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 util ize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled subst rate and products is achieved with a run time of 10 min and UV detecti on. 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 tak es advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluoresce in label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluores cence detection which provides essentially total freedom from interfer ence. Synthesis of fluorescein-labeled peptide substrate is accomplish ed by solid-phase peptide synthesis.