A radiometric assay for HIV-1 protease

✍ Scribed by Lawrence J. Hyland; Brian D. Dayton; Michael L. Moore; A.Y.L. Shu; J. Richard Heys; Thomas D. Meek

• Publisher: Elsevier Science
• Year: 1990
• Tongue: English
• Weight: 927 KB
• Volume: 188
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(90)90628-m

✦ Synopsis

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.