to the plane of the photosynthetic membranes, in the spectral region of 620–660 nm, fluorescence polarization ratios of 1.0 were observed, whereas at longer wavelengths the fluorescence polarization ratios were much higher, and similar to that of fully green chloroplasts. The same result was obtained with chloroplasts isolated from leaves fed by δ-amino levulinic acid. These data indicate that the emitting oscillators of the short and long wavelength protochlorophyll species are oriented at random with respect to the plane of thylakoid membranes. Isotropy of the protochlorophyll species is discussed in terms of isotropic structures containing Chi precursors.     

THE FLUORESCENCE EXCITATION SPECTRUM OF QUININE BISULFATE

Abstract— A new measurement of the fluorescence excitation spectrum of chromatographically homogeneous quinine bisulfate has been made using a spectrometer offering high resolution and high sensitivity. Comparison of this spectrum with the absorption spectrum of the compound indicates that its quantum yield is independent of the wave number of the exciting light within ± 5 per cent for 2.57 μ^-l v 5.0μ^-1.     

THE PHOTOCHEMISTRY OF SPECIFIC TRYPTOPHAN RESIDUES IN PROTEINS AS ANALYZED BY THE FLUORESCENT SCANNING OF TRYPTIC PEPTIDE MAPS

Abstract— The fact that most proteins contain several tryptophans hinders the investigation of the photochemistry of a particular indole residue. A method is presented here that can be used to investigate the photochemistry of specific tryptophan residues in proteins. It consists simply of separating the peptides of a proteolytically digested protein by TLC and then scanning the peptides at the fluorescent maximum of tryptophan. The assignment of the resultant peaks to a particular peptide is based on the chromatographic comparison of the scans with peptide maps.
Using this method, the photochemistry of the tryptophan residues in alpha crystallin, a major protein of lenses, was investigated. Under photolytic conditions that mimic the transmission characteristics of the cornea (>293 nm), it was found that there is a differential photolysis of the tryptophan residues in the protein; with Trp-9 in the N-terminal peptide photolyzing at a considerably faster rate than Trp-60. In addition to tryptophan, photolytic losses of tyrosine were assessed by scanning the peptide maps at the tyrosine fluorescent maximum. Only one tyrosine residue photolyzes under these conditions. The differential photolysis of the tryptophan residues is explained in part by the presence of residues in the vicinity of the indole moieties.     

AN EVALUATION OF CHARGE EFFECTS ON THE QUENCHING OF TRYPTOPHAN FLUORESCENCE IN SMALL PEPTIDES BY IODIDE ION

Abstract— Charge effects on the quenching of tryptophan fluorescence in small peptides by iodide ion have been analyzed by the conventional "static" quenching model and by a recently proposed competitive quenching model. The former involves a fit of the quenching data using two quenching parameters—one for dynamic and one for static quenching contributions. The latter model involves a single parameter fit in which the fitting parameter is the characteristic rate constant for quenching of the fluorescent state. Both models indicate a clear charge effect on the efficiency of quenching by iodide ion. However, the static model results are obscured by the interdependence of the two fitting parameters and the fact that the true physical meaning of the static parameter is uncertain. Rate constants derived from the competitive model can be converted into relative quenching efficiencies. These efficiencies, which vary by more than a factor of two for the molecules studied, are greatest when the positive charge is on the tryptophan and least when this residue contains a negative charge.     

TIME-RESOLVED FLUORESCENCE SPECTRA OF TRYPTOPHAN IN MONOMERIC GLUCAGON*

Abstract— The fluorescence decay of tryptophan-25 in monomeric glucagon at pH 8.2 was measured at a series of emission wavelengths using pulsed laser excitation and single photon counting techniques. Double exponential kinetics were consistently observed, with time constants 3.26 and 1.11 ns. This allowed the steady-state emission spectrum to be resolved into two components with differing intensities but similar emission maxima near 350 nm. Decay parameters were almost unchanged in the presence of 5.5 M guanidinium chloride. The dual emission is thought to reflect different conformers of the indole ring or of the peptide chain.     

CALCIUM-DEPENDENT FLUORESCENCE LIFETIMES OF INDO-1 FOR ONE- AND TWO-PHOTON EXCITATION OF FLUORESCENCE

Abstract— We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca²⁺. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in thc multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 × 10⁻⁴⁹ cm⁴ s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.     

LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.