Fluorescence‐based continuous assay for the aspartyl protease of human immunodeficiency virus‐1

FEBS Letters

Andrews, Glenn C.¹  Geoghegan, Kieran F.¹  Contillo, Leonard G.¹  Spencer, Robin W.¹  Danley, Dennis E.¹ 

[1] Pfizer Central Research, Eastern Point Road, Groton, CT 06340, U.S.A.

关键词: Human immunodeficiency virus;    Proteinase;    Fluorometric assay;    Energy transfer;    HIV-1;    human immunodeficiency virus-1;    AIDS;    acquired immune deficiency syndrome;    Boc;    tert-butoxycarbonyl;    Bzl;    benzyl;    Brz;    2-bromobenzyloxycarbonyl;    CHO;    formyl;    PAM;    2-pyridinealdoximine methiodide;    DMSO;    dimethylsulfoxide;    Dns;    (dansyl);    5-(dimethylamino)naphthalene-1-sulfonyl;    DCM;    dichloromethane;    DMF;    dimethylformamide;    TFA;    trifluoroacetic acid;    FAB-MS;    fast atom bombardment mass spectrometry;   

DOI  :  10.1016/0014-5793(90)80168-I

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

5-Dimethylaminonaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Trp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (λ_ex 290 nm, λ_em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations <60 μM, initial reaction velocity increased as a linear function of substrate concentration, with k _cat/K _M= 9700 M⁻¹s⁻¹. Limited solubility and internal fluorescence quenching precluded a determination of K _M for Dns-SQNYPIVW, but this was clearly > 100 μM.

【 授权许可】

Unknown