Fluorescence of proteins

Callis PR (1997) 1 La and 1 Lb transitions of tryptophan applications of theory and experimental observations to fluorescence of proteins. Meth Enzymol 278 113-150... 

J. M. Beechem and L. Brand, Time-resolved fluorescence of proteins, Annu. Rev. Biochem. 54, 43-71 (1985). 

K. K. Turoverov and I. M. Kuznetsova, Polarization of intrinsic fluorescence of proteins. 2. The studies of intramolecular dynamics of tryptophan residues, Mol. Biol. (Moscow) 17, 468-475 (1983). 

Although protein phosphorescence was in fact observed earlier than fluorescence, fluorescence of proteins is now widely used, whereas phosphorescence receives much less attention. The reason for this is that until recently it was thought that protein phosphorescence could only be observed in frozen samples, thereby limiting its use. The early literature provides clues that this need not be the case. Beccari reported in 1746 that phosphorescence was observed from a cold hand after it had been exposed to the sunlight/61 A comprehensive coverage of the early sightings of phosphorescence is found in the book by Harvey. 

The intrinsic UV fluorescence of proteins is dominated by the tryptophan indole rings. The absorption maximum is 280-290 nm with the fluorescence maximum ranging from 315-355 nm, depending on the local environment of the indole side-chains. Quantum yields range from 0.04 to 0.50 0.10 is a common value. As the local environment polarity or dielectric constant increases, the fluorescence maximum shifts up to 355 nm, such as for an indole ring in water or buffer. Trp moieties in highly hydro-phobic environments fluoresce at 315-320 nm. Thus the fluorescence emission maximum (and the quantum yield) provide indirect information as to the local environment of the Trp fluors. 

Detailed discussion of intrinsic fluorescence of proteins and factors that affect fluorescence emission by the aromatic amino acids (see pp. 618-663). 

In proteins, tryptophan, tyrosine, and phenylalanine are responsible for the absorption and fluorescence of proteins in the UV (Figures 1.2 and 7.10). 

Teale, F.W.J. (1960). The ultraviolet fluorescence of proteins in neutral solution. The Biochemical Journal, 76, 381-388. 

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... 

Chen, R. F. Extrinsic and Intrinsic Fluorescence of Protein, 467, in Practical Fluorescence, Theory, Methods and Techniques (ed. Guibault, G. G.) Marcel Dekker, New York 1973 Feitelson, J. J. Phys. Chem. 68, 391 (1964)... 

Interference by the fluorescence of proteins, compounds, or nonspecific adsorption of the tracer on proteins. 

Teale FWJ, Weber G. Ultraviolet fluorescence of proteins. Bio-chem. J. 1959 72 15. 

Eftink MR. Intrinsic fluorescence of proteins. In Topics in fluorescence spectroscopy. Lakowicz JR, ed. 2000. Kluwer Academic/Plenum Pubhshers, New York. pp. 1-15. 

H -tetramethylbenzidine in anionic-cationic mixed micelles has been studied in detail by ESR . The photochemistry of the semi-oxidised forms of eosin Y and rose bengal have been investigated in colloidal solutions. Relevant to the fluorescence of proteins is a study of fluorescence quenching of indolic compounds by amino-acids in SOS, CTAB, and CTAC micelles O Rate constants for proton transfer of several hydroxyaromatic compounds have been measured in a variety of surfactant solutions. Photoprotolytic dissociation does not require exit of the reactant molecules from the micelles. Micellar solutions can be used to improve the fluorescence determination of 2-naphthol by inhibiting proton transfer or proton inducing reactions z2. jpe decay of the radical pair composed of diphenylphosphonyl and 2,4,6-trimethyl benzoyl radicals in SDS is affected by magnetic... 

Chen, R.F. Extrinsic and intrinsic fluorescence of proteins. In Practical Fluorescence Theory, Methods and Techniques Guilbault, G.G., Ed. Marcel Dekker, Inc. New York, 1973 Ch. 12. 

Once you have pinned your molecule down, you need some way of observing what it gets up to. Fluorescence-based methods are usually the most sensitive here, because photomultipliers and CCD devices (see Chapter 2) can readily detect the single photons emanating from individual fluorescent groups. Moreover, the intrinsic fluorescence of proteins or other fluorescence probes is very sensitive to conformational changes and is well understood for bulk samples. 

Privat J. P, Wahl, P. and Auchet J.C, 1979, Rates of deactivation processes of indole derivatives in water-organic solvent mixtures—application to tryptophyl fluorescence of proteins. Biophysical Chemistry 9, 223-233. 

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... 

Diuing the past decade, the instrumentation for time-resolved fluorescence of proteins has advanced dramatically. The flashlamp light sources have been replaced by hi -repetition-rate (MHz) picosecond dye lasers, which provide both higher excitation intensities and raon rapid data acquisition. The dynode-chain PMTs have been replaced by MCP detectors, which provide much shorter single-photoelectron pulse widths than a dynode chain PMT. In con nnation. the new light sources and detectors provide instniment r ponse functions with half-widths near 100 ps, so that picosecond resolution can now be obtained. 

Although the measurement of fluorescence p>olari-zation and decay are still powerful tools in the study of the physical chemistry of proteins, the application of fluorescence labelling of proteins has lost some of its im H>rtance, since improved instrumentation permits measurement of the native fluorescence of proteins. Most of the fluorescent reagents developed for this purpose reacted with the amino groups of proteins. As these same reagents were applied for microanalytical purposes, initially only a few fluorescent labels were... 

One of the main advantages of CE over gel electrophoresis is that the separation is monitored by online, on-column, or end-column detection. In the most frequently employed UV absorption photometric detection, a small part (less than 1mm) of the capillary serves as a detection cell. Micromolar concentrations of proteins are detectable using the low UV detection wavelength of 200-220 nm. A higher sensitivity, up to nanomolar concentrations, is achieved with fluorescence, particularly laser induced fluorescence (LIE) detection. The disadvantage of the LIE detection of proteins is the necessity for their derivatization using a fluorogenic label. The native fluorescence of proteins, mostly due to the presence of aromatic amino acids residues, tryptophan, and tyrosine, can be utilized only when low UV laser... 

---