Protein partitioning in two-phase aqueous

N. L. Abbott, D. Blankschtein, and T. A. Hatton, On protein partitioning in two-phase aqueous polymer systems, Bioseparation 1990, 1, 191-225. 

Experimental Observations on Protein Partitioning in Two-Phase Aqueous Polymer Systems... 

Experimental observations such as those presented in Figures 1 and 2 motivated the philosophy behind our theoretical formulation (21). In particular, the correlation observed between the overall sizes of the proteins and their partitioning behavior suggested that a coarse-grained view of these systems, rather than an atom-by-atom account, may be sufficient to describe the nature of the interactions responsible for the observed partitioning behaviors of these proteins. This realization prompted our decision to pursue (21,24) a scaling account (20,32-34) of protein partitioning in two-phase aqueous polymer systems. 

We have reviewed our recent theoretical and experimental studies of the physical mechanisms responsible for protein partitioning in two-phase aqueous polymer systems (21-25). It is gratifying to see that from a common physicd basis, our theoretical... 

Because the partitioning behavior of proteins in two-phase aqueous polymer systems reflects the relative interactions between the proteins and the two coexisting polymer solution phases, and because the independent control of the polymer concentration in only one of the two coexisting phases is not possible, we have explored an alternative experimental technique, namely, the measurement of the partitioning of proteins between an entangled PEO solution phase and an aqueous (polymer-free) phase using a diffusion cell (24). 

SIV Sivars, U. and Tjemeld, F., Mechanism of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins, Biochim. Biophys. Acta, 1474, 133, 2000. 

The formation of two aqueous phases can be exploited in the recovery of proteins using liquid-liquid extraction techniques. Many factors contribute to the distribution of a protein between the two phases. Smaller solutes, such as amino acids, partition almost equally between the two phases, whereas larger proteins are more unevenly distributed. This effect becomes more pronounced as protein size increases. Increasing the polymer molecular weight in one phase decreases partitioning of the protein to that phase. The variation in surface properties between different proteins can be exploited to improve selectivity and yield. The use of more hydrophobic polymer systems, such as fatty acid esters of PEG added to the PEG phase, favors the distribution of more hydrophobic proteins to this phase. In Fig. 10.13, partition coefficients for several proteins in a dextran-PEG system are given [27]. 

The most popular method for measuring the polarity of a solute entails determination of the distribution constant between water and a water-immiscible solvent, e.g., octanol. However, because there is difficulty in dissolving proteins in the solvent, a two-phase aqueous system was developed (Shanbhag and Axelson, 1975). Albertson (1986) reported the construction of various aqueous phase systems for partitioning proteins, other macromolecules, and even cells. Recently, simpler aqueous biphase systems were selected for hydrophobic partitioning of proteins (Hachem et al., 1996). However, because of restrictions similar to those for HIC, as discussed above, it may be premature to replace the method used in Basic Protocol 5. The definition of hydrophobicity is based on the polarity of chemical compounds, which is closely related to the distribution between solvents of different polarities. This theory is similar to the elution mechanism of phase distribution chromatography as well as phase partition. However, complexity in the partition system and procedure hampers the broad use of the phase partition approaches. 

Liquid-liquid partitioning is a convenient and often economical method for bioseparations. L. Gu (personal communication, 1999) has shown that an acetonitrile-water system can be used for separation of proteins. This system partitions into two phases under subzero temperatures with the top phase containing more acetonitrile and water. The low temperature and the presence of water in both phases help reduce protein denaturation. An added advantage is that sample solution can be directly applied to reversed-phase high-performance liquid chromatography (HPLC) for further purification. Aqueous liquid-liquid partitioning is likely to remain an attractive choice for the separation of proteins, and exploration of new systems will continue. 

For the extraction of proteins, aqueous two-phase systems (ATPS) are preferred over organic solvents, which usually denature the proteins and render them biologically inactive. They consist of polyethylene glycol (PEG), and a salt (e.g., potassium phosphate) or dextran in water. At concentrations above a critical value, the mixture separates into two phases—one rich in PEG and the other in dextran or salt. In industrial systems, salts are more commonly used because they are relatively inexpensive as compared to dextran. The MW, charge and surface properties of the protein decide how the protein partitions in the system. The nature of the phase components, the MW of the polymer, and the concentration and type of salt used also affect the distribution. ... 

Lam H, Kavoosi M, Haynes CA et al. (2004) Affinity-enhanced protein partitioning in decyl a-D-glucopyranoside two-phase aqueous micellar systems. Biotechnol Bioeng 89(4) 381-392 Lambert PW, Meets JL (1983) The production of industrieil enzymes. PMl Trans R Soc Lond B 300 263-282... 

WAZ Waziri, S.M., Abu-Sharkh, B.F., and Ali, S.A., Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol), Biotechnol. Progr, 20, 526, 2004. 

LI Oliveira, M.C. de, Filho, M.A.N. de A., and Filho, P. de A.P., Phase equilibrium and protein partitioning in aqueous two-phase systems containing ammonium carbamate and block copolymers PEO-PPO-PEO, Biochem. Eng. J., 37, 311, 2007. 

JOH Johansson, H.-O., Magaldi, F.M., Feitosa, E., and Pessoa Jr., A., Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems, J. Chromatogr. A, 1178, 145, 2008. 

Separation by partition between two immiscible aqueous phases has been successfully applied to a large number of proteins, nucleic acids, and cellular particles such as mitochondria, chloroplasts, membrane vesicles, and whole cells. The phase systems are made up of two different water-soluble polymers, dextran and poly(ethylene glycol), dissolved in water and complemented with suitable buffers, salts, and sucrose. Highly selective adjustment of the distribution process is possible by linking an affinity... 

Fig. 5. Schematic presentation of protein partitioning in aqueous two-phase pol3rmer system formed by a smart (thermosensitive) poljmier. Reproduced from Ref. 140 with permission.

---