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. 

Fig. 12 a Quenching assay where QSY-7-labeled DNA strand (DNA-QTL) is presented to microsphere and microsphere-bound biotinylated ALF capture strand, b Competition assay for detection of target DNA. Variable amounts of ALF target are added to microsphere and bound ALF capture strand. This is then subjected to a fixed amount of QSY-7-labeled DNA. The amount of quenching is dependent on the amount of ALF target already bound on the microsphere. (Reprinted with permission from Ref. [33]. Copyright 2002 American Chemical Society)... 

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. 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

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

The azulenyl furanone 60 was a key intermediate in the synthesis of the monofunctional azulenyl squaraine dye NIRQ700 61 (NIRQ = near-infrared quencher) <2003TL3975>. The resulting nonfluorescent squaraine dye absorbed in the 600-700 nm range and can potentially be used to quench a number of available NIR fluorochromes in order to extend the spectrum of biological quenching assays. 

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. 

The quench assay described by Peppard et al. [68] to determine the activity of matrix metalloproteases (MMP) is based on a generic substrate PEGEAARK that is... 

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

The SA sensory polymer concept was first demonstrated in an analyte-induced quenching assay (Figure la) where the ligand to the receptor site was a quencher [4]. A key feature of the polymeric system was that the exciton migration caused greatly enhanced quenching relative to a chromophore bearing only one of the receptors. This is established by... 

Figure 5 Typical rapid chemical quench assay of single-nucleotide incorporation by Pol j3. (a) Single-turnover time course of DNA product formation fitted to a single exponential equation [DNAn+i] =A (b) Plot of rate versus...

Figure 13 Comparison of Pol /3 catalyzed single-nucleotide incorporation at 10% and 35% glycerol, pH 8.3. (a) 2-AP fluorescence stopped-flow assays show that both phases of the fluorescence change are slowed down at increased glycerol concentration, (b) In contrast, rapid chemical quench assays demonstrate that the rate of nucleotide incorporation remains unaffected by the altered glycerol concentration. Adapted with permission from M. Bakhtina M. P. Roettger S. Kumar ...

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. 

Microspheres with a capture ligand are presented to a quencher-labeled DNA strand. Quenching occurs if this strand is complementary and binds to the capture ligand. This is a direct quenching assay. 

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

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