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.