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The stability and structure of protein-containing water-in-oil (w/o) microemulsions were investigated by using the large protein immunoglobulin G (IgG, MW 155,000) in a mixture comprised of brine, sulfosuccinic acid bis [2-ethylhexyl]ester (sodium salt), and isooctane. We explored factors affecting the initial uptake of IgG into the w/o microemulsion and its subsequent release to a solid (precipitate) phase, and the kinetics of the latter process. Influences of such parameters as pH, ionic strength, and protein concentration on the solubilization and precipitation of bovine IgG in the organic phase are described. The structure and dynamics in microemulsions containing bovine IgG were probed by using dynamic light scattering, and it was found that the presence of IgG in the microemulsion induced strong attractive forces between the droplets. Based on results obtained by using these various experimental approaches, a model for protein solubilization and release is proposed. In this model, we propose the formation of clusters within which bovine IgG resides and which substantially slow the kinetics of protein release from the droplets to the precipitate phase.

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Changes in structure of protein molecules and water-in-oil (w/o) microemulsion aggregates were investigated using the large protein immunoglobulin G (IgG, MW 155,000) and an equivolume oil/water mixture composed of brine, sulfosuccinic acid bis [2-ethylhexyl]ester (sodium salt) (AOT), and isooctane. The protein solution in the microemulsion phase was metastable: over time this solution changed, as protein and w/o droplets aggregated and precipitated to the interface between aqueous and organic phases. Such factors as AOT concentration, temperature, and salt concentration were found to influence the protein and surfactant structures in the microemulsion. Protein conformation was probed using circular dichroism spectroscopy whereas the microemulsion structure was determined from dynamic light scattering measurements. Protein conformation and microemulsion structure were found to have significant effects on protein stability in the microemulsion. The stabilizing effects of clusters formed at higher salt and/or AOT concentrations are discussed. IgG adopts an intermediate denatured state in the microemulsion phase close to the alternatively folded state known as the A state, with well-defined contacts in the tertiary structure immediately after phase equilibration. The change in protein conformation with time accompanied by the cluster growth eventually leads to the protein and surfactant transfer into a third, solid middle phase from the organic solution.