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Fluorescence‐based continuous assay for the aspartyl protease of human immunodeficiency virus‐1
Author(s) -
Geoghegan Kieran F.,
Spencer Robin W.,
Danley Dennis E.,
Contillo Leonard G.,
Andrews Glenn C.
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80168-i
Subject(s) - protease , virology , fluorescence , human immunodeficiency virus (hiv) , chemistry , virus , biology , biochemistry , enzyme , quantum mechanics , physics
5‐Dimethylaminonaphthalene‐1‐sulfonyl‐Ser‐Gln‐Asn‐Tyr‐Pro‐Ile‐Val‐Trp (Dns‐SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus‐1. In intact substrate, fluorescence of Trp (λ ex 290 nm, λ em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease‐catalyzed cleavage at the Tyr‐Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations <60 μM, initial reaction velocity increased as a linear function of substrate concentration, with k cat / K M = 9700 M −1 s −1 . Limited solubility and internal fluorescence quenching precluded a determination of K M for Dns‐SQNYPIVW, but this was clearly > 100 μM.