Protein pMolecular Weight (kDa) 3.1 6.9 10.3 7.1 8.6 265 93 96 43 189 You load t

Question

Protein pMolecular Weight (kDa) 3.1 6.9 10.3 7.1 8.6 265 93 96 43 189 You load the protein mix onto a cation exchange column at pH 5. Next, you apply a "washing" step by passing through buffer at pH 5. Finally, for your elution step, you apply a pH gradient starting from pH 2.0 to pH 13.0. (A gradient buffer system allows you to gradually and continuously change the pH of vour mobile phase starting from pH 2 up to pH 13). Indicate the order in which proteins will exit / elute from the column during the elution step. Please explain your answer.

Explanation / Answer

Ion exchange resins are polymers that are capable of exchanging particular ions within the
polymer with ions in a solution that is passed through them. In a cation-exchange resin all the sites are negatively charged, so that only positive ions can be separated.

When the mixture to be separated is passed through the ion exchange column, the molecules that do not bind to the column are removed via washing the column with the starting buffer.

Elution is achieved through the continuous change of the ionic strength or the pH, i.e. the application of a gradient elution. During gradient elution, molecules with smaller net charges (i.e. the weaker-binding ones) will be the first to leave the column.

At the lowest pH all proteins will be positively charged because of the increase in the presence of H+ ions. As the pH is increased, they eventually lose their H+ ions and reach the isoelectric state where they are electrically neutral. If the pH is increased furthermore, they tend to lose more H+ ions and will attain a negative charge.

Generally, when the pH is greater than the pI the protein will be negatively charged and when the pH is less than the pI, the protein will be positively charged.

Protein A has a pI value of 3.1. When the washing buffer at pH 5 is used, the protein loses all its H+ ions and becomes negatively charged. Since, the cation exchange resin is also negatively charged, they get eluted because of the force of repulsion. At pH 5 all the other proteins will be positively charged and hence will remain bound to the resin.

While applying elution buffer from pH 2.0, no elution will occur till the pH reaches 6.9 which is the pI value of protein B. At this pH, the protein B becomes electrically neutral (no charge) and should detach itself from the resin and should get eluted first.

In the same way, as we increase the pH, the protein which reaches the isoelectric state next would be protein D which at the pH 7.1 becomes electrically neutral and should get detached from the resin and be eluted.

But since pI 6.9 and 7.1 are very nearby, they get eluted almost together. Here, protein D has a very low molecular weight and hence gets eluted first followed by protein B whose molecular weight is higher.

This will be followed by protein E which becomes electrically neutral at pH 8.6 and then protein C which gets eluted when the pH reaches 10.3

So the order of elution is A, D, B, E and C.

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