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 (λex 290 nm, λem 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−1s−1. Limited solubility and internal fluorescence quenching precluded a determination of K M for Dns-SQNYPIVW, but this was clearly > 100 μM.
