Tyrosine emission spectrum

Histone HI from the fruit fly Ceratitis capitata has two tyrosine residues. Jordano et al.(<)2> have observed two differences from calf thymus HI (1) the apparent quantum yield does not increase on protein folding and (2) there is a pH- and conformation-dependent shoulder at 340 nm in the emission spectrum. This group has attributed this 340-nm emission to tyrosinate.(97) Their studies demonstrate that the folding of histone HI from C. capitata is pH and ionic strength dependent. The possibility of tyrosinate formation at neutral pH is discussed in greater detail in Section 1.5.2. 

In 1971, adrenodoxin, an iron-sulfur protein with a single tyrosine residue and no tryptophan was shown to fluoresce at 331 nm upon 280-nm excitation at neutral pH/20 1 On cooling from room temperature to 77 K, the emission maximum shifts to 315 nm. The redox state of the iron does not have any effect on the tyrosine emission. From these results, an exciplex between the excited singlet state of tyrosine and an unidentified group was suggested as the cause of the anomalous emission energy/2031 Later studies have shown that the excitation spectrum is a red-shifted tyrosine spectrum, that removal of the iron to form the apoprotein has no effect on the emission, and that heat, low pH, guanidine hydrochloride, urea, and LiCl all cause the emission... 

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. 

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.

Model compound and difference spectral studies have been carried out on tyrosine and related compounds by Wetlaufer (1956), Laskowski (1957), Edelhoch (1958), Chervenka (1959), Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Foss (1961) (see also Section VI,Z)). Studies of the fluorescence of tyrosine—activation spectrum, fluorescence emission spectrum, and quantum yield—have been reported by Teale and Weber (1957). The pH-dependence of the fluorescence of tyrosine and related compounds was studied by White (1959) fluorescence-polarization studies from the same laboratory were reported by Weber (1960a) for simple compounds, and for proteins (Weber, 1960b). Teale (1960) has carried out extensive studies on the fluorescence characteristics of a score of proteins. 

We can notice also that there is no peak at 345 nm in the fluorescence emission spectrum, revealing that formation of tyronisate is not occurring within the protein. Therefore, interaction of tyrosine residues with the surrounding amino acids is weak. Interaction with the side-chains of the neighboring residues contributes to the formation of hydrogen bond with the tyrosine. This interaction, when it exists, induces a decrease in the fluorescence quantum yield of the tyrosines. 

P13 acts in the fission yeast cell division cycle. Addition of the proton to the kinase assay in vitro was able to rescue the defect in the Cdc2 mutant kinase activity P13 cont ns two tryptophans (Trp-71 and Trp-82) and seven tyrosine residues. The maximum of the fluorescence emission spectrum b localcd at 336 nm (>., 295 or 275 nm). Thus, tryptophan residues are responsiUe for the protdn fluorescence. Three lifetimes are found for the intensity dec, 0.6,2.9 and 6.1 ns, with fracUonal intensities of0.04,0.32 and 0.64, respectively. 

A very common method to put into evidence the fluorescence spectrum of each component is the decay associated spectra. This method allows combining the dynamic time-resolved fluorescence data with the steady-state emission spectrum. The fluorescence decays are globally or individually fitted to the n-exponential function (n being the number of species, tryptophan or tyrosine residues for example), and the decay associated spectra are constructed (Krishna and Periasamy, 1997). Figure 8.27 displays the decay associated spectra of Trp residues of ai-acid glycoprotein performed by Hof et al (1996). The authors found four fluorescence lifetimes instead of the three we obtained. They attributed the peak of 337 nm to Trp-122 with a location between the surface of the protein and its core. The determination of the degree of hydrophobicity around the Trp residues showed that Trp-25 residue is located in a hydrophobic environment and Trp-160 is the most exposed to the solvent. 

In Fig. 6A and 6B the aromatic region of the normal and the photo-CIDBP difference spectrum of gene-5 protein containing deuterated phenylalanyl residues Is shown. The photo-CIDBP difference spectrum obtained from the gene-5 protein tetranucleotlde complex Is given in Fig. 6C. The tyrosine emission signals are completely quenched. To ensure that the total amount of protein was ccinplexed to the... 

The indole group of tryptophan (Trp) has a higher molar extinction coefficient than the phenolic and phenyl side chains of tyrosine (Tyr) and phenylalanine (Phe), respectively (see Table 1, Chapter 12). Thus although its quantum yield is similar to that of lyr, its fluorescence emission is much mote intense. Furthermore, its excitation spectrum overlaps the emission spectrum of Tyr and therefore fluorescence resonance energy transfer (FRET, see Chapters 2 and 3) from lyr to Trp occurs readily when both residues are in close proximity (i.e. located in the same protein molecule) and favourably orientated. The intrinsic fluorescence of a protein is therefore dominated by the contribution from Trp... 

An additional emission band near 350 nm has been observed for lima bean trypsin inhibitor (LBTI).(173) The authors discussed both the possibility of contamination by tryptophan and excited-state tyrosinate formation. Since this 350-nm emission has a tyrosine-like excitation spectrum that is slightly shifted compared to that of the major 302-nm emission, it is also possible that the tyrosine residue in a fraction of the LBTI molecules could be hydrogen bonded. This model is supported by the observations that the phenol side chain is shielded from solvent and has an anomalously high pKa. 

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. 

The fluorescence emission maximum of CGTase is located at 338 nm, and its spectrum bandwidth is 55 nm (Figure 7.12a). Thus, both embedded and surface tryptophan residues contribute to protein fluorescence. Although CGTase contains many tyrosine residues, the absence of a shoulder or a peak at 303 nm (Figure 7.12b), when excitation is performed at 273 nm, suggests that tyrosine residues do not contribute to CGTase emission. 

Because the 2570 A band of phenylalanine is weak, it is often obscured in proteins by the much stronger tyrosine and tryptophan absorptions. It is occasionally visualized in protein spectra as ripples (fine structure) in the spectral region 2500-2700 A. These ripples can be amplified by the difference spectral technique, as is shown in Fig. 13. A typical phenylalanine difference spectrum, obtained in a comparison of the isoelectric amino acid with a solution of the same concentration at pH 1 is shown in Fig. 12. Difference spectra for phenylalanine in various solvents have been measured by Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Donovan et al. (1961). Fluorescence activation and emission spectra for phenylalanine were measured by Teale and Weber (1957). 

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