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?

Question

A.) the peptide will displace the enzyme and the subsequent fractions should contain a protein complex that includes the enzyme bound to the peptide
B.) the enzyme will remain bound to the column and none of the fractions will contain the enzyme of interest; only flow through will leave the column
C.) the peptide will disrupt the binding of the enzyme to the antibody because antibodies are generally much larger than enzymes; the enzyme will emerge in the next fractions, followed by the antibody
D.) the peptide will displace the enzyme, which will then be free to cleave the peptide; subsequent fractions will contain the enzyme and the peptide fragments, the latter of which will emerge first due to their smaller size
E.) the peptide will bind to the antibodies on the column, displacing the purified enzyme; they should obtain pure enzyme in the subsequent fractions

Answer 1

Ans: B.) the enzyme will remain bound to the column and none of the fractions will contain the enzyme of interest; only flow through will leave the column