Figure 2

ITC analysis of eicosanoid binding. For cysteinyl leukotriene experiments, the calorimeter cell was filled with P. papatasi D7 protein AGE83092 (A–C) or P. duboscqi ABI15936 (E–G) at 2 μM in Tris-buffered saline. The syringe contained 20 μM LTC₄ (A,E), LTD₄ (B,F) and LTE₄ (C,G) in the same buffer. Injections (10 µL) were spaced at 300 s intervals. For U46619 (D,H), the protein concentration in the cell was 5 µM for AGE83092 (D) and 10 µM for ABI15936 (H). The syringe concentration was 50 µM AGE83092 and 100 µM for ABI15936. Heats were recorded on a VP-ITC MicroCalorimeter and data were fit using a single binding site model on the MicroCal software package (Origin 7) for calculation of thermodynamic parameters. Titration curves are representative of at least 3 measurements. Thermodynamic parameters: Panel A K_a = 1.7 × 10⁸ M⁻¹, ΔH = −10.3 kcal/mol, N = 1.1 sites; Panel B K_a = 5.7 × 10⁸ M⁻¹, ΔH = −10.2 kcal/mol, N = 1.1 sites; Panel C K_a = 6.3 × 10⁷ M⁻¹, ΔH = −11.3 kcal/mol, N = 1.1 sites; Panel D K_a = 1.33 × 10⁶ M⁻¹, ΔH = −8.5 kcal/mol, N = 0.6 sites; Panel E K_a = 3.1 × 10⁸ M⁻¹, ΔH = −8.6 kcal/mol, N = 1.3 sites; Panel F K_a = 2.4 × 10⁸ M⁻¹, ΔH = −8.9 kcal/mol, N = 1.3 sites; Panel G K_a = 3.4 × 10⁷ M⁻¹, ΔH = −7.0 kcal/mol, N = 1.3 sites; Panel H K_a = 7.8 × 10⁵ M⁻¹, ΔH = −11.5 kcal/mol, N = 0.25 sites.