Peptides, tyrosine fluorescence

Cowgill pointed out that there are essentially two distinct quenching processes of tyrosine fluorescence resulting from association with the peptide bond.(3) Tyrosines affected by these mechanisms are classified in Table 1.3 as... 

It is interesting to note that the first demonstration of tyrosinate fluorescence in a protein was made by Szabo et al.au> with two cytotoxins from the Indian cobra Naja naja. While exhibiting different relative amounts of the two emission bands, both toxins had fluorescence at 304 and 345 nm, with the 304-nm band being greatly reduced on excitation at 290 nm. Since these proteins have three tyrosine residues and no tryptophan, it was concluded that the 345-nm emission band was due to tyrosinate. Furthermore, tyrosinate appeared to be formed in the excited state from a hydrogen-bonded ground-state complex based on the absorption spectra. Szabo subsequently reexamined these peptide samples and found that they were contaminated with tryptophan (A. G. Szabo, personal communication). While Szabo s approach to the demonstration of tyrosinate fluorescence was correct based on his initial data, his subsequent finding exemplifies an important caution if tyrosinate emission is suspected, every effort must be made to demonstrate the... 

Lawrence, D.S. and Q. Wang. 2007. Seeing is beheving peptide-based fluorescent sensors of protein tyrosine kinase activity. Chem. BioChem. 8, 373-378. 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

Poveda, J. A., Prieto, M., Encinar, J. A., Gonzalez-Ros, J. M. and Mateo, C. R, 2003, Intrinsic Tyrosine Fluorescence as a Tool To Study the Interaction of the Shaker B "Ball" Peptide with Anionic Membranes. Biochemistry, 42, 7124 -7132. 

In this work, the solutions of human serum albumin (HSA) (>96%, Sigma) and of bovine serum albumin (BSA) (>98%, MP Biomedicals) in a phosphate buffer (0.01 M, pH 7.4) have been used. The proteins concentrations were lO- (absorption spectra measurement) and 10- M (fluorescence measurement at the nanosecond laser fluorimeter). All of the experiments were performed at a temperature of 25 1 °C. The structure and biological functions of HSA and BSA can be found in (Peters, 1996). Tryptophan, tyrosine, and phenylalanine (with relative contents of 1 18 31 in HSA and 2 20 27 in BSA) are the absorption groups in these proteins (as in many other natural proteins). The tyrosine fluorescence in HSA and BSA (as in many other natural proteins) is quenched due to the effect of adjacent peptide bonds, polar groups (such as CO, NH2), and other factors, and phenylalanine has a low fluorescence quantum yield (0.03) (Permyakov, 1992). Therefore, the fluorescence signal in these proteins is determined mainly by tryptophan groups. In that case the fluorescence, registered in nonlinear and kinetic laser fluorimetry measurements, correspond to tryptophan residues (this fact will be used in Section 6.1). 

Haas et al.(m) have examined the fluorescence decay of tyrosine due to different Tyr-Pro conformations in small peptides to elucidate further the nature of the fluorescence change associated with Tyr-92. These peptides have acetyl groups at the amino terminus and /V-mcthylamidc groups at the carboxyl terminus. They found that whereas the dipeptide fluorescence decay requires a double-exponential fit, that of the tripeptide Tyr-Pro-Asn can be fit by a single exponential. By comparison of the average fluorescence decay time and steady-state quantum yield of the peptide to that of A-acetyltyrosine-A-methylamide, they found a relatively greater reduction in the steady-state quantum yield of the peptides. This is attributed to static quenching, which increased from 5 % in the dipeptide to 25 % in the tripeptide. The conformations of these peptides were also examined by NMR, but the results could be interpreted in terms of either cis-trans isomerization or other conformational isomerizations. 

There are several biologically important peptides which contain tyrosine but not tryptophan. These include small molecules with molecular weights of about 1000 or less. Molecules such as oxytocin, vasopressin, and tyrocidine A are cyclic, while others such as angiotensin II and enkephalin are linear. Schiller 19) has reviewed the literature up through 1984 on fluorescence of these and several other peptides. One major finding that has been reported recently is that the anisotropy and fluorescence intensity decays of many peptides are complex. This is especially evident in some of the tyrosine-containing peptides, and we expect that there will be considerable effort made over the next few years toward understanding the physical basis for these complex kinetics. 

X.-Y. Liu, K. O. Cottrell, and T. M. Nordlund, Spectroscopy and fluorescence quenching of tyrosine in lima bean trypsin/chymotrypsin inhibitor and model peptides, Photochem. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Detection of peptides in HPLC can be achieved by measuring natural absorbance of peptide bonds at 200-220 nm. Unfortunately at these wavelengths a lot of food components and also the solvents used for analysis absorb, demanding an intensive sample pretreatment and clean-up [129]. Peptides with aromatic residues can be detected at 254 nm (phenylalanine, tyrosine, and tryptophan) or 280 nm (tyrosine and tryptophan). Taking advantage of the natural fluorescence shown by some amino acids (tyrosine and tryptophan), detection by fluorescence can also be used for peptides containing these amino acids [106]. 

Tryptophan, tyrosine, and phenylalanine are the three natural amino acids that give rise to the intrinsic fluorescence of peptides in the ultraviolet region. Reliable, corrected fluorescence excitation and emission spectra of these aromatic amino acids were first published by Teale and Weber.M The fluorescence emission maxima of tryptophan, tyrosine, and phenylalanine in water are at 348, 303, and 282 nm, respectively. The photophysics and photochemistry of tryptophan and tyrosine have been comprehensively reviewed.1910 ... 

Fluorescence quantum yields of 0.13, 0.14, and 0.024 have been determined 15 for tryptophan, tyrosine, and phenylalanine in water, respectively, and these values have found general acceptance. Dissociation of the phenolic group of tyrosine at high pH results in strong fluorescence quenching. 16 The fluorescence quantum yield of a Trp, Tyr, or Phe residue contained in a peptide (tpPP) can be determined by comparison with the corresponding amino acid in H20 as standard on the basis of eq 6... 

Because of the low extinction coefficient and low fluorescence quantum yield of phenylalanine, phenylalanine fluorescence is not easily observed in tryptophan- or tyrosine-containing peptides and only very few fluorescence studies on Phe-containing peptides have been carried out. 

A large number of fluorescence decay measurements have been performed with proteins.127 Studies on the fluorescence decay of tyrosine and tryptophan and their derivatives, and on biologically active peptides containing intrinsic or extrinsic fluorophores have also been carried out and a few illustrative examples will be reviewed here. 

A number of studies on the fluorescence decay of tyrosine, tyrosine derivatives, and small tyrosyl peptides have been carried out. 36-38 Whereas the tyrosine zwitterion and tyrosine derivatives with an ionized a-carboxy group exhibited monoexponential fluorescence decay (x = 3.26-3.76 ns), double- or triple-exponential decay was observed in most other cases. As in the case of the tryptophan model compounds, the complex decay kinetics were again interpreted in terms of rotamer populations resulting from rotation around the C —Cp bond. There is evidence to indicate that the shorter fluorescence lifetimes may arise from rotamers in which the phenol ring is in close contact with a hydrated carbonyl group 36 37 and that a charge-transfer mechanism may be implicated in this quenching process. 39 ... 

This conclusion is borne out by other evidence. Fluorimetric alkaline titration gives a simple curve with a pX of 10.3 similar to the first part of the usual spectral titration. The phenolate ion is nonfluorescent. By itself the observed effect could represent quenching by nonfluorescent free tyrosines of fluorescent buried ones. This is particularly unlikely in view of the action of dioxane. The fluorescence of simple tyrosine peptides increases linearly with volume per cent of dioxane in the solvent. 

With RNase-S 40% dioxane appears to cause the dissociation of S-protein and S-peptide (308). The fluorescence change as a function of dioxane concentration shows a transition with a midpoint at about 28% dioxane. At 40% the transition is complete. All tyrosine residues appear to be exposed and all enzymic activity is lost. The quantum efficiencies of various samples in several solvents are shown in Table XVI. 

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