A FLUOROMETRIC ASSAY FOR HIV-PROTEASE ACTIVITY USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

被引:37
作者
TAMBURINI, PP
DREYER, RN
HANSEN, J
LETSINGER, J
ELTING, J
GOREWILLSE, A
DALLY, R
HANKO, R
OSTERMAN, D
KAMARCK, ME
YOOWARREN, H
机构
[1] Molecular Therapeutics, Inc., West Haven, CT 06516
关键词
D O I
10.1016/0003-2697(90)90095-Q
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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-SQNYPIV 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 peptideenzyme 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. © 1990.
引用
收藏
页码:363 / 368
页数:6
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