Question 6. For each of these methods of separating proteins, describe the princ

Question

Question 6. 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.

Question 7. 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?

Question 8. How is beta- Mercaptoethanol used in establishing the amino acid sequence of polypeptides? Why is it used to prepare the sample for gel-electrophoresis?

Question 9. How would you make 100mL of a 0.05M phosphate buffer at pH 7.5 using:

Na2HPO4*7H2O

NaH2PO4*1H2O

Question 5. Many intracellular proteins are required to interact or bind to other macromolecules within a cell in order to function properly. One class of such proteins is comprised of proteins that can bind DNA. Such proteins often have conserved structural features, called motifs, which interact with DNA. One such motif consists of two closely aligned a-helices (shown as cylinders below) that each have leucine-rich regions. This motif is referred to as a leucine zipper Figure by MIT OCW. a) Based on what you know about leucine, what type of interaction is likely to be holding the two halves of the "zipper" together? Circle one of the following: Hydrogen bonds Covalent Hydrophobic Ionic

Explanation / Answer

Question no. 5: As we know Leucine contains isobutyl moiety, the interaction holding leucine molecules together should be hydrophobic interaction as isobutyl group are hydrophobic in nature.

Question no. 6:

a) Ion Exchange Chromatography: As name suggests this technique is suitable to separate proteins which contains charged part.

for example: A given protein is positively charged and we need to separate it from mixtures of uncharged ones then in that case we can pass the given mixture through column containing negativlely charged substrates. The positively charged one will be strongly bounded by negativley charged substrate of column thus facilitating the separation.

b) Size - Exclusion chromatography: In this case, the guiding principle for separation is difference in size. Column consits of sieve of various size. Small sized particle can pass easily while larged ones will be excluded.

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