Problem:

Question 1: Separation of amino acids by ion-exchange Chromatography. Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion-exchange chromatography. Amino acids placed on a cation-exchange resin containing sulfate (-SO3-) groups flow down the column at different rates because of two factors that influence their movement

(1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and

(2) hydrophobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed,

determine which will be eluted first from the cation-exchange column by a ph 7.0 buffer.

a.) Asp and Lys

b.) Arg and Met

c.) Glu and Val

d.) Gly and Val

e.) Ser and Ala

Question 2: Note the structure of the heptapeptide:

NH2-ser-asp-gln-tyr-gly-cys-thr-COOH.

Required:

Question: Would you expect the peptide to dissolve well in water?

Question: Answer the same for the heptapeptide:

NH2-trp-ala-gly-met-val-leu-phe-COOH.

Question 3: At pH=7, do you expect the following peptides to bind a cation exchanger or an anion exchanger resins?

a) NH2-ser-glu-cys-tyr-ala-asp-lys-COOH

b) NH2-ala-arg-lys-thr-asn-glu-lys-COOH

Explain.

Question 4: List four different proteases that are used in peptide mapping, and provide their cleavage specificity.

Question 5: You have a solution in your hand that contains your protein. You want to sequence the protein. Use the PPT presentation included in Topic 5B to make a step by step list of how this process is carried out.

Question 6: The following polypeptide was digested with the proteolytic enzyme trypsin.

NH2-Met-Gln-Tyr-Thr-Ser-Gly-Asn-Arg-Trp-Ala-Gly-Val-Leu-Cys-Pro-Lys-Gln-Glu-Asp-Ser-Asp-Glu-Tyr-Thr-COOH

Required:

Question: Write the resulting peptide fragments and mark the amino acids who's side chains are expected to be charged at pH=7 (Write + or - charge)

Question: You are asked to separate the peptides from one another by a two-step column chromatography procedure. At your disposal are ion exchange columns (cation or anion, based on your choosing), and a hydrophobic column. Describe how you would carry out chromatography to accomplish this separation. Your description should explain how the various fragments separate from each other in each step

Please explain all the answers thoroughly.