Fluorescence of tryptophan

Toptygin D, Gronenbom AM, Brand L (2006) Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan. J Phys Chem B 110(51) 26292-26302... 

C. M. L. Hutnik, J. P. MacManus, D. Banville, and A. G. Szabo, Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102, J. Biol. Chem. 265, 11456-11464 (1990). 

Quenching of fluorescence of tryptophan residues, coenzyme fluoro-phores, or extrinsic probes buried in the interior of proteins by colli-sional quencher molecules diffusing through the protein matrix/7,25 27)... 

Fluorescence quenching has proven to be a powerful means to determine location of tryptophans. Small organic molecules, such as acetone, acrylamide, and amino acids, have been used to quench fluorescence of tryptophans which are exposed to the solvent.(50 51) These molecules apparently quench by close interaction and so provide a tool to determine the surface accessibility7 of tryptophan in a protein. 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

Unnatural amino acids can possess such diverse structures that there are a number of them that may be used for purposes previously unidentified. For instance, nitrophenylalanine (16), originally used as a distance probe to quench the fluorescence of tryptophan, has recently been found to stimulate potent immune responses for novel immunogenic applications. 

Maximum fluorescence of tryptophan occurs at lower wavelengths (<350 nm) and has greater intensity the lower the polarity of its environment. 

Maity H, Maiti NC, Jarori GK. Time-resolved fluorescence of tryptophans in yeast hexokinase-PI effect of subunit dimerization and ligand binding. Journal of Photochemistry and Photobiology B 2000, 55, 20-26. 

Figure 6. Time and wavelength resolved fluorescence of tryptophan (3 X in water at 5°C excited by a single 263 nm picosecond pulse. Wavelength range is from 310 nm in the lower track to 430 nm in the upper track. Full scale on the time axis is approximately 10 nsec. The abscissa is not exactly linear with time...

Rosbash, D.O. and Leavitt, D., Decalcification of bone with trifluoroacetic acid, Am. J. Clin. Pathol. 22, 914-915, 1952 Katz, J.J., Anhydrous trifluoroacetic acid as a solvent for proteins, Nature 174, 509, 1954 Uphaus, R.A., Grossweiner, L.I., Katz, J.J., and Kopple, K.D., Fluorescence of tryptophan derivatives in trifluoroacetic acid, Science 129, 641-643, 1959 Acharya, A.S., di Donato, A., Manjula, B.N. et al., Influence of trifluoroacetic acid on retention times of histidine-containing tryptic... 

Permyakov and Burstein (1977) measured the steady-state fluorescence of tryptophan in several proteins as a function of hydration. They suggested that hydration increases the flexibility of the protein. 

This chapter is devoted to describe the impact of metallic nanosphere to the multi-photon excitation fluorescence of Tryptophan, and little further consideration to multi-photon absorption process will be given, as the reader can find several studies in [11-14]. In section II, the nonlinear light-matter interaction in composite materials is discussed through the mechanism of nonlinear susceptibilities. In section III, experimental results of fluorescence induced by multi-photon absorption in Tryptophan are reported and analyzed. Section IV described the main results of this chapter, which is the effect of metallic nanoparticles on the fluorescent emission of the Tryptophan excited by a multi-photon process. Influence of nanoparticle concentration on the Tryptophan-silver colloids is observed and discussed based coi a nonlinear generalization of the Maxwell Garnett model, introduced in section II. The main conclusion of the chapter is given in secticHi IV. 

Total internal reflection fluorescence spectroscopy has been used to assay the fluorescence of tryptophan in proteins or of fluorescence markers. Morphine has been determined in this manner with a detection limit of 0.2 pmol/l on a quartz support bearing immobilized fluorescein-labeled antihapten (Kronick and Little, 1973). 

TIME-RESOLVED FLUORESCENCE OF TRYPTOPHAN AND ITS MOTION IN PROTEINS... 

Fluorescence of tryptophans in proteins also decayed with non-exponential functions, even though the proteins contained single tryptophan. The concept of rotamer was applied to explain the non-exponential decay profiles observed in proteins. 

This explanation for the origin of the non-exponential decays of tryptophan fluorescence, however, cannot rationalize the results of time-resolved fluorescence anisotropy in these proteins. Beec-hem and Brand review the time-resolved fluorescence of tryptophans in proteins (32). The following conclusions concerning time-resolved fluorescence can be derived i) fluorescence decays with at least two-lifetime components, when internal rotation of tryptophan is observed from the time-resolved fluorescence anisotropy, ii) fluorescence decays... 

Proteins which contain tryptophan fluoresce at about 350 nm when excited at 270-305 nm. The fluorescence of tyrosine at 305 nm is not visible in LDH this is normal for proteins which also contain tryptophan 223). The fluorescence of tryptophan residues in LDH is much higher in the native apoprotein than in the unfolded protein or its alkaline hydrolysate... 

Robbins et al. on tryptophan and 3-methylindole since powerful solid-state laser excitation was used. Jameson and Weber have resolved the fluorescence of tryptophan by phase and modulation fluorometry in terms of emission from the zwitterion and anion present in amounts determined by the pH of the solution. The forms interconvert more slowly than fluorescence processes and have similar absorption and emission spectra. Measurements were made with excitation frequencies of 6, 18, and 30 MHz in the pH range 8—10, in which the relative zwitterion concentration varies from 0.82—0.09. Resolved lifetimes were 3.1 0.4 ns for the zwitterion and 8.7 0.1 ns for the anion. The agreement with Gudgin et al. seems satisfactory. 

Silver.—In a 1 1 complex, Ag+ quenches the fluorescence of tryptophan and induces a three-fold increase in the phosphorescence quantum yield.13 This observation is attributed to an intramolecular heavy-atom effect in the complex. 

The intrinsic fluorescence of tryptophan also changes slightly with a variety... 

While it is possible to detect proteins based on the native fluorescence of tryptophan, that amino acid is the least abundant in yeast, representing 1.0% of the total amino acid content of yeast. Excitation of tryptophan requires expensive and temperamental UV lasers. Instead, it is usually more convenient to label the protein with a fluorophore that is excited by inexpensive and reliable lasers that operate in the visible portion of the spectmm. 

Proteins in foods such as milk have been the subject of much fluorimetric study involving measurement of the intrinsic fluorescence of tryptophan and tyrosine however, since proteins vary in their content of these amino acids, the fluorescence intensity also varies markedly from protein to protein and, for a single protein, with the experimental conditions. Also, the natural fluorescence of peptides is limited to peptide-containing tryptophans and tyrosines. However, the joint use of LC and derivatizing fluorogenic agents such as dansyl chloride, ninhydrin, fluorescamine, and o-phthaldialdehyde (OPA) has allowed the development of a number of methods for the determination of proteins, peptides, amino acids, and amines in food samples. 

Some fluorimetric methods for the individual determination of amino acids in foods have also been reported. Thus, the native fluorescence of tryptophan has been used for its determination in food and feed hydrolysates using ion-exchange chromatography. Also, 3-methylhistidine has been determined in meat and meat products using LC and precolumn derivatization with fluorescamine or postcolumn derivatization with OPA and 2-ME. 

When performing denaturant titrations using fluorescence, either Protocol 7 or 2 can be followed. When monitoring the fluorescence of tryptophan residues in a protein, excitation and emission wavelengths of 290 nm and 350 nm, respectively, are typical. It is important to also measure the fluorescence signal from the denaturant solution and to subtract this blank signal from the fluorescence signal from the protein solutions. 

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