Aim:

To purify hen egg white lysozyme from a given protein mixture using ion exchange chromatography.

Introduction:

Ion exchange chromatography is perhaps the most commonly employed chromatographic method for separating the proteins, polypeptides, nucleic acids, and other charged molecules. The principle underlying ion exchange chromatography is the reversible exchange of the charged molecules (ions) present in the solution with those electrostatically bound to the insoluble support medium. The molecules in ion exchange chromatography, therefore, get separated based on their charge. The stationary phase is constituted by the inert polymeric beads that are functionalized with ionizable groups. Based on the type of ionizable group present on the stationary phase, an ion exchange chromatographic column is termed as an anion exchanger (positively charged stationary phase) or a cation exchanger (negatively charged stationary phase). Quaternary amines are the most common functional groups for anion exchange while carboxyl and sulphopropyl are the most common cation exchanging groups (Table 24.1) Sulphonic and quaternary amino groups are completely ionized over a wide range of pH and are therefore termed as strong exchangers. Weak ion exchangers start losing their charge below/above certain pH (below pH 6 for cation exchangers and above pH 9 for anion exchangers).

Table 24.1 Ion exchangers and their functional groups

DEAE and CM continue to be the most widely used ion exchange materials for protein purification in the physiological pH range. Proteins have several ionizable groups; at neutral pH, amino terminus of the protein and the side chains of lysine and arginine carry +1 charge each while C-terminus of the protein and the side chains of aspartate and glutamate impart -1 charge each. As the charges at the protein termini cancel each other out, the net charge on the protein at physiological pH is determined by the number of arginine, lysine, aspartate, and glutamate residues.