Adhesion forces between protein layers studied by means of atomic force microscopy.

Adhesion forces between different protein layers adsorbed on different substrates in aqueous media have been measured by means of an atomic force microscope using the colloid probe technique. The effects of the loading force, the salt concentration and pH of the medium, and the electrolyte type on the strength, the pull-off distance, and the separation energy of such adhesion forces have been analyzed in depth. Two very different proteins (bovine serum albumin and apoferritin) and two dissimilar substrates (silica and polystyrene) were used in the experiments. The results clearly point out a very important contribution of the electrostatic interactions in the adhesion between protein layers.

Existence of hydration forces in the interaction between apoferritin molecules adsorbed on silica surfaces.

The atomic force microscope, together with the colloid probe technique, has become a very useful instrument to measure interaction forces between two surfaces. Its potential has been exploited in this work to study the interaction between protein (apoferritin) layers adsorbed on silica surfaces and to analyze the effect of the medium conditions (pH, salt concentration, salt type) on such interactions. It has been observed that the interaction at low salt concentrations is dominated by electrical double layer (at large distances) and steric forces (at short distances), the latter being due to compression of the protein layers. The DLVO theory fits these experimental data quite well. However, a non-DLVO repulsive interaction, prior to contact of the protein layers, is observed at high salt concentration above the isoelectric point of the protein. This behavior could be explained if the presence of hydration forces in the system is assumed. The inclusion of a hydration term in the DLVO theory (extended DLVO theory) gives rise to a better agreement between the theoretical fits and the experimental results. These results seem to suggest that the hydration forces play a very important role in the stability of the proteins in the physiological media.

Hydration forces between silica surfaces: experimental data and predictions from different theories.

Silica is a very interesting system that has been thoroughly studied in the last decades. One of the most outstanding characteristics of silica suspensions is their stability in solutions at high salt concentrations. In addition to that, measurements of direct-interaction forces between silica surfaces, obtained by different authors by means of surface force apparatus or atomic force microscope (AFM), reveal the existence of a strong repulsive interaction at short distances (below 2 nm) that decays exponentially. These results cannot be explained in terms of the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory, which only considers two types of forces: the electrical double-layer repulsion and the London-van der Waals attraction. Although there is a controversy about the origin of the short-range repulsive force, the existence of a structured layer of water molecules at the silica surface is the most accepted explanation for it. The overlap of structured water layers of different surfaces leads to repulsive forces, which are known as hydration forces. This assumption is based on the very hydrophilic nature of silica. Different theories have been developed in order to reproduce the exponentially decaying behavior (as a function of the separation distance) of the hydration forces. Different mechanisms for the formation of the structured water layer around the silica surfaces are considered by each theory. By the aid of an AFM and the colloid probe technique, the interaction forces between silica surfaces have been measured directly at different pH values and salt concentrations. The results confirm the presence of the short-range repulsion at any experimental condition (even at high salt concentration). A comparison between the experimental data and theoretical fits obtained from different theories has been performed in order to elucidate the nature of this non-DLVO repulsive force.

Langmuir-Blodgett films of biopolymers: a method to obtain protein multilayers.

In this work, we present a methodology for choosing the best experimental conditions for transferring protein Langmuir films onto solid substrates. As an example of applying the proposed methodology, we used monolayers of the protein bovine serum albumin, which is a very stable protein and is of great interest in the development of immunosensors. Langmuir-Blodgett (LB) films of this protein, on different solid substrates, were obtained and characterized as a function of pH, surface pressure, temperature, and contact angle. The compressibility modulus, the spreading entropy, and the fraction of desorbed protein sections were used as control parameters to find these conditions. A careful analysis of these parameters shows that there is a window on the values of these experimental parameters in which the LB films are best formed. Our methodology can be applied to other biomacromolecules to find the best conditions to form LB films from isotherm measurements.

Interactions, desorption and mixing thermodynamics in mixed monolayers of beta-lactoglobulin and bovine serum albumin.

The interactions between milk proteins, beta-Lactoglobulin (beta-Lg) and bovine serum albumin (BSA), at the air-water interface have been evaluated. The surface pressure (pi), molecular area (a) isotherms were obtained by compression of the monolayers at different pH and temperature. In the method used to calculate the interactions, the desorbed segments of the proteins into the aqueous subphase have been considered. Earlier, the desorbed segments have been estimated from the compressibility factor, z, as a function of the surface pressure (virial state equation). The main conclusion from this study is that for biopolymers it is not possible to apply only the mixing thermodynamics to evaluate the intermolecular forces. It is necessary to include the desorption phenomenon. From these results, we can conclude that the main interaction between both proteins is of electrostatic character.