Tryptophan emission spectra

FIG. 4 PCA similarity map defined by the principal components 1 and 2 for the tryptophan emission spectra. Samples were coded NHO, NHP, HOM, and HOP for raw, heated, homogenized, and homogenized -I- heated milks, respectively. Each label corresponds to a spectrum. 

Figure 3.2 shows the fluorescence and phosphorescence emission spectrum from tobacco mosaic virus coat protein. These spectra are fairly typical of the tryptophan emission spectra observed from proteins at room temperature. 

Figure 2. Emission spectrum of thionin. Tryptophan emission spectra of 6 pM thionin were obtained 30 pM DPPG at 42°C (ex=280 nm) and pH 8.0.

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

Plastocyanin from parsley, a copper protein of the chloroplast involved in electron transport during photosynthesis, has been reported to have a fluorescence emission maximum at 315 nm on excitation at 275 nm at pH 7 6 (2°8) gjncc the protein does not contain tryptophan, but does have three tyrosines, and since the maximum wavelength shifts back to 304 nm on lowering the pH to below 2, the fluorescence was attributed to the emission of the phenolate anion in a low-polarity environment. From this, one would have to assume that all three tyrosines are ionized. A closer examination of the reported emission spectrum, however, indicates that two emission bands seem to be present. If a difference emission spectrum is estimated (spectrum at neutral pH minus that at pH 2 in Figure 5 of Ref. 207), a tyrosinate-like emission should be obtained. 

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

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... 

A fluorescence emission spectrum is a record of fluorescence intensity vs wavelength for a constant intensity of exciting light. Excitation and emission spectra for a flavin and for the indole ring of tryptophan are both given in Fig. 23-13. The heights of the... 

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. 

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. 

The key step here is to construct both (t) and v/(f) and to determine tsc see Fig. 3c. When the difference of two maxima (vs(f) — v/(f)) reaches 0.5 cm-1, we consider solvation complete (t = tsc). The difference between vsc and vJS purely results from the mixture of two lifetime fluorescence emissions. The time evolution from vsc to vss could be very long. For tryptophan in water, the time-zero emission maximum (vo) is obtained at 322.1 nm and solvation is completed in 18 ps. Both vs and V merge at 346.6 nm, and the total Stokes shift is 2186 cm-1. However, it takes another 832 ps for the emission spectrum to reach the... 

Figure 8.1 Normalized fluorescence emission spectrum (>.ex = 295 nm with emission peak = 355 nm) (c), excitation spectrum obtained at >.em = 340 nm (b), and absorption spectrum (a) of L-tryptophan in phosphate buffer, pH 7.

Figure 8.6 clearly indicates that in the presence of free tryptophan in solution, there is no binding of TNS on the amino acid. However, in the presence of bovine serum albumin, TNS shows a fluorescence emission spectrum, indicating that TNS is bound to the protein. 

Figure 9.7 Experimental fluorescence emission spectrum of a mixture of L-tyrosine and L-tryptophan in water (line, spectrum a) and that approximated using Equation (9.1) ( , spectrum b). Substracting spectrum (b) from (a) yields a fluorescence spectrum (c) characteristic of tyrosine. Xex =260 nm.

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

Figure 3.18. (a) Kibbon diagram of acyl-CoA binding protein ACBP (Kraulis, P. J. 1991. J. Appl. Cryst. 24, 946>950), based on the MMR structure coordinates of Andersen and Poulsen (Andersen, K V, and Poulsen, F. M , 1993, J. Biomol. MMR 3,271-284.) The two tryptophan residues and the mutated C-terminal isoleucine are shown In ball and stick, (b) Absortance spectrum (solid line) and fluorescence emission spectrum with excitation at 280 nm (dashed line) of iS pM ACBP.I86C labeled with IAEDANS(in 20 mMMa-acetate, pH 5.3). 

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