Quantitation of Electrostatic and Hydrophobic Membrane Interactions by Equilibrium Dialysis and Reverse-Phase HPLC

Abstract

Equilibrium dialysis and reverse-phase HPLC have been used for the sensitive and precise quantitation of both electrostatic and hydrophobic interactions of pep-tides and small molecules with lipid bilayers. We show that hydrophobic solutes are rapidly and quantitatively released from lipid dispersions when loaded onto a C4 reverse-phase HPLC column equilibrated in water + 0.1% trifluoroacetic acid and that the lipid molecules have no interfering effect on the chromatography. Peptides interacting electrostatically with bilayers are released quantitatively when a higher ionic strength buffer (water + 2% ammonium acetate) is used. As little as 50 ng of solute can be accurately quantitated even in the presence of milligram amounts of lipid. We demonstrate the application of these methods to the hydrophobic interactions between indoles and lipid bilayers and to the electrostatic interaction between defensins, which are cationic antibiotic peptides, and anionic bilayers. The high sensitivity allows nondestructive quantitation of submicrogram amounts of precious solutes and the high precision allows the heat capacity change, an important thermodynamic parameter, to be obtained from the partitioning data.