Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.
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.
Betageri
R
R
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877-0368.
Hopkins
J L
JL
Thibeault
D
D
Emmanuel
M J
MJ
Chow
G C
GC
Skoog
M T
MT
de Dreu
P
P
Cohen
K A
KA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019793:Fluorescein; D005452:Fluoresceins; D016333:HIV Protease / Q000302:isolation & purification*; D017320:HIV Protease Inhibitors / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D005452:Fluoresceins; D017320:HIV Protease Inhibitors; D010455:Peptides; D016333:HIV Protease; D019793:Fluorescein