A simple, continuous fluorometric assay for HIV protease

MIHALY V. TOTH, GARLAND R. MARSHALL

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206 Scopus citations

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.

Original languageEnglish
Pages (from-to)544-550
Number of pages7
JournalInternational Journal of Peptide and Protein Research
Volume36
Issue number6
DOIs
StatePublished - Dec 1990

Keywords

  • HIV protease inhibitors
  • fluorogenic substrate
  • fluorometric assay

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