New fluorogenic substrates for renin

A simple and sensitive fluorometric assay was developed to test renin activity within several hours. Two new fluorogenic peptides, Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (octapeptide-MCA) and a succinyl derivative of the octapeptide-MCA were synthesized and used as a renin substrate. Renin cleaved the substrates at the Leu-Leu bond, releasing Leu-Val-Tyr-MCA. Three amino acids of this product were then successively split off by the auxiliary enzyme, leucine aminopeptidase, to liberate free 7-amino-4-methylcoumarin (AMC). The generation of the fluorescent 7-amino-4-methylcoumarin was proportional to renin concentrations up to 100 mGoldblatt U/tube. The optimal pH of renin reaction for both substrates was 6.5 to 7.0. As low as 5 mGoldblatt U of renin could be detected by this method. This method was applied to the assay of renin during its purification.