Fluorescence quenching assay

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Karvinen, J. et al. 2002. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase 3. J. Biomol. Screen. 1, 223-231. 

Protease is used to screen bead library for protease substrate positive beads in this fluorescent-quench assay fluoresce because the quencher is cleaved. 

Fluorescence intensity assays detect an increase or a decrease in the strength of a fluorescence emission. Based on the origin of this change in fluorescence, the two main classes are fluorogenic assays and fluorescence quench assays. 

A EXPERIMENTAL FIGURE 18-5 In vitro fluorescence quenching assay can detect phospholipid flippase activity of... 

FIGURE 9.8 Schematic of the fluorescence-quenching assay used to determine if micro-wave irradiation (MW) is capable of melting dsDNA. Open circle = fluorophore (fluorescein) closed circle = fluorescence quencher). (Reproduced with permission from Edwards, W. E Young, D. D. Deiters, A. Org. Biotnol. Chem. 2009, 7,2506-2508. Copyright Royal Society of Chemistry.)... 

S. Blincko, D. Colbert, W. G. John, and R. Edwards, A reliable non-separation fluorescence quenching assay for total glycated serum protein A simple alternative to nitroblue tetrazohum reduction, Xnn. Clin. Biochem., 37 (2000) 372-379. 

Cassie Baxter mode 2 Desorption/ionization on silicon, (DIOS) 4 Fluorescence quenching assay 6 Immunocapture and MALDI (iMALDI MS) 5... 

Organic fluorophores are commonly used as reporters in a large number of in vitro bioassays, such as quantitative immunoassays and fluorescence quenching assays for macromolecular interactions. High sensitivity has been realized with the use of organic dyes, but the spectral properties of QDs could lead to further improvements. Research in the appHcation of QDs for in vitro bioanalysis has been advanced primarily by Mattoussi and his coworkers at the U.S. Naval Research Laboratory [9,10], and can be divided into two areas immunoassays and biosensors. 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

Payre, N., Cottaz, S. and Driguez, H. (1995). Chemoenzymatic synthesis of a modified pentasaccharide as a specific substrate for a sensitive assay of alpha-amylase by fluorescence quenching. Angew. Chem. Int. Ed. Engl. 34, 1239-1241. 

Brown et al.68 have developed a cellulose plate with a fluorescent indicator. Compounds are developed in 3.0% (w/v) NHfc.Cl and detected by fluorescence quenching. These authors also use 0.5% mercaptoethanol in their mobile phase, but this is only to prevent oxidation of the labile reduced pteridines, which are not adequately protected by substitution at the N5 position. Since neutral or alkaline solutions of leucovorin are relatively stable in air, this precaution may not be required for routine assay. 

Quenching can actually be used to create analytical methods. An example is that of fluorescent antibody assay. Here, a fluor is bound to an antibody but the binding constant is less than that for the antibody-antigen pair for which the antibody was generated. Binding quenches the fluorescence of the fluor. When a solution containing the native antigen is mixed with the fluor complex, the fluor is released and can fluoresce normally. There are numerous examples of this approach. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

Meldal et al. developed a novel protease assay based on the long-range resonance energy transfer (FRET) [25] fluorescence quenched (EQ) pair 3-nitrotyro-sine/2-aminobenzoic acid. This served to characterize enzyme specificity by direct visual inspection of the resin beads (33). 

Selected entries from Methods in Enzymology [vol, page(s)] Design, 178, 551 immunoassay, 178, 542 production, 178, 531 purification, 178, 543 substrates and enzymatic assay, 178, 544 derivatization with spectroscopic probe, 178, 567 ester cleavage assays, 178, 565 fluorescence quenching binding assay, 178,... 

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

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