Tyrosine fluorescence, intrinsic

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

Proteins containing amino acids like tryptophan and tyrosine which intrinsically fluoresce upon excitation in the ultraviolet range can be employed, hence, the name total internal reflection intrinsic fluorescence-TIRIF, or... 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making de-... 

The fluorescence quantum yield of the protein can be estimated from the ratio Tq / tr, where Xo is the mean fluorescence lifetime equal to 1.8 ns and tr is the radiative lifetime of tyrosine equal to 2.7 ns (Lux et al. 1977 Wu et al. 1994). Of = 0.067. The fact that steady-state and lifetime measurements yield the same value of Of indicates that the three tyrosines of 029 SSB contribute to its intrinsic fluorescence. Therefore, ground-state deexcitation of the tyrosine fluorescence by the surrounding environment is weak. 

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. 

Lihertini, L. J. Small, E. W. The intrinsic tyrosine fluorescence of histone-Hl—steady-state and fluorescence decay studies reveal heterogeneous emission. Biophys. J., 1985, 47(6), 765-772. 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making derivatization a prerequisite for fluorescence detection. Derivatization has been described with naphthalene-2,3-dicarboxaldehyde-(S-mercaptoethanol for determination of substance P and its metabolites, fluorescamine, and 5-carboxyfluorescein succinimidyl ester for luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, and 3-endorphin. Kostel and Lunte compared various postcolumn reactor designs, whereas Advis et al. employed precolumn derivatization, among others. In order to improve sensitivity, laser light is frequently employed for exciting the fluorescent molecules referred to as laserinduced fluorescence (LIE) and provides a 500-1000 times improved sensitivity compared to UV detection. 

The absorption of the tyrosines in pig intestinal Ca2+-binding protein is reported to be shifted to longer wavelengths the intrinsic fluorescence, however, is in the normal energy region for tyrosine emission with a possibility of some emission from tyrosinate/143, 45) These results can be equally well explained by a ground-state, hydrogen-bonded complex. 

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. 

A third type of detector is the intrinsic or native fluorescence detector that utilizes native fluorescence properties of amino acids. The sensitivity of this detector is between UV/PDA and LIF detection. The advantage of this technique over pre-labeling is that there is no pre-labeling step required therefore, the sample preparation is relatively simple, and the sensitivity is improved over UV/LIF. However, the intrinsic fluorescence detection relies on the presence of Tryptophan (Try), Tyrosine (Tyr), Phenylalanine (Phe), and this detector has just become commercially available. 

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 ... 

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 suitable fiuorescent probe is an organic molecule, which must change its characteristic parameters with changes in its microenvironment and the parameter must be measurable when the probe is added to the system [54]. The fluorescent probes are categorized as either extrinsic, intrinsic, or covalently bound probes. The intrinsic probes allow a system to be observed without any chemical perturbation. This occurs when the system to be characterized has an in-built fluorescent chromophore unit like tryptophan, tyrosine and phenyl alanine in protein. In some cases the fluorophore is covalently... 

Some biomolecules are intrinsic fluors that is, they are fluorescent themselves. The amino acids with aromatic groups (phenylalanine, tyrosine,... 

Protein concentration can also be determined by measuring the intrinsic fluorescence based on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and/or phenylalanine. Usually tryptophan fluorescence is measured. The fluorescence intensity of the protein sample solution is measured and the concentration is calculated from a calibration curve based on the fluorescence emission of standard solutions prepared from the purified protein. This assay can be used to quantitate protein solutions with concentrations of 5 to 50 (J-g/ml. 

Proteins typically exhibit some level of ultraviolet-excited intrinsic fluorescence, due to the presence of aromatic amino acids tryptophan and tyrosine. These fluorogenic groups possess some inherent solvent sensitivity and, therefore, exhibit changes in net... 

One of the most interesting features of natural fluorescence results from the fact that the fluorescence response of a given molecule depends very much on their microenvironment. This feature can be used in order to gather information about the structure of complex molecules such as polypeptides and proteins, which may integrate several fluorescent amino acids residues such as tryptophan, tyrosine, and phenylalanine. Among these, tryptophan is the one that exhibits the highest quantum yield, which makes it a good candidate to be used as an intrinsic fluorescence reporter. 

Natural fluorescent labeling of proteins is derived from their primary structure, i.e. mainly from the type, number and occurrence of amino acids having fluorescent properties. For native (intrinsic) fluorescence of proteins tryptophan and tyrosine are specially responsible, although some other amino acids (phenylalanine, histidine, arginine) are fluorescent, too. The fluorescence contribution of these other amino acids is, however, extremely The fluorescence of tyrosine is normally... 

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