Mechanism of protein solubilization in sodium bis(2-ethylhexyl) sulfosuccinate water-in-oil microemulsion

We selected alpha-chymotrypsinogen A (CTN) as a model protein, and the mechanism for protein solubilization in a sodium bis(2-ethylhexyl) sulfosuccinate (AOT) water-in-oil microemulsion was investigated using the two-phase transfer method. The extraction of CTN by the microemulsion is composed of two processes; a fast extraction and a subsequent slow back-extraction process. The adsorption of AOT onto the surface of CTN through the electrostatic interaction between CTN cations and AOT anions converts CTN from a hydrophilic to a hydrophobic state. This adsorption is responsible for the fast extraction process. The adsorption of AOT due to a hydrophobic interaction with CTN in turn makes the CTN-surface hydrophilic. The slow back-extraction process is attributed to the adsorption due to the latter interaction. The presence of two adsorption modes is ascertained by cation-exchange and hydrophobic-interaction chromatography, and spectroscopic measurements. In the fast extraction stage, CTN is extracted to the microemulsion accompanied with large amounts of AOT and water when the CTN-to-AOT mole ratio is comparatively large, suggesting the formation of large clusters composed of many AOT, water and CTN molecules. With a decrease in the ratio, the large cluster is divided into finer aggregates. The distribution of CTN between the microemulsion and the aqueous phase was examined by altering the salinity and organic solvent species of the microemulsion phase in the fast extraction stage. Both effects of the salinity and the solvent species were ascribed to the size effect of the microemulsion droplets. The weak interaction between the microemulsion droplet surface and the tails of the AOT chian adsorbed on the protein plays an essential role in the solubilization of the guest proteins. Since the energy of the droplet deformation required for uptake of the guest protein exceeds this weak interaction, the droplet has the ability to recognize the size of the guest molecules.