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

• Published in: Biochemical and biophysical research communications Vol. 163; no. 2; pp. 1079 - 1085
• Main Authors: Nashed, Nashaat T., Louis, John M., Sayer, Jane M., Wondrak, Ewald M., Mora, Peter T., Oroszlan, Stephen, Jerina, Donald M.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 15.09.1989; Elsevier
• Subjects: AIDS/HIV; Analytical, structural and metabolic biochemistry; Avian Leukosis Virus - enzymology; Avian Myeloblastosis Virus - enzymology; Biological and medical sciences; Chromatography, High Pressure Liquid; Endopeptidases - analysis; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HIV Protease; HIV-1 - enzymology; human immunodeficiency virus; Hydrolases; Kinetics; proteinase; spectrophotometry; Spectrophotometry, Ultraviolet; Substrate Specificity; Oncovirinae; HIV-1 virus; Enzyme; Proteinase; Retroviridae; Avian myeloblastosis virus; Virus; Assay; Enzymatic activity; Type C oncovirus; Substrate specificity; Human immunodeficiency virus; Kinetic parameter; Spectrophotometry
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(89)92331-0

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO 2)-Pro-Val-Val-NH 2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO 2)-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 (Δε = 1000) and an increase at 316 nm (Δε = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 μl) 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 ofV max/K m were obtained.