DABCYL-GABA-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS (also called Renin Substrate I in some literature) contains a kidney that appears in the human N-terminal peptide Angiotensinogen, a cleavage site. The cleavage of the substrate takes place in particular on the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen. The fluorescent peptide substrate is used to continuously measure the proteolytic activity of human renin. The assay relies on FRET-mediated quenching of intramolecular fluorescence in the intact peptide substrate. The good energy overlap of the EDANS excited state and DABCYL absorption and the relatively long excited state lifetime of the EDANS fluorophore will lead to effective fluorescence quenching. The cleavage of the substrate by renin causes the peptidyl-EDANS fragment to be released from the vicinity of the DABCYL receptor, restoring the fluorescence of the EDANS part. This leads to an increase in fluorescence intensity over time, which is directly related to the degree of substrate cleavage. The kinetics of this substrate catalyzed by renin was proved to be consistent with a simple substrate inhibition model, and the substrate Km was approximately equal to 1.5 mM at physiological pH. After only incubating for 3-5 minutes, the substrate can detect renin as low as 30 ng/mL.
Wang GT, et al. (1993). A continuous fluorescence assay of renin activity. Anal Biochem 210, 351-9, Nakamura N, et al. (1991). Identification of the active site of human renin with use of new fluorogenic peptides. J Biochem (Tokyo) 109, 741-5, Murakami K, et al. (1981). New fluorogenic substrates for renin. Anal Biochem 110, 232-9.
Sequence (One-Letter Code)
Sequence (Three-Letter Code)
DABCYL - g - Abu - Ile - His - Pro - Phe - His - Leu - Val - Ile - His - Thr - EDANS