Proteins are most often fractionated by column chromatography, in which a mixture of proteins in solution is passed through a column containing a porous solid matrix. Different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separately as they flow out of the bottom of the column (Figure 8–8). Depending on the choice of matrix, proteins can be separated according to their charge (ion-exchange chromatography), their hydrophobicity (hydrophobic chromatography), their size (gel-filtration chromatography), or their ability to bind to particular small molecules or to other macromolecules (affinity chromatography).
Many types of matrices are available. Ion-exchange columns (Figure 8–9A) are packed with small beads that carry either a positive or a negative charge, so that proteins are fractionated according to the arrangement of charges on their surface. Hydrophobic columns are packed with beads from which hydrophobic side chains protrude, selectively retarding proteins with exposed hydrophobic regions. Gel-filtration columns (Figure 8–9B), which separate proteins according to their size, are packed with tiny porous beads: molecules that are small enough to enter the pores linger inside successive beads as they pass down the column, while larger molecules remain in the solution flowing between the beads and therefore move more rapidly, emerging from the column first. Besides providing applied to the top of applied reservoir of solvent
Figure 8–8 The separation of molecules by column chromatography. The sample, a solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose. A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. Because various components of the sample travel at different rates through the column, they are fractionated into different tubes.
a means of separating molecules, gel-filtration chromatography is a convenient way to estimate their size.
Affinity chromatography (Figure 8–9C) takes advantage of the biologically important binding interactions that occur on protein surfaces. If a substrate molecule is covalently coupled to an inert matrix such as a polysaccharide bead, the enzyme that operates on that substrate will often be specifically retained by the matrix and can then be eluted (washed out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically designed sequence can be immobilized in this way and used to purify DNA-binding proteins that normally recognize this sequence of nucleotides in chromosomes. Alternatively, specific antibodies can be coupled to a matrix to purify protein molecules recognized by the antibodies. Because of the great specificity of all such affinity columns, 1000to 10,000-fold purifications can sometimes be achieved in a single pass.
If one starts with a complex mixture of proteins, a single passage through an ion-exchange or a gel-filtration column does not produce very highly purified fractions, since these methods individually increase the proportion of a given protein in the mixture no more than twentyfold. Because most individual proteins represent less than 1/1000 of the total cell protein, it is usually necessary to use several different types of columns in succession to attain sufficient purity, with affinity chromatography being the most efficient (Figure 8–10).
Inhomogeneities in the matrices (such as cellulose), which cause an uneven flow of solvent through the column, limit the resolution of conventional column chromatography. Special chromatography resins (usually silica-based) composed of tiny spheres (3–10 μm in diameter) can be packed with a special apparatus to form a uniform column bed. Such high-performance liquid chromatography (HPLC) columns attain a high degree of resolution. In HPLC, the solutes equilibrate very rapidly with the interior of the tiny spheres, and so solutes with different affinities for the matrix are efficiently separated from one another even at very fast flow rates. HPLC is therefore the method of choice for separating many proteins and small molecules.