A radiometric assay for HIV-1 protease.
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.
Hyland
L J
LJ
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Dayton
B D
BD
Moore
M L
ML
Shu
A Y
AY
Heys
J R
JR
Meek
T D
TD
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D016333:HIV Protease / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014316:Tritium
D014316:Tritium; D016333:HIV Protease