Edans fluorescent donor

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. 

An example of this technology is the HIV protease assay shown in Fig. 9, which was published by Wang and co-workers [67]. The peptide substrate is labeled at the amino terminus with EDANS (5-((2 -aminoethyl)amino)naphthalene-l-sulfonic acid) as a donor fluorophore and at the carboxyl terminus with DABCYL (4-((4 -(di-methylamino)phenyl)azo)benzoic acid) as the acceptor chromophore. In the intact peptide, fluorescence resonance energy transfer (FRET) from EDANS to DABCYL results in quenching of the EDANS fluorescence. On cleavage of the peptide by HIV protease, the fluorescence of EDANS is restored. 

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. 

Fio. 5. (A) Use of FRET technique for measuring HIV protease activity. The polypeptide was Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Lys(DABCYL)-Arg. Because a simple system which could be commercialized was desired (fluorescence means HIV activity, no fluorescence means no HIV activity), the nonfluorescent acceptor was beneficial because it does not contribute signal after cleavage. This system also has the attribute that detailed information about the dyes and their energy transfer properties are not needed. (B) Good overlap between donor emission (maximum at 490 nm, excitation at 340 nm) and acceptor absorbance (maximum of 28,000 cm" ). (From Molecular Probes Catalog. )... 

---