Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: Application to human immunodeficiencyviral protease

A homogeneous time-resolved fluorescence (HTRF) assay has been developed fo r human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be meas ured. Potential substrates were evaluated by HTRF with the best results bei ng obtained using (LCB)K(4)AVSQN beta-Nap-PIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detecti on conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with appar ent first-order kinetics with a K-m of 37.8 +/- 8.7 mu M and a k(cat) of 0. 95 +/- 0.07 s(-1). Examination of the first-order rate constant versus enzy me concentration suggested a K-d of 9.4 +/- 2.7 nM for the HIV protease mon omer-dimer equilibrium. The HTRF assay was also utilized to measure the inh ibition of the enzyme by two known inhibitors. (C) 1999 Academic Press.