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

✍ Scribed by Paul P. Tamburini; Robert N. Dreyer; Jutta Hansen; Jack Letsinger; Jim Elting; Ann Gore-Willse; Robert Dally; Rudolf Hanko; David Osterman; Michael E. Kamarck; Heeja Yoo-Warren

Publisher
Elsevier Science
Year
1990
Tongue
English
Weight
883 KB
Volume
186
Category
Article
ISSN
0003-2697

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✦ Synopsis

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.

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