A fluorometric assay for HIV-protease activity using high-performance liquid chromatography |
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| Authors: | P P Tamburini R N Dreyer J Hansen J Letsinger J Elting A Gore-Willse R Dally R Hanko D Osterman M E Kamarck |
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| Affiliation: | Molecular Therapeutics, Inc., West Haven, Connecticut 06516. |
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| 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. |
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