2. Academic Validation
  3. Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV

Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV

  • Biochem Biophys Res Commun. 1989 Sep 15;163(2):1079-85. doi: 10.1016/0006-291x(89)92331-0.
N T Nashed 1,  J M Louis,  J M Sayer,  E M Wondrak,  P T Mora,  S Oroszlan,  D M Jerina
Affiliations

Affiliation

  • 1 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
PMID: 2551268 DOI: 10.1016/0006-291x(89)92331-0
Abstract

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of Peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.

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