You accidently mixed 2 precious proteins. Protein A is a large, positively-charged molecule while Protein B is a small, negatively-charged molecule at physiological pH.
(i) Propose 2 methods to separate these proteins. Be sure to explain the order in which these proteins elute.
(ii) Is it possible for protein B to contain Lysines or Arginines? Explain why
(iii) If you were to convert protein A to a zwitterion, would you increase or decrease pH. Why?

i) Exclusion chromatography and ion-exchange chromatography.

ii) Yes.

iii) I would increase pH.

Explanation:

i) Since proteins A and B differ in both size and charge, we can separate them via exclusion chromatography or ion-exchange chromatography.

In exclusion chromatography, the column is packed with porous beads that are composed of dextran polymers, agarose, or polyacrylamide. The larger protein (in this case protein A) will elute first because it is too big to fit into the pores of the matrix, and it will pass by the pores, therefore flowing through the column. On the other hand, smaller proteins (in this case protein B) will enter the pores increasing the retention time in the column, therefore they will elute later.

In ion-exchange chromatography, like a cation-exchange chromatography, we can separate protein A which is positively charged, using a stationary phase that is negatively charged. Protein A will bind to the matrix and the protein B (negatively charged) will elute first. To separate protein A from the column, we can run a higher concentration of ions through the column or change the pH of the column, then protein A will elute.

ii) Yes. What gives the net charge of the protein is the combination of all the charges of its amino acids at a given pH. That's why basic aminoacids, like Lysine and Arginine, will be present and not make the protein positively-charged as long as there are enough acid amino acids.

iii) The isoelectric point is the pH at which a molecule carries no net electrical charge. At this point we have the molecule as a zwitterion. Given that we have a positively charged protein, we should increase the pH to provide negative charges.

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