New Method for the Measurement of Binding Constants for Amphiphiles with a Very Small Solubility in Aqueous Media

Abstract

The aqueous solubility of the monomeric form of most amphiphiles is relatively small and above a certain concentration, the critical aggregation concentration (CAC), they tend to form aggregates where the contact between their non-polar moieties and water is minimized. The affinity of the amphiphiles to hydrophobic environments, such as proteins or lipid bilayers, may be obtained by equilibrium titration with the binding agent but its correct evaluation requires the use of amphiphile concentrations below their CAC which, at times, can be extremely low. In this work we develop a method where the partition of amphiphiles between water and lipid bilayers may be accurately measured for amphiphiles with a CAC in the sub-nanomolar range. The method is based on the physical separation of the bound and free amphiphile using size exclusion chromatography and quantification of the bound amphiphile by HPLC. The high sensitivity of the method relies on an efficient increase in the concentration of the amphiphile by a minimum factor of 25 at the HPLC column, by injection of a very large volume coming from the size exclusion column and, for fluorescent amphiphiles, on the quantification in a solvent where it shows a very high fluorescent quantum yield. The equilibrium partition is performed at the required temperature and the physical separation between both fractions of amphiphile is performed at low temperature to guaranty that the equilibrium is not displaced. The method was implemented for the fluorescent amphiphile NBD-C16 for which the desorption from POPC lipid bilayers is a very slow process (k-=7.4×10−5 s−1 at 4°C (Cardoso, R., Master Thesis, Coimbra 2008)) conducing to less than 5% deviation from equilibrium during the 10 min required for separation of the two amphiphile fractions.