Amino acids, aromatic, natural fluorescence

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. 

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 ... 

The most obvious fluorescent compound in milk is riboflavin, which absorbs strongly at 440-500 nm and emits fluorescent radiation with a maximum at 530 nm. Riboflavin in whey is measured easily by fluorescence (Amer. Assoc. Vitamin Chemists 1951). Proteins also fluoresce because of their content of aromatic amino acids. Part of the ultraviolet radiation absorbed at 280 nm is emitted at longer wavelengths as fluorescent radiation. A prominent maximum near 340 nm is attributable to tryptophan residues in the protein. Use of fluorescence for quantitation of milk proteins was proposed by Konev and Kozunin (1961), and the technique has been modified and evaluated by several groups (Bakalor 1965 Fox et al. 1963 Koops and Wijnand 1961 Porter 1965). It seems to be somewhat less accurate than desired because of difficulties in disaggregating the caseinate particles and in standardizing instruments. It also involves a basic uncertainty due to natural variations in the proportions of individual proteins which differ in tryptophan content. 

Online photodiode array detection and OPA-derivatization have been used to corroborate the peptidic nature of the peaks obtained by RP-HPLC and to identify the aromatic amino acid residues contained in wine peptides (104a). Figure 3 shows the flowchart proposed by the authors for the interpretation of both spectral data and OPA-fluorescence response. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

The steady state luminescence from naturally occurring macromolecules has been extensively studied in the past to investigate the effect of li t on the properties of biological sterns and to reveal some aspects of conformation and stmcture (see reviews ° )). Certainly much useful information has been obtained about the type and behaviour of excited state of biopolymers from such measurements. However, the complex and heterogeneous nature of these systems have often led to ambiguities in interpretation of the results. The fluorescence from proteins, for example, has largely been attributed to the constituent aromatic amino acids phenylalanine, tyrosine and tryptophan. Many workers have used the observation that the emission characteristics of these amino acids are sensitive to the environment sur-... 

Certain aromatic analogues of natural amino acids can be used as potential fluorescent probes of peptide structure and dynamics in complex environments. The research team of M.L. McLaughlin undertook the gram scale synthesis of racemic 1- and 2-naphthol analogues of tyrosine. The synthesis of the 1-naphthol tyrosine analogue started with the Gattermann formylation of 1-naphthol using the Adams modification to afford the formylated product 4-hydroxy-1-naphthaldehyde in 67% yield. 

The environmental sensitivity of the fluorescence and phosphorescence of phenylalanine, tryptophan and tyrosine, and their side chains, is often examined when considering the macromolecular luminescence of natural peptides and proteins. Therefore, lower-lying singlet and triplet states of toluene, aniline and phenol have been extensively studied as the simplest models of the proteins mentioned above, respectively. Knowledge of the various aspects of electronic spectra of the corresponding aromatic amino acids is often exploited to probe those of the proteins137. In other words, accurate information on both... 

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

One of the most useful applications of fluorescence is in the routine determination of certain important molecules in body fluids for diagnostic purposes. Some such molecules are naturally fluorescent, but others must be chemically treated to form fluorescent products. For example, the amino acids tyrosine, tryptophan, and phenylalanine are all measured fluorometrically. Both tyrosine and tryptophan possess aromatic rings that absorb intensely and therefore have an intense natural fluorescence. Tyrosine is excited at both 225 and 280 nm, and emits at 303 nm tryptophan is excited at 220 and 280 nm and emits at 438 nm [34]. 

Thousands of fluorescent probes are known, uid it is not practical to describe them all. This ch rto contains an overview of the various types of fluorO ores, dieir spec-tnd propoties, and their applications. Fluoro tores can be Ixoadly divided into two main classes, intrinsic and extrin-ric. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of die sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine. and numerous odier substances. 

Intrinsic fluorophores are naturally occurring whereby the intrinsic fluorescence originates within the aromatic amino acids such as tryptophan, tyrosine, and phenylalanine. The indole groups of tryptophan residues are the dominant source of UV absorbance/emission in proteins. 

Most of the fluorescence in biological organisms comes from natural fluorophores such as the aromatic amino acids tryptophan, phenylalanine, and tyrosine NADH, picolinic acid, and flavins. 

UV spectroscopy in molecular beams involves either laser-induced fluorescence (LIP) or resonance-enhanced multiphoton ionization (REMPI) methods. The latter method has the advantage that the resulting ionized molecules can be mass-analysed in a TOP mass spectrometer. The application of either REMPI or LIP spectroscopy requires the molecule of interest to incorporate a UV chromophore, such as an aromatic moiety. The DNA and RNA bases adenine, guanine, cytosine, thymine and uracil are aromatic molecules with well-known UV absorptions. Of the 20 naturally occurring proteinogenic amino acids, 3 - phenylalanine, tyrosine and tryptophan - feature an aromatic side chain, as do many neurotransmitter molecules. To study molecules that lack a UV chromophore, such as peptides without Trp, Tyr and Phe residues and carbohydrates, a UV chromophore needs to be chemically attached [47, 48, 74]. 

Fluorescence detectors are based on filter-fluorimeters or spectrofluori-meters. They are more selective and can be up to three orders of magnitude more sensitive than UV absorbance detectors. The detector responds selectively to naturally fluorescing solutes such as polynuclear aromatics, quinolines, steroids and alkaloids, and to fluorescing derivatives of amines, amino acids and phenols with fluorogenic reagents such as dansyl chloride (5-(dimethylamino)-l-naphthalene sulfonic acid). 

Though all three natural amino acids with aromatic side chains exhibit fluorescence, in most cases only the tryptophan residues are detected due to the efficient electronic energy transfer occuring in the sequence phenylalanine tyrosine tryptophane. 

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