Phenylalanine fluorescence, intrinsic

Intrinsic tryptophan, tyrosine, and phenylalanine fluorescence quenching induced by gold NPs was recorded on a Cary Eclipse fluorescence spectrophotometer (Varian). Excitation was performed at 280 nm. Fluorescence emission was measured at 25°C in DPBS (without Ca and Mg " ) containing various concentrations of gold NPs. The protein concentration was fixed at 0.01 mg/mL. Nanoparticle concentration... 

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 ... 

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. 

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... 

The nucleotides and nucleic acids are generally non-fluorescent. However, some notable exceptions are known. Phenylalanine transfer RNA from yeast (tRNA ) contains a single highly fluorescent base, called the Y-base, which has an emission maximum near 470 nm. The presence of this intrinsic fluorophore has resulted in numerous studies of tRNA by fluorescence spectroscopy. Regarding the non-fluorescent nucleic acids, it should be noted that they do fluoresce, but with very low yields and with short decay times. 

The majority of naturally occurring compounds are nonluminescent, including nucleic acids (DNA/RNA), mono- and polysaccharides, hpids, and most of the small biomolecules. Proteins contain in their structure three amino acids—phenylalanine, tyrosine, and tryptophan— that fluoresce in the UV range. Due to its long-wave emission and relatively low abundance, tryptophan is commonly used as an intrinsic luminescent probe to study proteins. Tryptophan also exhibits phosphorescence at room temperature. 

Intrinsic protein fluorescence originates with the aromatic amino acids. tryptophan (trp), tyrosine (tyr). and phenylalanine (phe) (Figure 3.1). The indole groups of tryptophan... 

Because of their spectral properties, RETcan occur fiom phenylalanine to tyrosine to tryptophan. Also, blue-shifted tryptophan residues can transfra the excitation to longer-wavelength tryptophan residues. In fact, energy transfer has been repeatedly observed in proteins and is one reason for the minor contribution of phenylalanine and tyrosine to the emission of most (Hoteins. The anisotropy displi ed by tyrosine and tryptophan is sensitive to both ov l rotational diffusion of proteins and the extent of segmental motion during the excited-state lifetimes. Hence, the intrinsic fluorescence of proteins can provide considerable information about protein structure and dynamics and is often used to study protein folding and association reactions. In this chapter, we present examples of protein... 

The intrinsic tryptophan fluorescence emission spectra of Sac7 and Sso7 (5 p, Af protein in 10 mM KH2PO4, pH 6.8) are obtained with excitation at 295 nm using 4 nm excitation and emission slit widths. Excitation at 295 nm prevents contributions from the two tyrosine and two phenylalanine residues. An emission maximum at 350 nm is similar to that of free tryptophan and indicates significant solvent exposure, consistent with the NMR solution structure. Addition of double-stranded DNA [e.g., duplex poly[d(GC)]] leads to quenching of tryptophan fluorescence by nearly 90%. A blue shift of the emission maximum to 340 nm is also observed. 

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. 

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... 

Photoluminescence can be used to detect an analyte in three ways (1) the analyte itself is intrinsically fluorescent (direct sensing) (2) the analyte can be tagged with a fluorophore label or (3) the analyte interacts with a luminescent probe. Direct sensing and fluorophore-tags are widely used in biomedical applications to probe cell environments. Many proteins are intrinsic fluorophores due to the presence of the aromatic amino acids tryptophan, phenylalanine and tyrosine. Analytes such as pH, CO2, NH3, O2 and various cations and anions can be measured indirectly using luminescence probes. 

Macromolecules may or may not fluoresce. Those that do are considered to contain intrinsic fluors. The common intrinsic fluors for proteins are tryptophan, tyrosine, and phenylalanine (the same three groups that absorb UV radiation). Macromolecules that have no intrinsic fluors can be made fluorescent by adding an extrinsic fluor to them. This is done by the process of chemical coupling or sample binding. The most common extrinsic fluors for proteins are l-aniline-8-naphthalene sulfonate, l-dimethylaminonaphthalene-5-sulfonate, dansyl chloride, 2-p-toluidyl-naphthalene-6-sulfonate, rhodamine, and fluorescein. The most common extrinsic fluor for nucleic acids are various acridienes (acridine orange, proflavin, acriflavin) and ethidium bromide. 

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