Figure 3.
Binding affinities of PDs measured by fluorescence polarisation. (A) Representative binding curve of PDs (PD3) titrated with PDEδ. The FP value was measured in PBS buffer (pH 7.2) containing 8 concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 μM) of PDEδ mixed with 0.5 μM PDs. Ex: 440 nm, Em: 526 nm; (B) Kd value and half-life of each compound; (C, D) Kinetic trace of PD3 and PD7 in the competition assay. PD3 was shown as a compound with the longest retention time (slow dissociation), whereas PD7 was shown as a compound with the shortest retention time (fast dissociation). The half-life was obtained by a competition binding assay using excess deltarasin. The optimal concentration of PDEδ for the competition assay was determined using the Kd value and PD concentration (0.5 μM) for making more than 68% of the binding complex. Ex: 430 nm, Em: 520 nm.