A continuous fluorometric assay for the feline immunodeficiency virus protease.
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.
Fitzgerald
M C
MC
Scripps Research Institute, La Jolla, California 92037, USA. mfitz@scripps.edu
Laco
G S
GS
Elder
J H
JH
Kent
S B
SB
D004279:DNA, Viral; D010450:Endopeptidases / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology*; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D004279:DNA, Viral; D010455:Peptides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C040239:4-nitrophenylalanine; D010649:Phenylalanine; D010450:Endopeptidases