Renin FRET Substrate I

DABCYL-GABA-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS (also called Renin Substrate I in some literature) contains a renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen. This fluorogenic peptide substrate is used to continuously measure the proteolytic activity of human renin. The assay relies upon FRET-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate is shown to be consistent with a simple substrate inhibition model with a substrate Km approximately equal to 1.5 mM at physiological pH. The substrate can detect renin as low as 30 ng/mL after an incubation of only 3-5 min.

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