Biophysical characterization of the interaction of O-acylcholines with the major bovine seminal plasma protein, PDC-109.

Abstract

Thermodynamic parameters characterizing the interaction of O-lauroylcholine (OLC) and O-myristoylcholine (OMC) with PDC-109 have been determined by isothermal titration calorimetric studies. Titration of OLC or OMC with PDC-109 resulted in exothermic peaks, the magnitude of which decreased with subsequent injections, showing saturation of the binding sites at high protein concentration and indicating that the binding process is associated with negative enthalpy change (ΔH° < 0). Association constant (Ka), Stoichiometry (n), and thermodynamic parameters, ΔH°, ΔS° and ΔG° for the binding of OLC to PDC-109 have been obtained at different temperatures, with the values obtained at 25 °C being 5.08 × 105 M−1, 0.47, −4.46 kcal mol−1, 11.13 cal mol−1 K−1 and −7.78 kcal mol−1, respectively. Values of thermodynamic parameters obtained at different temperatures indicate that the binding of PDC-109 to O-lauroylcholine is governed by a negative enthalpy contribution. The interaction of O-lauroylcholine with PDC-109 was also studied by monitoring changes in the protein intrinsic fluorescence emission intensity. The association constant, Ka, for the interaction of PDC-109 with O-lauroylcholine from fluorescence studies at 25 °C was estimated to be 5.46 × 104 M−1, which is somewhat lower than the Ka value determined by ITC. Circular dichroism spectroscopic studies indicate that interaction of O-lauroylcholine with PDC-109 induced changes in the secondary and tertiary structure of PDC-109. Differential scanning calorimetric studies revealed that the thermal unfolding temperature of PDC-109 increases in the presence of OMC, indicating that binding of O-acylcholines stabilizes the protein structure.