Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 2. Effect of entrained micelle-poor domains.

Abstract

Unlike the partitioning behavior of hydrophilic, water-soluble proteins, the partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained using the excluded-volume theory developed recently by our group. A central assumption underlying the excluded-volume theory--that macroscopic phase separation equilibrium is attained--was therefore challenged experimentally and theoretically. Photographs of the two-phase aqueous C10E4 micellar system were taken for different volume ratios to demonstrate that the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase decreases with a decrease in the volume ratio. Partitioning experiments were then conducted with the model virus bacteriophage P22 and the model protein cytochrome c at different operating temperatures for different volume ratios. For bacteriophage P22, the measured viral partition coefficient at each temperature decreased by about an order of magnitude when the volume ratio was decreased from 10 to 0.1, which clearly indicated that entrainment is an important factor influencing viral partitioning. For cytochrome c, the measured protein partition coefficient did not change, which demonstrated that this entrainment effect negligibly influences protein partitioning. A new theoretical description of partitioning was also developed that combines the excluded-volume theory with this entrainment effect. In this theory, one fitted parameter--the volume fraction of entrained micelle-poor domains in the macroscopic, top, micelle-rich phase--is used to account for the entrainment. To fit this parameter, only a single partitioning experiment is required for a given volume ratio, irrespectively of the partitioning solute. The new theoretical description of partitioning yielded very good quantitative predictions of the viral partition coefficients. Accordingly, it can be concluded that the primary mechanisms governing viral partitioning in the two-phase aqueous C10E4 micellar system are the entrainment of micelle-poor (virus-rich) domains in the macroscopic, top, micelle-rich phase and the excluded-volume interactions that operate between the viruses and the micelles.