A CONTINUOUS FLUOROMETRIC ASSAY FOR THE FELINE IMMUNODEFICIENCY VIRUSPROTEASE

A novel fluorogenic substrate for continuous feline immunodeficiency v irus (FIV) protease (PR) assay was developed in which 2-aminobenzoic a cid (Abz) and p-nitrophenylalanine (F(NO2)) were used as the fluoresce nt donor and acceptor, respectively. The 14-amino-acid fluorogenic sub strate of sequence RALTK(Abz)VQ similar to F(NO2)VQSKGR (similar to in dicates cleavage site) was modeled after a naturally occurring FIV PR capsid/nucleocapsid cleavage site in the gag polyprotein. The 2-aminob enzoyl group was attached to the epsilon amino group of a lysine (K:(A bz)) in position P3 and the F(NO2) is in position P1' in order to prom ote efficient intramolecular quenching prior to cleavage by FIV PR. We measured a K-m of 33 +/- 6 mu M and a k(cat) of 0.29 +/- 0.02 s(-1) f or the enzymatic hydrolysis of this fluorogenic substrate by FIV PR un der the conditions of our assay (0.05 M sodium citrate/0.1 M sodium ph osphate buffer, pH 5.25, 0.2 M NaCl, 0.1 mM EDTA, and 1 mM dithiothrei tol). This assay affords a rapid and convenient means for quantitating FIV PR activities and promises to be useful for judging the relative strength of inhibitors. (C) 1997 Academic Press.