Proteins fluorescent probes

Morii, H. Ichimiua, K. Uedaira, H. Asymmetric inclusion by de novo designed proteins fluorescence probes studies on amphiphilic a-helix bundles. Proteins Struct., Fund, Genet. 1991,11,133-141. 

Kolubayev T, Geacintov N E, Paillotin G and Breton J 1985 Domain sizes in chloroplasts and chlorophyll-protein complexes probed by fluorescence yield quenching induced by singlet-triplet exciton annihilation Biochimica Biophys. Acta 808 66-76... 

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

From the results obtained, it was found that compound 10a showed very high fluorescence intensity in the presence of the BSA and BSA/SDS mixture ( F 0.27) together with a noticeable emission enhancement. The presence of dimethyl indo-lenyl increased the affinity of the dyes to both native and denatured proteins. The authors proposed compound 10a for further studies as fluorescent probes for protein detection. 

Due to the fact that biarsenical-TC complex is stable under the denaturing conditions typically used for gel electrophoresis of proteins and has a molecular weight of less than 2 KDa, when bonded to the biarsenical dye [157], Kottegoda and collaborators [161] studied the biarsenical dyes, as fluorescent probes for in vitro, cellular peptide, and proteins studies using capillary electrophoresis. 

Yang WC, Schmerr MJ, Jackma R et al (2005) Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe. Anal Chem 77 4489 1494... 

Wu XL, Tian M, Fie HZ et al (2009) Synthesis and biological applications of two novel fluorescent proteins-labeling probes. Bioorg Med Chem Lett 19 2957-2959... 

Kamoto M, Umezawa N, Kato N, Higuchi T (2009) Turn-on fluorescent probe with visible light excitation for labelling of hexahistidine tagged protein. Bioorg Med Chem Lett 19 2285-2288... 

The sulfo-gmup containing squaraine-taurine probe 8 [62] displayed a very high affinity for BSA and other blood proteins. This probe exhibits low quantum yields and short fluorescence lifetimes in water and a significant increase of these characteristics upon binding to proteins. 

Squaraine dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e were used to measure different proteins such as BSA, HSA, ovalbumin, avidin from hen egg white, lysozyme, and trypsin (Fig. 12) [58]. It is difficult to predict correlations between the dyes structures and the affinity or sensitivity of the dyes for different proteins. All squaraine probes exhibit considerable fluorescence increases in the presence of BSA. Dicyanomethylene-squaraine 41c is the brightest fluorescent probe and demonstrates the most pronounced intensity increase (up to 190 times) in presence of BSA. At the same time, the fluorescent response of the dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e in presence of other albumins (HSA and ovalbumin) is, in general, significantly lower (intensity increases up to 24 times). Dicyanomethylene-squaraine 41a and amino-squaraines 39a and 39b are the most sensitive probes for ovalbumin. Dyes 41d, 10b, and 41e containing an A-carboxyalky I -group demonstrate sufficient enhancement (up to 16 times) in the presence of avidin. Nevertheless, the presence of hydrolases like lysozyme or trypsin has only minor effects on the fluorescence intensity of squaraine dyes. 

Umezawa K, Citterio D, Suzuki K (2008) Water-soluble NIR fluorescent probes based on squaraine and their application for protein labeling. Anal Sci 24 213-217... 

Sytnik A, Kasha M (1994) Excited-state intramolecular proton transfer as a fluorescence probe for protein binding-site static polarity. Proc Natl Acad Sci USA 91 8627-8630... 

The aim of this Chapter is to review a method by which fluorescence properties of organic dyes can, in general, be predicted and understood at a microscopic (nm scale) by interfacing quantum methods with classical molecular dynamics (MD) methods. Some review of our extensive applications [1] of this method to the widely exploited intrinsic fluorescence probe in proteins, the amino acid tryptophan (Trp) will be followed by a discussion of electrochromic membrane voltagesensing dyes. 

Sackett, D. L. and Wolff, J. (1987). Nile red as a polarity-sensitive fluorescent-probe of hydrophobic protein surfaces. Anal. Biochem. 167, 228-234. 

Kenner, R. A. and Aboderin, A. A. (1971). A new fluorescent probe for protein and nucleoprotein conformation. Binding of 7-(p-Methoxyben-zylamino) -4-nitrobenzoxadiazole to bovine trypsinogen and bacterial ribosomes. Biochemistry 10, 4433 1440. 

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

SAMSA-fluorescein, 5- [2(and 3)-5-(acetylmercapto)-succinoyl]amino fluorescein, is a fluorescent probe containing a protected sulfhydryl group. In its protected state, the compound is unre-active. The acetyl-protecting group can be removed by treatment with dilute NaOH at pH 10.0 (Figure 9.9). The resulting free sulfhydryl derivative can be used to label thiol-reactive crosslinkers or to couple with sulfhydryl residues on proteins and other molecules. After activating... 

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