A radiometric assay for HIV-1 protease
- Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA)
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.
- OSTI ID:
- 6277868
- Journal Information:
- Analytical Biochemistry; (USA), Vol. 188:2; ISSN 0003-2697
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PEPTIDE HYDROLASES
RADIOASSAY
AMINO ACID SEQUENCE
ENZYME ACTIVITY
ION EXCHANGE CHROMATOGRAPHY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
CHROMATOGRAPHY
ENZYMES
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
MOLECULAR STRUCTURE
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques