Title | Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay. | ||
Author | Hopkins, J L; Betageri, R; Cohen, K A; Emmanuel, M J; Joseph, C R; Bax, P M; Pallai, P V; Skoog, M T | ||
Journal | J Biochem Biophys Methods | Publication Year/Month | 1991-Sep |
PMID | 1658106 | PMCID | -N/A- |
Affiliation | 1.Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut 06877. |
The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7\' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.