A fluorometric assay for HIV-protease activity using high-performance liquid chromatography

Anal Biochem. 1990 May 1;186(2):363-8. doi: 10.1016/0003-2697(90)90095-q.

Abstract

A rapid sensitive method for the quantification of in vitro HIV-protease activity has been developed on the basis of the endoproteolytic conversion of N-Dns-SQ-NYPIV to N-Dns-SQNY. The use of the N-dansyl group as a fluorescence label was shown to not significantly alter the apparent kinetic parameters for the peptide-enzyme interaction. Using fluorescence detection, the dansylated product and unconverted substrate are detected in a single rapid (3 min) isocratic reverse-phase HPLC separation in quantities as low as 0.2 pmol. The method is highly reproducible and suited to a variety of applications including the analysis of large sample numbers and rigorous enzymological studies.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid*
  • Dansyl Compounds
  • Endopeptidases / analysis*
  • Fluorescent Dyes
  • Fluorometry*
  • Gene Products, pol / analysis*
  • HIV Protease
  • Molecular Sequence Data
  • Peptide Fragments / metabolism
  • Recombinant Fusion Proteins / analysis
  • Retroviridae Proteins / analysis*
  • Substrate Specificity

Substances

  • Dansyl Compounds
  • Fluorescent Dyes
  • Gene Products, pol
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Retroviridae Proteins
  • Endopeptidases
  • HIV Protease
  • dansyl chloride