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.

an individual’s sex (i.e., whether they are a male or female) is determined by the sex chromosomes. most people have two sex chromosomes, one that is inherited from their mother and one that is inherited from their father. typically, females have two x chromosomes (xx) and males have one x chromosome and one y chromosome (xy). conditions caused by changes (“mutations”) in genes located on the x chromosome are considered x-linked.

x-linked recessive inheritance

most x-linked conditions are recessive. this means that in a person with two x chromosomes (most females), both copies of a gene (i.e., one on each x chromosome) must have a change or mutation whereas in a person with one x chromosome (most males), only one copy of a gene must have a mutation. a female with a mutation in one copy of a gene on the x chromosome is said to be a “carrier” for an x-linked condition. a male with a mutation in a gene on the x chromosome is typically affected with the condition. because females have two copies of the x chromosome and males have only one x chromosome, x-linked recessive diseases are more common among males than females. however, x-linked recessive diseases can occur in both males and females.

for x-linked recessive disorders, an unaffected carrier mother who has a mutation in a gene on the x chromosome can transmit either the x chromosome with this mutation or a “normal” x chromosome to her children. if the father is unaffected, none of her daughters will be affected and all of her daughters will be unaffected—since they will inherit at least one normal x chromosome from their father. however, each daughter will have a 50% chance of being an unaffected carrier like her mother and a 50% chance of both x chromosomes being normal.

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