DABCYL

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. 

Following previous work, Hagiwara and collaborators [71] recently prepared 5 -terminal acridone-labeled DNAs, using the succinimidyl ester 24 of the acridone acetic acid 23 reported before [69], and evaluated their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with 3 -dabcyl-tagged DNA... 

FRET probes have not only been generated to measure the phospholipase activity but to study its substrate specificity as well. Several substrates of PLA2 with a variety of head groups and labeled with a BODIPY dye and a Dabcyl quencher were created by Rose et al. and tested against different PLAs in cells to determine substrate specificity and intracellular localization [137], The specificity of PLA2 isoforms towards the number of double bonds in the sn2 position was evaluated with a small series of PENN derivatives. It was demonstrated that the cytosolic type V PLA2 preferred substrates with a single double bond [138],... 

Dabcyl-N-succinimidyl ester aminofluorescein Texas Red hydrazide... 

Fig. 23 (a) DNAzyme-based sensor design with two dabcyl quenchers and a FAM fluorophore (top) and mechanism of operation (bottom), (b) Fluorescence response before and after complete cleavage through Pb2+ inset contains the corresponding image of the DNAzyme probe in the absence (left) and presence of Pb2+ (after 2 min of reaction time, right). (Reprinted with permission from [150]. Copyright 2003 American Chemical Society)... 

Ac-DED(EDANS)EE-aAbu-Lac-SK(DABCYL) Ac-DD(EDANS)MEE-aAbu-Lac-SK(DABCYL)... 

EDANS = 5-[(2-aminoethyl)amino]naphthalenesulfonic acid DABCYL = 4- [4-(dimethylamino)phenyl]azo benzoic acid... 

Dichlorophenoxyacetic acid 522 DABCYL 820 Dairy products 671 Decoupler 831, 839-840, 856 Dehydrogenation of o-diphenol 371, 372... 

Of the three Edans/Dabcyl peptides synthesized, peptide F3 exhibited much better solubility in aqueous solution than the first two, and thus was tested for 3C... 

Tsourkas et al. (2003) reported dual FRET molecular beacon assays, where the donor probe was labeled with either Eu3+ or Tb3+ complex of DTPA-csl24-ethylenediamine (and no quenchers attached). For the Eu3+ complex, the acceptor probe was Cy5-labeled (and no quenchers attached) and for the Tb complex, the acceptor probe was labeled with Cy3 or ROX as a fluorophore and with dabcyl as a quencher. They demonstrated that these pairs of probes detected DNA targets ( 50-mer) with high S/N. 

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used EDANS and AMC 350 and 500 nm RhllO 485 and 535 nm TAMRA 535 and 595 nm PT14 405 and 450 nm. 4 = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. RhllO = rhodamine 110. yE = glutamic acid attached to RhllO via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-l-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label. 

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. 

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