Affinity Partitioning in Aqueous Two Phase Systems

purification. If, however, the protein of interest (the target protein) has a very high K value and is mainly in the upper phase and all the contaminating proteins have very low K values so that they are in the bottom phase, an effective and selective extraction can be obtained in a single or a few partitioning steps. This type of partitioning has been made possible by using affinity ligands restricted to the upper phase.

The composition of the phases when two polymers like dextran and polyethylene glycol (PEG) are dissolved together in water depends on the amount of the polymers and their molecular weights. The concentration of the polymers in two phases of a given system can be found in the phase diagram for the temperature being used. A typical phase diagram is shown in Figure 1.

The line that connects the points in the diagram representing the compositions of the top and bottom phases of a system is called the tie-line. Each system with a total composition (percentage of each polymer) belonging to the same tie-line will have the same phase compositions. The smaller the tie-line, the more similar are the two phases in their composition. The greatest difference in composition of the top and bottom phases is therefore obtained by using high polymer concentrations.

The partitioning of proteins and also of membranes and particles depends on the polymer concentration of the system. The K value of a protein will be the same for all systems belonging to the same tie-line.

Concentration of dextran (%w/w)

Figure 1 Phase diagram for the system dextran 500 (500 000 Da), PEG 8000 (8000 Da), and water at 230C. Polymer compositions above the curved line (bimodal curve) give two liquid phases. All two-phase systems with their total composition on the same straight line (tie-line) have the same composition of top phase (A) and bottom phase (■). The systems differ in phase volume ratio depending on their position on the tie-line. The indicated total compositions (•) give systems with more top phase (three to five times) than bottom phase.

Concentration of dextran (%w/w)

Figure 1 Phase diagram for the system dextran 500 (500 000 Da), PEG 8000 (8000 Da), and water at 230C. Polymer compositions above the curved line (bimodal curve) give two liquid phases. All two-phase systems with their total composition on the same straight line (tie-line) have the same composition of top phase (A) and bottom phase (■). The systems differ in phase volume ratio depending on their position on the tie-line. The indicated total compositions (•) give systems with more top phase (three to five times) than bottom phase.

The partition coefficient will, in most cases, decrease with the length of the tie-line, i.e. by using higher concentrations of the two polymers the material will accumulate more in the lower phase. Another way to affect the partitioning of proteins is by addition of salts to the system. Their effect depends on the type of cation and anion introduced with the salt. Negatively charged proteins show increasing K values when the cation is changed in the series:

For the anion the partition coefficient increases in the following order:

ClO4~ < SCN" < T < Br" < Cl" < CH3W

The highest K value of negatively charged proteins will then be obtained with the salt tetrabutylam-monium hydrogenphosphate and the lowest K value with potassium perchlorate. Proteins with zero net charge (at their isoelectric points) are not affected by salts while positively charged proteins behave in an opposite manner to the negatively charged ones. For a number of proteins the log K values are nearly a linear function of their net charge (Figure 2).

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