Abstract
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-NH 2, which is based on the p17–p24 cleavage site found in the viral polyprotein substrate Pr55 gag . 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.
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