Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease

Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 2) between the ( p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of ≤20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05–0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.