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...

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Bibliographic Details
Published inInternational journal of peptide and protein research Vol. 36; no. 6; p. 544
Main Authors Toth, M V, Marshall, G R
Format Journal Article
LanguageEnglish
Published Denmark 01.12.1990
Subjects
Amino Acid Sequence
Fluorometry - methods
HIV Protease - metabolism
Humans
Kinetics
Molecular Sequence Data
Substrate Specificity
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Summary: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.
ISSN:0367-8377
DOI:10.1111/j.1399-3011.1990.tb00994.x