Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease.
A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-beta-naphthylamide). Fluorescence due to the release of Pro-MeO-beta-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kappa cat for the hydrolysis of N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta- naphthylamide by HIV-1 protease were calculated to be 2.0 +/- 0.2 mM and 75 +/- 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.
Tyagi
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SC
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794.
Carter
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CA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D009842:Oligopeptides; C074742:N(alpha)-benzoyl-arginyl-glycyl-phenylalanyl-prolyl-methoxy-beta-naphthylamide; D016333:HIV Protease