Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV.
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.
Nashed
N T
NT
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Louis
J M
JM
Sayer
J M
JM
Wondrak
E M
EM
Mora
P T
PT
Oroszlan
S
S
Jerina
D M
DM
D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000032:analysis*; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D010450:Endopeptidases; D016333:HIV Protease