Question

Question 3. Draw the tripeptide: Val-Ser-Ala. Include a phosphoryl group on the amino acid sidechain that is most likely to undergo phosphorylation. Question 4. A protein retained on an ion exchange chromatography column is usually eluted off the column by (circle the correct answer) A. increasing the salt concentration in the buffer B. adding the protein's free ligand. C. changing the temperature of the elution buffer. D. allowing the retained protein to naturally come off the column after the non-specifically bound proteins have first passed through the resin. Question 5. After performing several purification steps on your favorite protein, you prepare the following chart: procedure fraction volume total protein activity specific activity (ml) (mg) (units) cell extract 1,400 100,000 400,000 column #1 280 4,000 16,000 column #2 140 1,000 10,000 column #3 10 25,000 25 A. Calculate the specific activity of the sample after each step B. After which column is the protein the most pure?__ C. Which column increased the purification the least amount?

Answer 1

3. Val -Ser- Ala tripeptide can be drawn using pepdraw:

но HN, IZ

4. An Ion exchange matrix would be charged positive (Anion exchange) or negative (cation exchange) so that molecules to be purified that carry a net positive or negative charge interact by binding to the matrix. If the net charge of a protein in a mixture of other proteins in negatively charged whereas the rest of the proteins in the mixture is positively charged, then in an anion exchange column, the negatively charged protein would bind whereas the other proteins would move out of the column without binding. The bound protein is then dislodged from the matrix by passing through a salt solution such as NaCl, where the Cl- ions would take the place of the protein. The right answer here is option A.

Adding the protein's free ligand would only facilitate the binding of the ligand to the already bound protein and would not help in dislodging the protein. Ligands are used in affinity chromatography,not ion exchange. option B would be wrong. Changing the temperature might prove detrimental to the protein, especially if it is an enzyme it would lose its activity when treated at high temperatures. The principle of ion exchange separation does not involve heat treatment. option c would be wrong. option D would be wrong too as the protein would stay put once it is bound to the matrix and left undisturbed.

5 Given the list of purification events for an enzyme:

procedure fraction	volume (ml)	total protein (mg)	activity (units)	specific activity (units / mg protein)	Fold purity
cell extract	1400	100,000	400,000	4	-
column#1	280	4000	16000	4	1.0
column#2	140	1000	10000	10	2.5
column#3	10	25	25000	1000	100

A. Specific activity of an enzyme is defined as the units per mg protein. It is calculated by diving the total activity by the total protein. Specific activity is highest after purification with column #3.

B. The protein is most pure after passing through column#3. The fold purity calculated above tells us how pure the enzyme has become after each step of purification. After purification with column#3 the protein is 100-fold pure than when in the previous step.

C. The least amount of purification is obtained after purification with column#1, the specific activity is the same as that of the cell extract which means the protein is not purified at all.