Many biological processes, such as regulation, catalysis, and complex formation, depend on the capacity of biopolymers to interact with apolar groups, i.e., their surface or "effective" hydrophobicity (1). The determination of surface hydrophobicity is therefore important for understanding the mechanism of interaction of a biopolymer with its ligands. The determination of surface hydrophobicity is also important for designing a method for the purification or the concentration of biopolymers, especially proteins.
This chapter describes the usefulness of affinity partitioning in aqueous two-phase systems containing Dextran and Poly(ethylene glycol) (PEG), where a fraction of the latter is replaced by PEG to which hydrophobic groups have been covalently coupled, for the aforementioned purposes (2-4). Comparison of affinity partitioning in the presence of PEG carrying a hydrophobic group with that in the presence of PEG carrying the same group but unsaturated or with a terminal-charged (e.g., COO- or NH3+) or polar (e.g., OH) group, permits further characterization of the binding site (5). This chapter also describes the use of hydrophobic affinity data for the characterization of conformational changes in proteins (4,6,7).
Shanbhag and Axelsson (2) have described the theoretical basis of hydrophobic affinity partitioning. In effect, one measures the partition coefficient (K') in the presence and (K) in the absence of the PEG-coupled hydrophobic ligand (PEG-Lff). The respective partition coefficient is given by log K = log K'0 + Y Z (1)
From: Methods in Biotechnology, Vol. 11: Aqueous Two-Phase Systems: Methods and Protocols Edited by: R. Hatti-Kaul © Humana Press Inc., Totowa, NJ
where K0 and K0 are the charge (Z)-independent values of the partition coefficients, in the presence and absence of PEG-LH, respectively, y' and y are the corresponding values of the interfacial potential in the two-phase system, and Z is the net charge of the partitioned molecule. At a constant pH, Z is constant and if y is not affected by the presence of PEG-LH i.e., y' = y (a plausible assumption), the difference, A log K, between the partition coefficient in the presence respectively the absence of PEG-LH , is given by
The difference, A log K, is a measure of the affinity of the biopolymer for LH (if A log K > 0) or repulsion of the biopolymer by LH (if A log K < 0). When A log K = 0 the biopolymer is unaffected by the presence of PEG-LH in the system. If A log K is plotted as a function of concentration of PEG-LH, usually expressed as a percent of the total PEG in the two-phase system, the plot will resemble a binding curve (as exemplified in Fig. 1).
It should be noted, that the partition curve provides only a relative and not an absolute measure of the affinity or the repulsion of a biopolymer for the hydrophobic ligand bound to PEG.
If PEG in PEG-Lff is the dominating part, the latter will be soluble in water in spite of the bound hydrophobic ligand. This is in contrast to the methods depending on the binding of hydrophobic probes, e.g., fluorescence probes, which will have to carry polar or charged groups to be soluble in water. These groups on the probe could affect the binding, which, therefore, might not reflect the true hydrophobicity. Hence, if the aforementioned precautions are taken, hydrophobic affinity partitioning (A log K) should yield a better measure of surface hydrophobicity of a biopolymer, a type of hydrophobic site, and should be useful in comparing different conformational or oligomerization states of the same biopolymer or in comparison between two biopolymers.
Further, the knowledge of the A log K in a system containing a particular PEG-Lff should facilitate the design of a method for the separation of the biopolymer concerned from a mixture if the A log K for it is very much different.
The preparative use of hydrophobic affinity partitioning can be exemplified by the specific extraction of serum albumin from human plasma (8). For a general use of partitioning for preparative purposes, the readers should refer to refs. 9 and 10.
Examples of the use of hydrophobic affinity partitioning, mostly for characterization of the surface hydrophobicity of proteins are recorded in Table 1.
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