Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.
J Biochem Biophys Methods
; 27(3): 191-7, 1993 Oct.
Article
em En
| MEDLINE
| ID: mdl-8258639
ABSTRACT
The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Protease de HIV
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Biochem Biophys Methods
Ano de publicação:
1993
Tipo de documento:
Article