title: A simple, continuous fluorometric assay for HIV protease.
abstract: Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site-derived hexapeptide substrate. Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH2, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO2-Phe at the P1' position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96-well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.
authors.affiliation: Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: M V
authors.initials: MV
authors.lastname: Toth
authors.forename: G R
authors.initials: GR
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D005470:Fluorometry / Q000379:methods; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D016333:HIV Protease