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.
Duke Scholars
Altmetric Attention Stats
Dimensions Citation Stats
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- ortho-Aminobenzoates
- Substrate Specificity
- Phenylalanine
- Peptides
- Kinetics
- Immunodeficiency Virus, Feline
- Fluorometry
- Endopeptidases
- DNA, Viral
- Biochemistry & Molecular Biology
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- ortho-Aminobenzoates
- Substrate Specificity
- Phenylalanine
- Peptides
- Kinetics
- Immunodeficiency Virus, Feline
- Fluorometry
- Endopeptidases
- DNA, Viral
- Biochemistry & Molecular Biology