A method for probing the affinity of peptides for amphiphilic surfaces.
We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.
D000327:Adsorption; D001053:Apolipoproteins / Q000032:analysis; D001665:Binding Sites; D008555:Melitten / Q000032:analysis; D008563:Membrane Lipids; D010455:Peptides / Q000032:analysis*; D010713:Phosphatidylcholines; D010743:Phospholipids; D011107:Polylysine / Q000032:analysis; D011137:Polystyrenes; D012710:Serum Albumin, Bovine / Q000032:analysis; D013499:Surface Properties; D014753:Vinyl Compounds
D001053:Apolipoproteins; D008563:Membrane Lipids; D010455:Peptides; D010713:Phosphatidylcholines; D010743:Phospholipids; D011137:Polystyrenes; D014753:Vinyl Compounds; D008555:Melitten; D011107:Polylysine; D012710:Serum Albumin, Bovine; C004985:divinyl benzene