New fluorogenic substrates for N-arginine dibasic convertase

N-Arginine dibasic (NRD) convertase is a recently described peptidase capab le of selectively cleaving peptides between paired basic residues. The char acterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp a nd Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoy l moiety that is quenched in the intact peptide by a 2,4-dinitrophenyl moie ty. Cleavage by NRD convertase at the Arg-Arg sequence results in an increa se of fluorescence. NRD convertase cleaves these peptides efficiently and w ith high specificity as observed by both HPLC and fluorescence spectroscopy . The rate of hydrolysis of the fluorogenic substrates is proportional to e nzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic param eters for the fluorescent peptides (K-m values of similar to 1.0 mu M, and V-max values of similar to 1 mu M/(min . mg) are similar to those obtained with peptide hormones as substrates. (C) 1999 Academic Press.