Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.
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.
Hopkins
J L
JL
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut 06877.
Betageri
R
R
Cohen
K A
KA
Emmanuel
M J
MJ
Joseph
C R
CR
Bax
P M
PM
Pallai
P V
PV
Skoog
M T
MT
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D005452:Fluoresceins; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D012229:Rhinovirus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins
D005452:Fluoresceins; D010455:Peptides; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases