A fluorometric assay for HIV-protease activity using high-performance liquid chromatography.
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.
Molecular Therapeutics, Inc., West Haven, Connecticut 06516.
P P
PP
Tamburini
R N
RN
Dreyer
J
J
Hansen
J
J
Letsinger
J
J
Elting
A
A
Gore-Willse
R
R
Dally
R
R
Hanko
D
D
Osterman
M E
ME
Kamarck
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D010450:Endopeptidases / Q000032:analysis*; D005456:Fluorescent Dyes; D005470:Fluorometry; D015684:Gene Products, pol / Q000032:analysis*; D016333:HIV Protease; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012191:Retroviridae Proteins / Q000032:analysis*; D013379:Substrate Specificity
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D015684:Gene Products, pol; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D010450:Endopeptidases; D016333:HIV Protease; C029038:dansyl chloride