Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease |
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Authors: | T A Tomaszek V W Magaard H G Bryan M L Moore T D Meek |
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Affiliation: | Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406. |
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Abstract: | Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (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 less than 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. |
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