Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer

Abstract

Synthesis of fluorogenic peptide substrate of HIV-I protease Dns-SQNYPIVWL which corresponds to the p17/p24 cleavage site for HIV-l protease have been performed. This fluorogenic substrate was based on the fluorescence resonance energy transfer between donor – Trp residue, and acceptor – dansyl group in the intact peptide. Hydrolysis of substrate by recombinant HIV-I protease resulted in the time-dependent increase of Trp fluorescence and decrease of dansyl fluorescence measured at 350 and 500 nm, respectively, due to the break of resonance energy transfer between donor and acceptor fluorophors. Hydrolysis of fluorogenic peptide substrate was studied also by reversed phase HPLC and two peptide fragments after cleavage of substrate have been detected. Kinetic constants of hydrolysis for this fluorogenic peptide substrate by HIV-I protease were calculated from Lineweaver – Burk plots: K