Enzyme Technology
Ion-exchange chromatography
Enzymes possess a net
charge in solution, dependent upon the pH and their structure and isoelectric
point. In solutions of pH below their isoelectric point they will be positively
charged and bind to cation exchangers whereas in solutions of pH above their
isoelectric point they will be negatively charged and bind to anion exchangers.
The pH chosen must be sufficient to maintain a high, but opposite, charge on
both protein and ion-exchanger and the ionic strength must be sufficient to
maintain the solubility of the protein without the salt being able to
successfully compete with the protein for ion-exchange sites. The binding is
predominantly reversible and its strength is determined by the pH and ionic
strength of the solution and the structures of the enzyme and ion-exchanger.
Normally the pH is kept constant and enzymes are eluted by increasing the
solution ionic strength. A very wide range of ion-exchange resins, cellulose
derivatives and large-pore gels are available for chromatographic use.
Ion-exchange
materials are generally water insoluble polymers containing cationic or anionic
groups. Cation exchange matrices have anionic functional groups such as
-SO3−, -OPO3− and -COO− and anion
exchange matrices usually contain the cationic tertiary and quaternary ammonium
groups, with general formulae -NHR2+ and -NR3+.
Proteins become bound by exchange with the associated counter-ions.
Ion-exchange
polystyrene resins are eminently suitable for large-scale chromatographic use
but have low capacities for proteins due to their small pore size. Binding is
often strong, due to the resin hydrophobicity, and the conditions needed to
elute proteins are generally severe and may be denaturing. Nevertheless such
resins are a potential means of concentrating or purifying enzymes.
Ion-exchange
cellulose and large pore gels are much more generally suitable for enzyme
purification and, indeed, many were designed for that task. A variety of charged
groups, anionic or cationic, may be introduced. The practical level of
substitution of cellulose is limited as derivatisation above one mole per
kilogram may lead to dissolution of the cellulose. Consequently, proteins may be
eluted from them under mild conditions. Ion-exchange cellulose can be used in
both batch and column processes but on a large scale they are used mainly
batchwise. This is because the increased speed of large-scale batchwise
processing and the avoidance of the deep-bed filtering characteristics of
columns outweigh any advantage due to the increase in resolution on columns.
Careful preparation before use and regeneration after use is essential for their
effective use.
Batchwise operations involve stirring the pretreated and
equilibrated ion-exchanger with the enzyme solution in a suitable cooled vessel.
Adsorption to the exchangers is usually rapid (e.g., less than 30 minutes) but
some proteins can take far longer to adsorb completely. Stirring is essential
but care must be taken not to generate fine particles (fines). Unadsorbed
material may be removed in a variety of manners. Basket centrifuges are a
particularly convenient means of hastening the removal of the initial
supernatant and the elution of the adsorbed material. This is usually done using
stepwise increases in ionic strength and/or changes in pH but it is possible to
place the exchangers, plus adsorbed material, in a column and elute using a
suitable gradient. However, while ion-exchange cellulose are widely used for
column chromatography on the laboratory scale, their compressibility causes
difficulty when attempts are made to use large scale columns.
Some of the
problems with derivatised cellulose may be overcome using more recently
introduced materials. Derivatives of cross-linked agarose (Sepharose CL-6B) and
of the synthetic polymer Trisacryl have high capacities (up to 150 mg protein ml−1) yet are not significantly compressible. In addition, they do not change
volume with pH and ionic strength which allows them to be regenerated without
removal from the chromatographic column.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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