A fluorogenic assay for endothelin-converting enzyme

The potent vasoconstrictor peptide endothelin-1 is proposed to arise via proteolysis of a precursor molecule, "big endothelin," at a unique cleavage site. To aid in the identification of a putative endothelin-converting enzyme, we have developed an assay that mimics the relevant cleavage reaction. The assay takes advantage of the intramolecular fluorescence energy transfer between the scissile-site tryptophan and a dansyl moiety present in the same synthetic substrate. Cleavage of the peptide chain separates the fluorophore and quencher, resulting in an increase in fluorescence. The assay has been validated using chymotrypsin as a model protease and has been employed in the identification of novel endothelin-converting enzyme activities.