A continuous fluorometric assay for the feline immunodeficiency virus protease.
Journal Article (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 approximately F(NO2)VQSKGR (approximately 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 microM and a kcat 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, SB
Published Date
- December 1997
Published In
Volume / Issue
- 254 / 2
Start / End Page
- 226 - 230
PubMed ID
- 9417781
Electronic International Standard Serial Number (EISSN)
- 1096-0309
International Standard Serial Number (ISSN)
- 0003-2697
Digital Object Identifier (DOI)
- 10.1006/abio.1997.2407
Language
- eng