Phenylalanine fluorescence

Phenylalanine reacts with ninhydrin in the presence of a dipeptide (usually glycyl-L-leucine or L-leucyl-L-alanine) to form a fluorescent product. The fluorescence is enhanced and stabilized by the addition of an alkaline copper reagent to adjust the pH to 5.8 and the resulting fluorescence is measured at 515 nm after excitation at 365 nm see Procedure 10.2. 

The fluorescence resulting from an idcnm.il treatment ot social standard solutions of phenylalanine (0.1-1.0 mmol 1 ) is used to calculate the test concentration. 

A biosensor was designed where a dehydrogenase and an enlarged coenzyme are confined behind an ultrafiltration membrane. The amino acid is determined indirectly, by measuring the fluorescence of the reduced coenzyme (kex 360 nm, kfl 460 nm) produced in reaction 22, with the aid of an optical fiber. The coenzyme is regenerated with pyruvate in a subsequent step, as shown in reaction 23. This biosensor was proposed for determination of L-alanine and L-phenylalanine for monitoring of various metabolic diseases and for dietary management363. 

The aromatic amino acids each have two major absorption bands in the wavelength region between 200 and 300 nm (see reviews by Beaven and Holiday(13) and Wetlaufer(14). The lower energy band occurs near 280 nm for tryptophan, 277 nm for tyrosine, and 258 nm for phenylalanine, and the extinction coefficients at these wavelengths are in the ratio 27 7 l.(14) As a result of the spectral distributions and relative extinction coefficients of the aromatic amino acids, tryptophan generally dominates the absorption, fluorescence, and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids. 

Eisinger(55) also noted that it is difficult to obtain accurate data with phenylalanine as the donor and either tryptophan or tyrosine as the acceptor. The source of this problem is the weak S, - S0 absorption of phenylalanine compared to that of tyrosine or tryptophan, which leads to considerable experimental uncertainty in measuring the sensitized acceptor emission. This error may account for the finding of Kupryszewska et al.<56> that the sensitization of the acceptor fluorescence was less than the quenching of the donor fluorescence in their study of phenylalanine-to-tyrosine energy transfer... 

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. 

Figures Selective protein modification using a keto amino acid, p-acetyl-L-phenylalanine. (a) Labeling of fluorescein hydrazide to the Z domain protein. Only the mutant protein containing p-acetyl-L-phenylalanine was labeled and became fluorescent, (b) A general method for preparing glycoprotein mimetics with defined glycan structure.

Dillard, C. J., and A. L. Tappel. Fluorescent products from reaction of peroxidiz-ing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine. Upids 8 183-189, 1973. 

Results of Wulf et al (7) show that carrot roots obtained from a supermarket contain myristicin Imperator variety carrots contain an average of 15 parts per million (ppm). Recently harvested, unprocessed carrots only rarely contain myristicin (8). The presence of myristicin in supermarket carrots and its absence in recently harvested ones indicate that its increased concentration may have been induced by some elicitor following harvest. Solar radiation after harvest, or fluorescent lighting during display, may function as such an elicitor. Light is known to produce ethylene and is an activator of phenylalanine ammonia-lyase, one of the regulatory enzymes responsible for phenylpropanoid biosynthesis in plants (9). 

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. 

Studies of nuclear magnetic resonance spectra (Chapter 3) and of polarization of fluorescence (Chapter 23), have shown that there is rapid though restricted rotational movement of side chains of proteins in solution. Even buried phenylalanine and tyrosine side chains often rotate rapidly whereas movement of the... 

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. 

The aromatic amino acids also have fluorescence emissions when excited by light in the UV range. Table Bl.3.3 gives the excitation wavelength, fluorescence emission wavelength, and quantum yield (Q) for tryptophan, tyrosine, and phenylalanine. The quantum yield is the ratio of photons emitted to photons absorbed. Typically, phenylalanine fluorescence is not detected in the presence of tyrosine and tryptophan due to low Q. Furthermore, tyrosine fluorescence is nearly completely quenched if the tyrosine residue is ionized or near an amino group, a carboxyl group, or a tryptophan residue (Teale, 1960 Freifelder, 1982). Therefore, tryptophan fluorescence is what is customarily measured. 

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