Protein partitioning driven by excluded-volume interactions in an aqueous nonionic micellar-gel system

The separation and purification of biomolecules in aqueous media driven by excluded-volume interactions is a well-established concept. In this article we propose a new separations method, based on excluded-volume principles, consisting of an aqueous micellar-gel system (AMGS). Specifically, an outer aqueous phase containing cylindrically shaped micelles of the nonionic surfactant n-decyl tetra (ethylene oxide) (C10E4) is physically separated from an inner aqueous phase defined by the interior volume of gel beads, from which the micelles are completely excluded because of their shape and size. In the AMGS, the concentration of the micelles outside the gel beads is sufficiently high that the volume excluded to a biomolecule in the solution external to the gel beads is much larger than that within the gel beads. Accordingly, when biomolecules are introduced into the AMGS, they partition preferentially into the gel-bead phase, according to their sizes, as a result of the greater effect of the excluded-volume interactions with the C10E4 micelles present in the aqueous phase outside the gel beads. The new AMGS is more versatile and adaptable than the conventional two-phase aqueous C10E4 micellar system because the micelle volume fraction is independent of the temperature and because the effects of entrainment are eliminated. After demonstrating the experimental feasibility of creating the new AMGS, the three proteins myoglobin, ovalbumin, and BSA-FITC, and the enzyme G6PD, were partitioned in the AMGS and their partitioning behavior was found to follow the experimental excluded-volume trends dictated by the interactions of the biomolecules with the C10E4 micelles. Specifically, the measured partition coefficients of the four biomolecules into the micellar phase were found to be less than unity and to decrease with increasing biomolecule size. A theoretical description of the partitioning behavior of the biomolecules in the new AMGS was formulated, based on excluded-volume considerations, and the predicted biomolecule partition coefficients were found to compare favorably with those measured for the four biomolecules studied.