Use of a Phosphotyrosine-Antibody Pair as a General Detection Method in Homogeneous Time-Resolved Fluorescence: Application to Human Immunodeficiency Viral Protease

Abstract

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for 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 measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNβ-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50of 7.7 ± 0.3 nM under optimized detection 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 apparent first-order kinetics with aKmof 37.8 ± 8.7 μM and akcatof 0.95 ± 0.07 s−1. Examination of the first-order rate constant versus enzyme concentration suggested aKdof 9.4 ± 2.7 nM for the HIV protease monomer–dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.