Question

Investigators wish to purify an enzyme—a serine protease—using affinity chromatography. They attach to the matrix of an affinity column an antibody molecule that binds specifically to a short sequence of amino acids located in the enzyme’s active site. When they apply a mixture of proteins to the column, the protease adheres to the column and the other proteins pass through. To extract their purified enzyme from the column, the investigators add a large excess of the peptide that the antibody recognizes. What should they expect to occur after this treatment?

Answer 1

After adding an excess of the specific peptide to the column, investigators should expect the serine protease to be displaced from the antibody due to competitive binding, allowing them to collect the purified enzyme.

Step-by-step explanation:

Purification of Serine Protease Using Affinity Chromatography

When using affinity chromatography to purify an enzyme such as a serine protease, investigators can attach an antibody that specifically binds to a sequence of amino acids in the enzyme's active site to the column's matrix.

During the purification process, the enzyme adheres to the column while other proteins do not.

To elute the bound serine protease, investigators can add an excess of the specific peptide recognized by the antibody.

This competitive process should result in the displacement of the serine protease from the column, allowing its collection in a now purified form.

The logic behind this method is based on the principle of competitive binding, where the free peptide competes with the bound enzyme for the antibody attached to the column.

Because a large excess of the peptide is added, it outcompetes the enzyme's binding to the antibody, thus releasing the enzyme into the solution where it can be collected.