Protein extraction into the bicontinuous microemulsion phase of a Water/SDS/pentanol/dodecane winsor-III system: Effect on nanostructure and protein conformation.

Bicontinuous microemulsions (BµEs), consisting of water and oil nanodomains separated by surfactant monolayers of near-zero curvature, are potentially valuable systems for purification and delivery of biomolecules, for hosting multiphasic biochemical reactions, and as templating media for preparing nanomaterials. We formed Winsor-III systems by mixing aqueous protein and sodium dodecyl sulfate (SDS) solutions with dodecane and 1-pentanol (cosurfactant) to efficiently extract proteins into the middle (BµE) phase. Bovine serum albumin (BSA) and cytochrome c partitioned to the BµE phase at 64% and 81% efficiency, respectively, producing highly concentrated protein solutions (32 and 44gL-1, respectively), through release of water and oil from the BµEs. Circular dichroism spectroscopic analysis demonstrated that BSA underwent minor secondary structural changes upon incorporation into BµEs, while the secondary structure of cytochrome c and pepsin underwent major changes. Small-angle x-ray scattering (SAXS) results show that proteins promoted an increase of the interfacial fluidity and surface area per volume for the BµE surfactant monolayers, and that each protein uniquely altered self-assembly in the Winsor-III systems. Cytochrome c partitioned via electrostatic attractions between SDS and the protein's positively-charged groups, residing near the surfactant head groups of BµE monolayers, where it decreased surfactant packing efficiency. BSA partitioned through formation of SDS-BSA complexes via hydrophobic and electrostatic attractive interactions. As the BSA-SDS ratio increased, complexes' partitioning favored BµEs over the oil excess phase due to the increased hydrophilicity of the complexes. This study demonstrates the potential utility of BµEs to purify proteins and prepare nanostructured fluids possessing high protein concentration.