[PEG] and [Dx] are the overall concentration of PEG and Dx (before phase separation). The densities (p in units of g/cm3) of each phase were measured with U-shaped oscillator densitometer, and the interfacial tensions were measured with a spinning drop tensiometer (Kruss Site 04, Hamburg, Germany). All systems are at 25°C.

between the polymers increases.

2. Polymer hydrophobicity. The polymers become less compatible when the hydrophobicity of one of them is increased. This can be done by derivatiz-ing the polymer. For example, two phases are formed by hydroxypropyl

Dx and Dx.

3. Temperature. The effect of this variable is small. In PEG/Dx mixtures, less polymer is needed at low temperatures. On the other hand, the phase transition in PEG-salt systems is facilitated by high temperatures.

Figure 1. Equilibrium curve for a P(EO-PO)/NaCl system at 25°C. Points to the left the equilibrium curve correspond to one phase, and points to the right correspond to 2 liquid phases at equilibrium Point B representing the composition of the top phase, point A the composition of the bottom phase, and points O and P two possible overall concentrations of P(EO-PO) and NaCl. An overall concentration equal to O yields V-/V b = 1, and an overall concentration equal to P yields V-/V b = 1/3.

Figure 1. Equilibrium curve for a P(EO-PO)/NaCl system at 25°C. Points to the left the equilibrium curve correspond to one phase, and points to the right correspond to 2 liquid phases at equilibrium Point B representing the composition of the top phase, point A the composition of the bottom phase, and points O and P two possible overall concentrations of P(EO-PO) and NaCl. An overall concentration equal to O yields V-/V b = 1, and an overall concentration equal to P yields V-/V b = 1/3.

4. Polydispersity of the polymers. Commercial grade polymers have a wide distribution of molecular weights. Therefore, phase separation occurs in a small range of polymer concentrations changing the binodal curve to a bin-odal zone. If the polymer molecular weight distribution is too wide, more than two phases can be formed.

5. Ionic Strength. The length of the tieline increases when salts are added to PEG-salt systems. In PEG/Dx/water systems the ionic strength does not have an appreciable influence on the position of the binodal.

1.1. Distribution of Proteins in Aqueous Two-Phase Systems

The separation power of a given aqueous 2-phase system is given by the partition coefficient, Kp, of a protein. Kp is defined as the ratio of the protein concentration in the top and bottom phases. This partition coefficient depends on the difference in chemical potential of the protein between top and bottom phases, and therefore, it is a function of the chemical nature of the polymers, the protein, added electrolytes, and temperature. It is usually manipulated by changing the pH (13,14) or the temperature, by adding salts or affinity ligands, or by changing the molecular weight of the polymers or their concentration.

Manipulation of the pH is of primary importance in partitioning studies of proteins. It is convenient to split the partition coefficient into 2 contributions, one that is independent of pH, and the other that is proportional to the net charge of the protein. In so doing, electrostatic and nonelec-trostatic contributions to the partition coefficient can be conveniently (but artificially) separated. This approach predicts that the logarithm of the partition coefficient is a linear function of the charge of the protein. In the analysis of partitioning data at differ ent pHs, we must consider any change in the physical properties of the proteins due to changes in the pH and how these changes may affect the protein partition coefficient. For example, whereas lysozyme does not change its conformation over the pH range 1.2 to 11.3 in dilute salt solutions at moderate temperatures, it polymerizes reversibly at pH above 5 (45). This change in the molecular weight of the protein will change the partition coefficient.

The molecular weight and concentration of the phase-forming species also affect the partition coefficient strongly (13). For example, in a PEG/Dx system, low PEG molecular weight favors the partitioning of proteins into the PEG-rich phase. The effect of polymer concentration on the partition coefficient is also well known. If Kp is smaller than 1, an increase in either one of the phase-forming species decreases Kp. Similarly, if Kp is larger than 1, an increase in either of the phase-forming species increases Kp. The amount of the phase-forming species also affects the volume ratio between the phases. For example, in Figure 1, a system of total composition O has a top:bottom volume ratio of 1, whereas a system of total composition P has a top:bottom volume ratio of 1/3. Therefore, the targeted protein can be not only purified, but also concentrated in a single step.

Generally, the partition coefficient increases with increasing temperature in Dx/ PEG systems between 4° and 40°C (12). The partition coefficient of small and hydrophilic proteins is only slightly affected by changes in temperature, whereas the partition coefficient of bigger and more hydrophobic proteins is strongly affected by temperature changes. High temperatures (around 40°C) may be used to minimize protein association, whereas low temperatures may be desirable to maintain protein stability.

Salts have unequal affinities for the top and bottom phases of aqueous 2-phase sys tems (25). This uneven partition of salts between the 2 phases affects the chemical potential of the protein in each phase and thus its partition coefficient. The following mental exercise helps to understand this phenomenon. Consider 2 phases at equilibrium in which a salt has been previously partitioned. The different affinity of the salt for the bottom and top phases creates 2 distinct ionic atmospheres in both phases. Picture a charged protein molecule at infinite distance from the phases. Bring the protein and try to insert it in each of the 2 phases. Since the protein is going to see different ionic atmospheres in both phases, the work needed to insert it in one or the other phase will be different. This difference in the work of insertion of a charged protein in each of the 2 phases at equilibrium is proportional to the electrochemical potential difference of the protein between them. Consequently, the partition coefficient, which is a function of that potential difference, is strongly affected by the type and concentration of salts and by the charge on the protein.

In general, the partition coefficient of proteins away from their isoelectric point depends on both the type and concentration of cation and anion (25). For example, for positively charged proteins the partition coefficient in PEG/Dx systems is higher in potassium chloride than in potassium phosphate; the reverse is true for negatively charged proteins. The effect of the cation on the partition coefficient of positively charged proteins is K approximately equal to Cs greater than Na greater than Li, whereas the reverse order holds for negatively charged proteins. It is possible to correlate the partition coefficient of a protein with its charge and with the type of salt. Johansson (25) found that, log Kp = log K0 + yZ [Eq. 1]

where, K0 depends on the particular protein and phase-system, but it is indepen dent on protein's charge, Z is the charge of the protein, and y depends on the types and concentration of polymers, temperature, and types of electrolytes. Values of y are plotted in Figure 2 for various salts (values are for Dx-500/PEG-8000 at 4°C). The net effect of a salt can be calculated by

Y = Ycation - Yanion. For exa^e the addition of tetrabutyl ammonium phosphate yields a Y = -79, whereas the addition of potassium perchlorate yields a Y = 30. This figure can be used to select the appropriate electrolyte to move the desired protein to either the top or bottom phases. A pH away from the isoelectric point of the protein must be selected, since the partition coefficient of proteins at their isoelectric point is quite insensitive to salt type and concentration (see Equation 1).

The partition coefficient of proteins can be manipulated by adding an affinity lig-and or by using liquid—liquid chromatography, counter—current extraction, or by a combination of the first two. Affinity lig-ands, such as PEG-palmitate (42) or PEG-red (24), have been routinely used to improve the selectivity of aqueous 2-phase extraction. In affinity partitioning (26), it is customary to define Aln Kp = ln KL - ln K0 (KL and K0 are the partition coefficients of the protein with and without added affinity ligand, respectively) to quantify the enhancement in partitioning as a result of the addition of the affinity lig-and. Liquid—liquid chromatography, with or without the addition of an affinity lig-and, can be used to improve the selectivity of aqueous 2-phase systems. For example, we have used liquid—liquid chromatogra-phy with an immobilized Dx-rich phase to purify formate dehydrogenase (FDH) from Candida boinidi with and without the addition of PEG-red as an affinity ligand (50). Another attractive alternative technique is the use of metal affinity aqueous 2-phase extraction (1,40,55). In this technique, a small portion (less than 1%) of the total PEG is replaced by PEG-iminodi-acetic acid (PEG-IDA), which chelates divalent cations like copper or zinc. Histidine groups on the protein surface recognize these metals, and the strength of the binding is proportional to the number of histi-dine groups on the protein surface.

One of the most common analytical uses of aqueous 2-phase systems is the determination of isoelectric points by determining cross-partitioning points (3, 41,51,53). The method uses the different sensitivity of the partition coefficient on the kind of salt used and determines the pH at which the partition coefficient is independent of salt type. If the protein of interest does not interact with contaminating materials in the solution, its isoelectric point can be determined without prior protein purification (other than desalting).

The determination of isoelectric points of proteins by cross-partitioning is straight forward. Two sets of 2-phase systems (usually Dx/PEG) covering a wide pH range are prepared. One set contains NaCl, whereas the other set contains Na2SO^ The pH dependence of the partition coefficient of proteins in the presence of NaCl is usually different from that in the presence of Na2SO^ So, the partition coefficient versus pH curves cross each other at a pH (cross-point) that usually agrees with the isoelectric point of the proteins. Discrepancies between the isoelectric points determined by cross-partitioning and by isoelectric focusing can be due to confor-mational changes of the protein in the phases system or by interactions between the polymers and the proteins.

The knowledge of the hydrophobicity of a protein is useful for the design of reverse phase chromatography units, for the understanding of protein—ligand interactions, and for the understanding of protein

Figure 2. Effect of cation and anion on the partition coefficient of proteins. The values of y obtained from this figure must be used with Equation 1.

folding and refolding. The usual way of measuring hydrophobicity of a solute is to measure the free energy of transfer of that solute from water into an organic solvent. Because of the need to use an organic solvent, these methods are not well suited to measure hydrophobicities of polar and flexible biological molecules. Aqueous 2-phase systems have been used to determine the relative hydrophobicity of various proteins (56). The overall idea is to "calibrate" a series of aqueous 2-phase systems (Dx/ Ficoll® and Dx/PEG have been used) by partitioning a series of small solutes (amino acids) of increasing hydrophobicity. It has been found that the partition coefficient of amino acid i, correlates with the partition coefficient of glycine by (56):

where HF is known as the hydrophobicity factor (a constant for a given 2-phase system) and RHj is the hydrophobicity of solute i relative to glycine. For a protein, the surface hydrophobicity of the i protein relative to molecule j, HSFj is given by:

The technique has been used to determine the hydrophobicity of several hemo-proteins including hemoglobin, apomyo-globin, and cytochrome c (56). The change of surface properties caused by pH-induced denaturation of some of these proteins has been investigated by this technique (56). For example, it has been found that the difference in hydrophobic index (expressed in equivalent CH2 groups) for denatured and native cytochrome c is 146, which indicates the exposure, as the protein denatures, of hydrophobic residues otherwise buried in the interior of the polypeptide.

It also is possible to follow protein-protein and protein-ligand association by 2-phase partitioning (29). The basic principle here is the strong dependence of the partition coefficient with the molecular size of the solutes. For example, Petersen (38,39) found that the partition coefficient in a Dx/PEG system of cytochrome c oxidase was 20, whereas that of cytochrome c was 0.275 and used these differences to show that both oxidized and reduced cytochrome c formed a 1:1 complex with the oxidase. In another study, Middaugh and Lawson (32) determined the association constant of hemoglobin by using aqueous 2-phase systems. They found that the partition coefficients of oxyhemoglobin and methemoglobin produced a sigmoidal curve when they were plotted against protein concentration. From these plots, they determined the dimer-tetramer association constant for these proteins.

The potential uses of this technique are vividly pictured in the following examples. PEG-coated liposomes are a current alternative to increase the stability of liposomes. The behavior of the modified liposomes will depend on their surface properties. Because the partition behavior of a particle is a signature of its surface properties, partitioning of PEG-coated liposomes in aqueous 2-phase systems can be used to anticipate their behavior in the blood stream. For example, Moribe et al. (34) used aqueous 2-phase systems to detect surface differences of PEG-coated liposomes. They partitioned the coated and uncoated liposomes (after exposing them to plasma) in two kinds of systems, charge sensitive (5% PEG-8000, 5% Dx T-500, and 0.11 M NaPO4, pH 7.0) and charge insensitive (0.01 NaPO4, 5% PEG-8000, 5% Dx T-500, and 0.15 M NaCl). Here charge sensitive or insensitive refers to systems in which the charge of the substance to be partitioned either affects its partition behavior or not. They concluded that in spite of PEG being a steric barrier for the interaction between plasma proteins and the liposomes, a weak interaction remains between the PEG-coated liposomes and plasma proteins. Berggren et al. (6) used P(EO-PO)/Reppal 2-phase systems to study the hydrophobicity of a series of proteins. They partitioned in these systems several salts, Na2PO4, NaCl, and NaClO4, and several proteins of different hydropho-bicity, myoglobin, cytochrome c, lysozyme, bovine serum albumin, and P-lactoglobu-lin. They were able to correlate the partition coefficients of these proteins with their tryptophan content.

1.2. Batch and Continuous Partitioning

Most of the times, aqueous 2-phase partitioning is carried out in test tubes, so it is a batch operation. Attempts have been made, however, to evolve the technique into a continuous operation, mostly to improve the purification factor of a given protein. One that we personally recommend is liquid—liquid partitioning chromatography. In liquid—liquid partitioning chromatography, one phase (for example the Dx-rich phase in a PEG/Dx system) is immobilized on a convenient support, and the other phase (in this case the PEG-rich phase) is used as the mobile phase.

The column is made of agarose or silica diol beads whose surface is derivatized by growing a hydrophilic polymer (polyacryl-amide) on it. The Dx-phase is retained, and the silica diol or agarose beads become impermeable to the proteins as determined in our laboratory. The PEG-rich phase is used as the mobile phase. The elution times suggest that the partitioning into Dx-phase is significant. For a column used by us, of 2.5 cm in diameter and 40 cm length (about 196.3-mL volume), the continuous phase (PEG) is about 10 mL, and the Dx-phase is about 5 mL. If the Dx-phase is assumed to coat the beads uniformly, then the ratio (radius of the bead with the coat)/(radius without the coat) is about 1.009. For a bead of radius 10 pm, the film thickness is about 900 nm.

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