A continuous fluorometric assay for the feline immunodeficiency virus protease

Journal Article

A novel fluorogenic substrate for continuous feline immunodeficiency virus (FIV) protease (PR) assay was developed in which 2-aminobenzoic acid (Abz) and p-nitrophenylalanine (F(NO2)) were used as the fluorescent donor and acceptor, respectively. The 14-amino-acid fluorogenic substrate of sequence RALTK(Abz)VQ F(NO2)VQSKGR (~ indicates cleavage site) was modeled after a naturally occurring FIV PR capsid/nucleocapsid cleavage site in the gag polyprotein. The 2-aminobenzoyl group was attached to the epsilon amino group of a lysine (K(Abz)) in position P3 and the F(NO2) is in position P1 in order to promote efficient intramolecular quenching prior to cleavage by FIV PR. We measured a K(m) of 33 ± 6 μM and a k(cat) of 0.29 ± 0.02 s-1 for the enzymatic hydrolysis of this fluorogenic substrate by FIV PR under the conditions of our assay (0.05 M sodium citrate/0.1 M sodium phosphate buffer, pH 5.25, 0.2 M NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol). 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.

Full Text

Duke Authors

Cited Authors

  • Fitzgerald, MC; Laco, GS; Elder, JH; Kent, SBH

Published Date

  • 1997

Published In

Volume / Issue

  • 254 / 2

Start / End Page

  • 226 - 230

PubMed ID

  • 9417781

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1006/abio.1997.2407