Hydrophobic interaction chromatography

Of the 20 amino acids commonly found in proteins, eight are classified as hydrophobic, due to the non-polar nature of their side chains (R groups, Figure 6.12). Most proteins are folded such

Table 6.3 Functional groups commonly attached to chromatographic beads in order to generate cation or anion exchangers

Group name

Group structure

Exchanger type

Diethylaminoethyl (DEAE)

—O—(CH2)2—NH+— (CH2—CH3)2


Quaternary ammonium (Q)



Quaternary aminoethyl (QAE)



Carboxymethyl (CM)



Methyl sulfonate (S)



Sulfopropyl (SP)



that the majority of their hydrophobic amino acid residues are buried internally in the molecule and, hence, are shielded from the surrounding aqueous environment (Chapter 2). Internalized hydrophobic groups normally associate with adjacent hydrophobic groups. A minority of hy-drophobic amino acids, however, are present on the protein surface and, hence, are exposed to the outer aqueous environment. Different protein molecules differ in the number and types of hydrophobic amino acid on their surface, and hence on their degree of surface hydrophobicity. Hydrophobic amino acids tend to be arranged in clusters or patches on the protein surface. Hy-drophobic interaction chromatography fractionates proteins by exploiting their differing degrees of surface hydrophobicity. It depends on the occurrence of hydrophobic interactions between the hydrophobic patches on the protein surface and hydrophobic groups covalently attached to a suitable matrix.

The most popular hydrophobic interaction chromatographic beads (resins) are cross-linked agarose gels to which hydrophobic groups have been covalently linked. Specific examples include alanine coo"

valine coo"

leucine coo"

isoleucine coo"

h3n+—°fc-h h3n+—ac-h h3n+—ac-h h3n+—"t-h ch3

tryptophan methionine phenylalanine proline coo"



'c —c i h^f NC"nh \ / C = C / \ hh ch2 ch2 s ch3


h v ac ^ h ^ / s ^ n+ c nh c —c h /c c\ h h h h h

Figure 6.12 Structural formulae of the eight commonly occurring amino acids that display hydrophobic characteristics



Figure 6.13 Chemical structure of (a) phenyl and (b) octyl sepharose, widely used in hydrophobic interaction chromatography

octyl- and phenyl-sepharose gels, which contain octyl and phenyl hydrophobic groups respectively (Figure 6.13).

Protein separation by hydrophobic interaction chromatography is dependent upon interactions between the protein itself, the gel matrix and the surrounding aqueous solvent. Increasing the ionic strength of a solution by the addition of a neutral salt (e.g. ammonium sulfate or sodium chloride) increases the hydrophobicity of protein molecules. This may be explained (somewhat simplistically) on the basis that the hydration of salt ions in solution results in an ordered shell of water molecules forming around each ion. This attracts water molecules away from protein molecules, which in turn helps to unmask hydrophobic domains on the surface of the protein.

Protein samples, therefore, are best applied to hydrophobic interaction columns under conditions of high ionic strength. As they percolate through the column, proteins may be retained via hydrophobic interactions. The more hydrophobic the protein, the tighter the binding. After a washing step, bound protein may be eluted by utilizing conditions that promote a decrease in hydro-phobic interactions. This may be achieved by irrigation with a buffer of decreased ionic strength, inclusion of a suitable detergent, or lowering the polarity of the buffer by including agents such as ethanol or ethylene glycol.

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins.

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