Immobilized artificial membrane (IAM) liquid chromatography as a model for antimicrobial peptide partitioning into cell membranes: An evaluation
Non-covalent immobilized artificial membrane reverse-phase high performance liquid chromatography was previously evaluated as a means whereby elution times for antimicrobial peptides from columns mimicking the lipid bilayers of different membrane systems might be used as a fast-screening method to compare relative binding effectiveness. Such a system would aid in the development of antimicrobial peptides that bind preferentially to model pathogenic systems and leave the host’s membranes reasonably unaffected. A non-covalent approach allows for flexibility in membrane composition but was found to be inadequate for analysis of most peptides due to significant lipid loss at high acetonitrile concentrations. A covalent approach where phosphatidylcholine was amide-linked to the silica surface was examined to evaluate its use as a fast-screening method and compare its data to that collected from the non-covalent columns. Initial work with a 1-cm column proved ineffective due to problems with balancing flow rates with retention times, and work was shifted to a longer 10-cm column. Results suggested that peptides bind much more strongly to covalent columns than non-covalent ones, with the binding especially enhanced by the presence of cationic residues. These columns had lipid packing densities much lower than true membranes, indicating that the peptides were partitioning deep into the bonded phase of the columns rather than into the interfacial region of the phosphate head groups, as expected in situations of biologically-relevant lipid packing densities.
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