Question

In order to learn more about the PAH enzyme, it was necessary to purify it. PAH has been isolated from both rats and humans. In the rat, three isozymes of PAH have been identified in the liver. Their molecular weights are identical, but their charges are different, as demonstrated by isoelectric focusing. The pI values are 5.2, 5.3 and 5.6. DEAE-cellulose (anion exchange) chromatography was one of the steps in the purification procedure of the enzymes. Predict the order of elution of these isozymes from the DEAE-cellulose column. What pH buffer would you choose in running the column? ?

Answer 1

Chromatography is a physical method for the separation of compounds or mixtures. The separation is based on differential partitioning of the compound in two different immiscible phases, mobile and stationary phase. The mobile phase (a liquid or a gas) travels through the stationary phase (a liquid or a solid) in a defined direction and the partition or distribution of components between the two phases depends on adsorption, ionic interactions, diffusion, solubility or specific interactions.

Anion exchange chromatography is used for the separation of charged molecules. In this technique, stationary (solid) phase consists of an insoluble matrix with covalently attached positively charged groups (cation) like (Diethylaminoethyl).

Since the pI values of PAH isozymes are 5.2, 5.3 and 5.6, the protein will bind to the anion exchanger (DEAE-cellulose) at pH > 5.6 (like Tris buffer saline pH 7.5) because at this pH the proteins will have a net negative charge. The bound molecules will be eluted by decreasing the pH of elution buffer till it reaches the pI of different isozymes (at that pH isozymes will possess no net charge and will be eluted). The first eluted enzyme will be the isozyme with pI 5.6 then the isozyme with pI 5.3 and last the isozyme with pI 5.2