Your browser does not support JavaScript!

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.


Home | Back | Next

This page was established in 2004 and last updated by Martin Chaplin
on 6 August, 2014