Interactions between adsorbed lactoferrin layers measured directly with the atomic force microscope

Abstract

Lactoferrin is an important protein found in tear fluid and has been implicated in the fouling of contact lenses. The interaction forces between lactoferrin layers, adsorbed onto hydrophobic model substrates, as a function of electrolyte concentration, solution pH and protein concentration were investigated using an atomic force microscope (AFM) where the geometry of the scanning tip was modified by attaching a colloid particle. Bovine lactoferrin adsorbed strongly and modified the interaction forces to include both a long-range electrostatic repulsive force, which could be described using the DLVO theory, and a short-range additional repulsion due to compression of the adsorbed protein layers. The adsorption of lactoferrin was verified by XPS analysis of the surfaces and the adsorbed layer was determined to be present as a full monolayer by AFM imaging. At smaller separation distances, deviations from theoretical predictions indicated that there was an adsorbed layer of at least 5 nm on both surfaces. DLVO theory could be used successfully to describe the longer ranged interaction forces in background electrolyte concentrations of 0.0002 and 0.001 M KNO 3, at various solution pH values. This analysis yielded effective diffuse electrical double layer potentials, which when plotted against the solution pH, suggested that the point of zero charge (pzc) of the protein coated surfaces was around pH 8, in agreement with literature values of the pI for lactoferrin. At higher electrolyte concentrations, 0.15 M KNO 3, the interaction forces could no longer be described using DLVO theory. The interaction forces between the adsorbed lactoferrin layers were purely repulsive, and the range and magnitude varied as a function of solution pH. Variations in the forces as a function of solution pH reflected conformational changes that depend on short ranged electrostatic interactions between the peptide residues within the protein molecules and the peptide residues and the substrate as well as van der Waals and hydrophobic interactions. The range of the interaction forces and the high compressibility of the adsorbed layers indicated that a significant number were present in an end-on orientation.