483 Lecture 6 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part C
1. Ion Exchange Chromatography - Part A - Types of Ion Exchangers
Ion exchange chromatography depends on the ionic character of a protein/enzyme and since this property of an enzyme will change with the pH, it also depends on the pH of buffer used to dissolve the enzyme. In addition, the ionic strength of the solution or its total salt concentration is important in making ion exchange chromatography work.
There are two basic types of ion exchangers: those for binding positively charged ions or cations, which display on their surface negatively charged groups; and those for binding negatively charged ions or anions, which display on their surface positively charged groups.
The ion exchanger is composed of the solid support material, which for enzymes and proteins must be a "hydrogel" or polymer composed for easily hydrated groups like cellulose consisting of polymers of sugar molecules. On the surface of the polymeric support material ionic groups are displayed which have been covalently linked to the polymer support. Here are models of ion exchangers:
DEAE-cellulose or DEAE-C has a cellulose bead as the support material which has been derivatized with a positively charged amino group displaying a positive charge into the solution.
CM-cellulose or CMC has a cellulose bead as the support material which has been derivatized with a negatively charged carboxylic acid group displaying a negative charge into the solution.
Both these ion exchangers have changing degrees of ionization depending on the pH:
Thus, the pH range over which you can use the ion exchangers is limited by these properties. For example, the effective pH range for CMC is above pH 4, while it is below pH 9 for DEAE-C.
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