The ability of water-soluble, globular proteins to tune surfactant/oil/water self-assemblies has potential for the formation of biocompatible microemulsions and also plays a role in protein function at biological interfaces. In this work, we examined the effect of the protein alpha-lactalbumin on Aerosol-OT (AOT) phase structures in equivolume mixtures of oil and 0.1 M brine. In this pseudo-ternary system, surfactants are free to move to either oil or water phase to adopt phase structures close to the spontaneous curvature of the surfactants. Using small-angle X-ray scattering, we observed that addition of this protein changed the spontaneous curvature of the surfactant monolayer substantially. In the absence of protein, AOT adopted a negative spontaneous curvature to form spherical w/o microemulsion droplets. When less than 1 wt % of alpha-lactalbumin was added into the system, the w/o droplets became nonspherical and larger in volume, corresponding to an increase in water uptake into the droplets. As the protein-to-surfactant ratio increased, protein, surfactant, and oil increasingly partitioned toward the aqueous phase. There the protein triggered the formation of o/w microemulsions with a positive spontaneous curvature. These protein-containing structures exhibited significant interparticle attraction. We also compared the influence of two oil types, isooctane and cyclohexane, on the protein/surfactant interactions. We propose that the more negative natural curvature of the AOT/cyclohexane monolayer in the absence of protein prevented protein incorporation within organic phase structures and consequently pushed the system self-assembly toward aqueous aggregate formation.

Self-assembly of the anionic surfactant AOT with the protein alpha-lactalbumin in isooctane/brine mixtures results in phase structures whose type, size, and shape differ considerably from those formed by the surfactant alone. Small-angle X-ray scattering was used to determine the size and shape of these structures for 5.4 < pH < 11.2 and 0.25, 0.33, and 0.4 wt % NaCl. All pH values were above the reported isoelectric point for the protein. The composition of the system (except for salt) was fixed, with 2.5 wt % surfactant in equivolume mixtures of oil and water and either 0 or 0.4 wt % protein. Under these conditions, AOT in the absence of protein always formed spherical, water-in-oil (w/o) microemulsion droplets in the organic phase with no self-assembly in the aqueous phase. In the presence of alpha-lactalbumin, self-assembled structures were formed in both aqueous and organic phases, and the size and shape of these was tuned by both pH and [NaCl]. Protein-surfactant interaction was weakest at the most alkaline pH, with protein-free, spherical droplets forming in the organic phase and surfactant-decorated soluble protein clusters forming in the aqueous phase. As pH was decreased, protein increasingly partitioned to the organic phase and droplets became ellipsoidal and much larger in volume, with these effects enhanced at lower salt concentration. Aqueous structures were also strongly affected by pH, shifting from prolate protein/surfactant aggregates at alkaline pH to oil-in-water, oblate microemulsion droplets at neutral pH. At acidic pH and higher salt concentration, self-assembly shifted toward a third, anisotropic aqueous phase, which contained discoid bilayer structures. It is proposed that hydrophobic attraction causes association of the protein with the surfactant monolayer, and pH and [salt] tune the system via the protein by modifying electrostatic repulsion and monolayer curvature.