Enzyme Technology
Affinity chromatography
This is a term which now covers a variety of methods of
enzyme purification, the common factor of which is the more or less specific
interaction between the enzyme and the immobilised ligand. In its most specific
form, the immobilised ligand is a substrate or competitive inhibitor of the
enzyme. Ideally it should be possible to purify an enzyme from a complex mixture
in a single step and, indeed, purification factors of up to several
thousand-fold have been achieved. An alternative, equally specific approach is
to use an antibody to the enzyme as the ligand. Such specific matrices, though,
are very expensive and cannot be generally employed on a large scale.
Additionally, they often do not perform as well as might be expected due to
non-specific binding effects. In general, affinity chromatography achieves a
higher purification factor (with a median value in reported purifications of
about ten fold) than ion-exchange chromatography (with a median performance of
about three fold), in spite of it generally being used at a later stage in the
purification when there is less purification possible.
A less specific approach,
suitable for many enzymes, is to use analogues of coenzymes, such as NAD+, as the
ligand. This method has been used successfully but has
now been superceded by the employment of a series of water soluble dyes as
ligands. These are much cheaper and, usually by trial and error, have been found
to have surprising degrees of specificity for a wide range of enzymes. This
dye-affinity chromatography was allegedly discovered by accident, certain
enzymes being found to bind to the blue-dyed dextran used, as a molecular weight
standard, to calibrate gel exclusion columns.
Another fortuitous discovery was
hydrophobic interaction chromatography, found when it was noted that certain
proteins were unexpectedly retained on affinity columns containing hydrophobic
spacer arms. Hydrophobic adsorbents now available include octyl or phenyl
groups. Hydrophobic interactions are strong at high solution ionic strength so
samples need not be desalted before application to the adsorbent. Elution is
achieved by changing the pH or ionic strength or by modifying the dielectric
constant of the eluent using, for instance, ethanediol. A recent introduction is
cellulose derivatised to introduce even more hydroxyl groups. This material (Whatman
HB1) is designed to interact with proteins by hydrogen-bonding. Samples are
applied to the matrix in a concentrated (over 50% saturated, > 2M) solution
of ammonium sulphate. Proteins are eluted by diluting the ammonium sulphate.
This introduces more water which competes with protein for the hydrogen-bonding
sites. The selectivity of both of these methods is similar to that of fractional
precipitation using ammonium sulphate but their resolution may be somewhat
improved by their use in chromatographic columns rather than batchwise.
Careful
choice of matrices for affinity chromatography is necessary. Particles should
retain good flow and porosity properties after attachment of the ligands and
should not be capable of the non-specific adsorption of proteins. Agarose beads
fulfil these criteria and are readily available as ligand supports (see also
Chapter 3). Affinity chromatography is not used extensively in the large-scale
manufacture of enzymes, primarily because of cost. Doubtless as the relative
costs of materials are lowered, and experience in handling these materials is
gained, enzyme manufacturers will make increased use of these very powerful
techniques.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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