PROBLEM SET 3: CHAPTER 3 AMINO ACIDS, PEPTIDES, AND PROTEINS
1) For each of these methods of separating proteins, describe the principle of the method,
namely what property of proteins allows their separation by this technique.
a) ion-exchange chromatography; b) size-exclusion chromatography; c) affinity
chromatography
Ans: Separates on the basis of charge
Separates on the basis of size
Separates on the basis of affinity to ligands
2) If two polypeptides have the same molar mass, name two methods you could employ to
separate them. What is the principle of the method of separation?
Ans: Ion-exchange chromatography – separates on the basis of charge
Isoelectric focusing – separates on the basis of pI
Affinity chromatography – separates on the basis of affinity to ligands
Ammonium sulfate fractionation – separates on the basis of differential aggregation
Hydrophobic interaction chromatography – separates on the basis of hydrophobicity
3) How are the reagents below used in establishing the amino acid sequence of polypeptides?
Edman reagent
Ans: To determine AA sequence at the N-terminal – sequentially cleaves AA residues when the
procedure is repeated
Mercaptoethanol
Ans: To cleave intra- and inter-molecular (S-S) disulfide bonds to remove tertiary and
quaternary structure
Trypsin
Ans: To cleave proteins to smaller peptides at defined locations – C-end of lys and arg