Substrate V, Mca-RPPGFSAFK(Dnp), is a very sensitive internally quenched fluorescent substrate for endothelin-converting enzyme-1 (ECE-1) a membrane-bound zinc metallopeptidase that is related to neprilysin in amino acid sequence. The kcat/Km value for the hydrolysis by ECE-1 was 1.9 x 10⁷ M⁻¹s⁻¹ and 1.7 x 10⁶ M⁻¹s⁻¹ for the hydrolysis by neprilysin. This FRET substrate was hydrolyzed rapidly by stromelysin 1 (MMP-3) with kcat/Km= 218000 M⁻¹s⁻¹ and very slowly by gelatinase B (MMP-9) (kcat/Km= 10100 M⁻¹s⁻¹). There was no hydrolysis by MMP-1 and MMP-2. Mca-RPPGFSAFK(Dnp) has also found use as substrate for insulin-degrading enzyme (IDE) and ACE2.