Protein fluorescer

Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

Several lines of evidence suggest a close link between protein fluorescence and free radicals in diabetes ... 

Several studies have demonstrated that treatment of diabetic patients with the sulphonylurea, gliclazide, is associated with a fall in lipid peroxidation, protein fluorescence and beneficial effects on platelet function (Florkowski et al., 1988 Jennings et al., 1992). These changes were seen to be independent of changes in giycaemic control. 

Maddalo SL, Zimmer M (2006) The role of the protein matrix in green fluorescent protein fluorescence. Photochem Photobiol 82 367-372... 

Bastiaens, P. I. and Jovin, T. M. (1996). Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein fluorescent-labeled protein kinase C beta I. Proc. Natl. Acad. Sci. USA 93, 8407-12. 

Mayer, L.M., Schick, L.L., Loder, T.C. 1999. Dissolved protein fluorescence in two main estuaries. Marine Chemistry, 64, 171-179. Miano, T.M., Senesi N., 1992. Synchronous excitation fluorescence spectroscopy applied to soil humic substances chemistry. Sci Total Environ, 117/118, 41-51. 

Besides the formation of luminescent silver clusters in fixed cells, likely due to the presence of proteins, fluorescent silver clusters have been synthesized using proteins as templates. In 2008, Pal et al. reported on the use of an enzyme, bovine pancreatic a-chymotrypsin (CHT) as biotemplate during the chemical reduction of... 

S. S. Lehrer, Solute perturbation of protein fluorescence. The quenching of other tryptophan fluorescence of model compounds and of lysosome by iodide ion, Biochemistry 10, 3254-3263 (1971). 

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

J. R. Lakowicz and G. Weber, Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale, Biochemistry 12, 4171-4179 (1973). 

J. R. Lakowicz, G. Laszko, I. Gryczynski, and H. Cherek, Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry, J. Biol. Chem. 261, 2240-2245 (1986). 

The intersystem crossing process has opposite effects on the yields of fluorescence and phosphorescence since it depletes the singlet state and populates the triplet state. It is commonly known that heavy ions, such as iodide and bromide, increase intersystem crossing by spin-orbit coupling.(1617) For proteins, fluorescence can be quenched as phosphorescence yield is enhanced. 8,19) However, although the phosphorescence yield is increased, the lifetime is decreased. This effect arises because spin-orbit coupling, which increases the intersystem crossing rate from 5, to Tt, also increases the conversion rate from T, to S0. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

The high sensitivity and selectivity of fluorescence detection make this the obvious choice for improving detection of proteins. Three approaches have been used direct detection of intrinsic protein fluorescence, indirect fluorescence detection, and protein derivatization for fluorescence detection. 

A simple alternative to direct detection of intrinsic protein fluorescence detection is the technique of indirect fluorescence detection proposed by Kuhr and Yeung.20 In this approach, the analysis buffer contains a fluorescent anion that produces a high background fluorescence signal. Nonfluorescent analyte anions displace the fluorescent species, producing a zone of reduced signal. Sensitivity in indirect fluorescence detection is determined by the dynamic reserve (ratio of signal... 

Fluorescence energy transfer. Panel (a) shows the fluorescence characteristics of a hypothetical protein A (excitation and emission maxima at 290 nm and 350 nm, respectively). In (b). Ligand B fluoresces with an emission maximum at 450 nm when excited at 350 nm. Panel (c) shows that the formation of the protein-ligand complex can be monitored using an excitation wavelength of 290 nm and recording the decrease in protein fluorescence at 350 nm or the increase in ligand fluorescence at 450 nm... 

Even lower temperatures have been used to study possible intermediate stages in the formation of the acyl enzyme. A tetrahedral intermediate (with a covalent bond between the substrate carbonyl carbon atom and the oxygen atom of the active site serine) (Fig. 2) had been suggested by analogy with nonenzymatic reactions. With rapid reaction techniques, spectrophotometric evidence has been obtained for an additional intermediate before the acyl enzyme in the case of chromophoric substrates. By using first the protein fluorescence emission (Fink and Wildi, 1974)... 

M. J. Hardman, Biochem. J. 197, 773 (1981) Pressure-jump combined with protein fluorescence changes are used to study LADH catalyzed reduction of acetaldehyde. The results show that the rate determining step is isomerization. 

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