Effect of α-Lactalbumin on the Phase Behavior of AOT−Brine−Isooctane Mixtures:  Role of Charge Interactions

Abstract

We have found that both electrostatic and hydrophobic interactions are involved in the ability of the protein alpha-lactalbumin (alpha-LA) to affect the self-assembly of the anionic surfactant sodium bis(ethylhexyl) sulfosuccinate (AOT, 3.5 wt %) in equivolume mixtures of organic and aqueous solutions. The composition and size of AOT phase structures that form in the presence of 0.35 wt % protein were evaluated as a function of pH and ionic strength. In the absence of protein, AOT forms water-in-oil microemulsion droplets for all pH and salt concentrations studied here. The presence of the protein in the water-in-oil microemulsion phase boosts water solubilization and droplet size, as the spontaneous curvature of the surfactant interface becomes less negative. Aggregates of protein, surfactant, and oil also form in the water-continuous phase. The size and composition of structures in both phases can be tuned in the presence of protein by varying the pH and ionic strength. alpha-LA induces the appearance of an anisotropic surfactant phase at pH <5.8. At intermediate salt concentrations, a third isotropic, viscous aqueous phase appears that contains 55-60% of the protein, 10-14% of the surfactant, and significant amounts of oil. Circular dichroism and fluorescence spectroscopy indicate that the protein contains enhanced alpha-helical secondary structure when self-assembling with surfactant, and has a loosened tertiary structure. The protein does not interact with the surfactant as an unfolded random coil. Although the conformation of alpha-LA in aqueous salt solutions is known to depend on pH, when self-assembling with AOT the protein adopts a structure whose features are quite pH insensitive, and likely reflect an intrinsic interaction with the interface.

α-Lactalbumin−AOT Charge Interactions Tune Phase Structures in Isooctane/Brine Mixtures

“…For isooctane, the peak at 1.1 ppm was used, whereas for AOT, peaks at 1.2 and 3.9 ppm were used for the aqueous and organic phase concentrations, respectively. Optical properties were observed using a polarized light microscope (Nikon Laposhot 2A) . Samples were contained in glass capillaries used for X-ray scattering measurements and directly placed on the microscope stage to be observed.…”

“…Small angle X-ray scattering from these aqueous phases suggested that these structures were small oil-in-water microemulsion droplets . Other studies indicated that conditions of pH and ionic strength had a strong influence on the AOT phase behavior modifications induced by αLA but did not obtain detailed information on the size, shape, or interparticle interactions of the resulting phase structures.…”

“…These structures are tunable on the basis of the curvature properties of the surfactant monolayer within the structure. We have previously found that αLA has a profound influence on the morphology of these self-assembled structures, changing their size, shape, and even the nature of the discrete and continuous fluids. − Protein molecules are zwitterionic, highly heterogeneous polymers whose tertiary structure is a result of a combination of electrostatic, hydrophobic, H-bonding, and other interactions within the chain. We have become interested in the factors allowing αLA to participate intimately in the surfactant self-assembly process, creating nanostructures whose morphology and other properties differ markedly from those formed in the absence of the protein.…”

“…However, in equivolume mixtures of oil and water where the surfactant monolayer is less constrained, a more substantial effect of protein on the type and structure of surfactant assemblies may be observed. Our previous studies have provided evidence of such significant effects of protein incorporation on the basis of changes in phase behavior of Aerosol-OT/oil/brine mixtures upon the addition of the protein αLA. − Aerosol-OT (AOT) is a two-tailed, anionic surfactant that forms w/o microemulsions in the absence of protein. When αLA was added to this ternary system at neutral pH, most of the protein partitioned to the w/o microemulsion phase despite unfavorable electrostatic interactions with AOT. , There, the protein substantially boosted the amount of water solubilized in the microemulsion , and increased the droplet size. , At the same time, new self-assembled structures appeared in the aqueous phase along with a considerable quantity of isooctane, AOT, and αLA .…”

“…Given the low isoelectric point for αLA (∼4.5), the net charge on the protein should be negative in our studies and thus should be charged similarly to the anionic surfactant. Despite this, association between the surfactant and the protein is observed even at very high pH values . The contribution of electrostatic attraction between AOT and cationic residues present on the protein must be considered as well as a role for hydrophobic interactions.…”