A simple, continuous fluorometric assay for HIV protease.
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.
Toth
M V
MV
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri.
Marshall
G R
GR
D000595:Amino Acid Sequence; D005470:Fluorometry / Q000379:methods; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D016333:HIV Protease