Tryptophan, fluorescence spectra

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

To detect possible conformational changes caused by acid activation, tryptophan fluorescence spectra were measures. When excited 295 nm, the emission spectrum of the protyrosinase exhibited as average emission wavelength of 339 nm. By comparison, in the spectrum of the acid-activated tyrosinase, the emission intensity was decreased by 62%, and there was a red shift of the average emission wavelength to 346 nm (Figure 41). These results indicate that tryptophan residues are brought into a polar environment by acid activation. 

Figure 41. Tryptophan fluorescence spectra of the protyrosinase (pro-TY) (—) and acid- activated tyrosinase (acid-TY) (—).

Figure 7.13 Tryptophan fluorescence spectra of CGTase after 18 h of nitration with 0, 0.25, 0.5, 1, 2, 4, and 8 mM TNM. Xex = 295 nm. Source Villette J.R., Helbecque, N., Albani, J.R., Sicard, RJ. and Bouquelet, S.J. (1993). Biotechnology and Applied Biochemistry, 17, 205-216. Reprinted with permission from Portland Press.

SSB contains 3 tyrosine residues at positions 50, 57, and 76 and lacks of tryptophan. Fluorescence spectra of 029 SSB free in solution and complexed to ssDNA, obtained at pH 7.5, are displayed in Fig. 3.12 ( ex, 276 nm and 308 nm). One can notice that both excitation and emission spectra intensities decrease in presence of ssDNA. The absence of spectra overlap between the excitation and emission indicates that energy transfer between tyrosine residues in 029 SSB is almost nonexistent. 

Tryptophan fluorescence spectrum. The emission spectrum appears at longer wavelengths as compared to the absorption spectrum. 

Apolipoproteins undergo changes in secondary protein structure when combined with phosphatidylcholine (Morrisett et al., 1977b). The circular dichroic spectrum and the blue-shifted tryptophan fluorescence spectrum are consistent with an amphipathic structure. Since lipoprotein lipase also can undergo hydrophobic association with phospholipids (Voyta et al., 1980), as indicated by blue-shifted tryptophan fluorescence, it seems probable that the interaction of the enzyme with the apoprotein activator at the lipid-water interface involves extensive lateral protein protein interactions (Smith and Scow, 1979). 

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

When tryptophan is dissolved in water, it shows the fluorescence characteristics illustrated in O Figure 5-2. However, one especially useful property of tryptophan fluorescence is that its emission spectrum is highly sensitive to the polarity of its environment. In less polar solvents (alcohols, alkanes, etc.), the emission... 

Fluorescence of an aqueous solution (10 iulM) of tryptophan. Superimposed on the figure is its absorbance spectrum (dashed line). Tryptophan fluorescence shows an excitation maximum at about 280 nm and broad fluorescence emission at 350-360 nm... 

Fluorescence Spectrum Fluorescence spectroscopy Tryptophan environment, presence of fluorescent ligands or impurities.b... 

If the absorbance spectrum of a small molecule overlaps the emission spectrum of tryptophan and if the distance between them is small, quenching will be observed. If binding of such a molecule to a protein quenches tryptophan fluorescence, tryptophan must be in or near (<2 nm away from) the binding site. 

Figure 9.1 shows the fluorescence spectrum of tryptophan residues of cytochrome b2 core dissolved in 6 M guanidine, pH 7, in the presence of 30 mM 2-mercaptoethanol. The emission peak is located at 357 nm, indicating that the protein is completely denatured (spectrum a). The fluorescence emission spectra of successive aliquots (0.55 /uM) of free tryptophan added to the protein solution are also shown (spectra b-g). 

Figure 9.7 shows the experimental fluorescence spectrum of a mixture of L-tyrosine and L-tryptophan in water (line, spectrum a). The presence of a small shoulder around 303 nm indicates that L-tyrosine contributes to the fluorescence emission. Analysis of the data using... 

Equation (9.1) yields a fluorescence spectrum (squares, spectrum b) that does not match with the experimental one, especially within the region where tyrosine emits. The calculated spectrum (b) corresponds to that of tryptophan alone. Subtracting spectrum (b) from (a) yields a spectrum with a maximum at 303 nm corresponding to that of tyrosine. 

Figure 18.4 Fluorescence spectrum of the Tryptophan solution excited by a) one, b) two and c) three-photon absorption process. Insets show the excitation intensity dependence of the fluorescence.

Thus, knowledge of the transition moment direction of a phenol band could help in interpreting the fluorescence spectrum of a tyrosine chromophore in a protein in terms of orientation and dynamics. The absorption spectrnm of the first excited state of phenol was observed around 275 nm with a fluorescence peak aronnd 298 nm in water. The tyrosine absorption was reported at 277 nm and the finorescence near 303 nm. Fluorescent efficiency is about 0.21 for both molecules. The fluorescent shift of phenol between protic and aprotic solvents is small, compared to indole, a model for tryptophan-based protein, due to the larger gap between its first and second excited states, which resnlts in negligible coupling . ... 

Acid Denaturation. LADH loses activity and zinc at pH 5 while still in the dimeric state 177,182). At lower pH dissociation occurs into subunits 182-184) and there are drastic changes in the protein fluorescence spectrum 185-187) and the fluorescence polarization spectrum 182). Different time dependences for the changes of the tyrosine and tryptophan difference fluorescence peaks are observed 187), which is consistent with a slower quenching of the buried Trp-314 (Section II,C,3,c) compared to the more exposed tyrosines. This interpretation implies that a partial unfolding of the tertiary structure occurs prior to the dissociation into subunits at acid pH. 

The large environmental dependence of the ll-(3-hexyl-l-indolyl)undecyltri-methylammonium bromide fluorescence lifetime and fluorescence spectrum has led to its continued use as a fluorescence probe for micellar systems.76 Fluorescence-detected circular dichroism, a technique which appears to have considerable potential, has been applied to the determination of the c.d. spectra of L-tryptophan and L-cystine and two macromolecular systems.78... 

A substantial literature exists describing experimental and theoretical studies of the photophysical processes of indoles. In part, this reflects the well-established use of the magnitude of the Stokes shift seen in fluorescence spectrum of the indole chromophore to probe the local environment of tryptophan residues in biological molecules [4]. However, it also reflects the complexity of indole photophysics and the fact that some aspects remain controversial. Only an overview is presented here, because a proper discussion of the photophysics of the indole ring would easily fill this chapter. Unfortunately, no recent, comprehensive review of the topic is available however, early publications have been summarized [4,5], and recent papers in this area provide leading references and excellent brief reviews of subsequent work [6]. 

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