A RADIOMETRIC ASSAY FOR HIV-1 PROTEASE

被引：21
作者：
HYLAND, LJ
DAYTON, BD
MOORE, ML
SHU, AYL
HEYS, JR
MEEK, TD
机构：
[1] SMITHKLINE BEECHAM PHARMACEUT,DEPT PEPTIDE CHEM,KING OF PRUSSIA,PA 19406
[2] SMITHKLINE BEECHAM PHARMACEUT,KING OF PRUSSIA,PA 19406
来源：
ANALYTICAL BIOCHEMISTRY | 1990年 / 188卷 / 02期
关键词：
D O I：
10.1016/0003-2697(90)90628-M
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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. © 1990.
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页码：408 / 415
页数：8
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