(323c) Characterization of Chemical Affinities and Interactions with Lipid Bilayers Using Electrokinetic Techniques

Efficient methods to study small molecule and macromolecule interactions with lipid bilayers are instrumental to the study of biophysical chemistry. Efficient and affordable methods require the use of small sample quantities, rapid turnaround of results, and relatively inexpensive instrumentation. Many of the industry standard techniques for studying these interactions require large sample sizes or require days, if not weeks, to generate results. These techniques also often require expensive optical fluorescence or nuclear magnetic resonance spectrometers.

Electrokinetic chromatography (EKC) is a variation of capillary electrophoresis (CE). In this form of chromatography, analytes are separated via selective interactions with a pseudostationary phase (PSP) dispersed within the CE background electrolyte (BGE). This technology allows for fast, selective, and efficient separations that require only nanoliter sample volumes. The utility of EKC to characterize PSP and PSP–solute interactions has been recognized since its introduction. A major application of EKC has been to measure the affinity between solutes and a PSP as a proxy for affinity for biological lipid bilayers or for octanol–water partition coefficients (Po/w). The measurement of lipophilicity, as described by P_O/W, is important because metabolic clearance rates and biotransport properties can be correlated to lipophilicity. P_o/w is also a useful measure of potential bioaccumulation and ecotoxicity.

Nanodiscs are nanometer scale disc-shaped phospholipid bilayer assemblies encircled by synthetic styrene–maleic acid copolymers for stabilization. The copolymers interact with the hydrophobic edges of the nanodisc on the inner side and the surrounding aqueous medium on the outer side, serving to stabilize the nanodiscs in aqueous dispersions. The use of copolymers in the synthesis of nanodiscs allow for nanodiscs to be generated inexpensively in sufficient quantity for use in EKC and furthermore, the anionic maleic acid group serves the purpose of imparting negative charge and electrophoretic mobility on the nanodiscs, making them suitable as a PSP. The phospholipid composition of the nanodiscs can also be varied to mimic specific biological systems. These phospholipid bilayer structures thus have extraordinary and unique potential to simulate biological membranes. It is demonstrated here that the nanodiscs present useful constructs to study membrane affinities by EKC.

Nanodiscs composed of three different lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine were studied by electrokinetic chromatography. These lipids have the same head group, but differing alkyl chain length or degrees of unsaturation. Thirty-eight compounds with varying functional groups, size, and with published values for P_o/w were used to determine the effectiveness of these nanodiscs for indirect measurement of P_o/w and to characterize chemical interactions with the bilayers. Retention factors (k) for these were measured and correlations between log(P_o/w) and log(k) were determined through linear regression. It is clear that there are at least two different classes of solute probes with different relationships between log (P_o/w) and log (k). The primary difference between the two groups of solutes was found to be their hydrogen bond donor strength. Hydrogen bond donating compounds were more retained than expected. It is possible that solutes with more acidic hydroxyl groups versus those with more basic amine groups may have slightly different trends, but more solutes with amine functionalities would need to be analyzed to determine if a significantly different trend is present.

In order to gain a better understanding of why the affinities of different classes of compounds correlate separately with P_o/w, linear solvation energy analysis (LSER) was employed to study the free energy for transfer of a solute from the BGE to the nanodisc. This model allows for the nanodiscs’ solvation properties to be compared as the different lipids compose the lipid bilayer of the nanodisc. Based on the LSER results it appears that changes in the alkyl tail length and degree of unsaturation do not lead to statistically significant changes in the solvation characteristics of the nanodiscs. Another characteristic determined from the LSER is that the nanodisc system is a stronger hydrogen bond acceptor than the octanol. The nanodisc bilayer may be more able to accept hydrogen bonds because of the multiple carbonyls or the quaternary ammonium group located in the lipid head group, or the negative charge on the phosphate could allow for electrostatic interactions with acidic hydrogen. The LSER data suggests that a significant portion of the solute interaction occurs with the head group of the lipid because changes to the tail structure of the lipid do not lead to significant changes.