1.  Knowing that it’s there. Why is an assay necessary to purify a protein? ✓ 4

2.  Pair bonding. Match the terms with the descriptions. ✓ 4

3.  Salting out. Why do proteins precipitate at high salt concentrations? ✓ 4

4.  Salting in. Although many proteins precipitate at high salt concentrations, some proteins require salt to dissolve in water. Explain why some proteins require salt to dissolve.

5.  Competition for water. What types of R groups would compete with salt ions for water of solvation?

6.  Column choice.

(a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion-exchange or molecular exclusion chromatography be most appropriate for separating the products? Explain. ✓ 4

(b) Suppose that the peptide had, instead, been digested with chymotrypsin. What would be the optimal separation technique? Explain.

7.  Frequently used in shampoos. The detergent sodium dodecyl sulfate (SDS) denatures proteins. Suggest how SDS destroys protein structure.

8.  Making more enzyme? In the course of purifying an enzyme, a researcher performs a purification step that results in an increase in the total activity to a value greater than that present in the original crude extract. Explain how the amount of total activity might increase. ✓ 4

10.  Charge to mass.

(a) Proteins treated with a sulfhydryl reagent such as β-mercaptoethanol and dissolved in sodium dodecyl sulfate have the same charge-to-mass ratio. Explain. ✓ 4

(b) Under what conditions might the statement in part a be incorrect?

(c) Some proteins migrate anomalously in SDS–PAGE gels. For instance, the molecular weight determined from an SDS–PAGE gel is sometimes very different from the molecular weight determined from the amino acid sequence. Suggest an explanation for this discrepancy.

11.  Push back. During ultracentrifugation, the force opposing centrifugation is the buoyant force, equal to . Using the equation for buoyant force, show why a dense particle moves more rapidly than does a less dense one in ultracentrifugation.

12.  A special attraction. What unique property of the estrogen receptor allows for its identification and purification?✓ 5

13.  Many or one. Differentiate between polyclonal and monoclonal antibodies. ✓ 5

14.  A falling out. Explain how immunoprecipitation can be used to purify proteins. ✓ 5

15.  Don’t you mean who? What is an ELISA, and how is it used? ✓ 5

16.  John Wayne’s favorite. Describe western blotting. ✓ 5

17.  A question of efficiency. The Edman method of protein sequencing can be used to determine a sequence of proteins no longer than approximately 50 amino acids. Why is this length limitation the case? ✓ 5

18.  Divide and conquer. The determination of the mass of a protein by mass spectrometry often does not allow its unique identification among possible proteins within a complete proteome, but determination of the masses of all fragments produced by digestion with trypsin almost always allows unique identification. Explain.

19.  Quaternary structure. A protein was purified to homogeneity. Determination of the mass by gel-filtration chromatography yields 60 kDa. Chromatography in the presence of urea yields a 30-kDa species. When the chromatography is repeated in the presence of urea and β-mercaptoethanol, a single molecular species of 15 kDa results. Describe the structure of the molecule.

20.  Protein solubility. The isoelectric point (pI) of a protein is the pH at which a protein has no net charge. The pI is also the pH at which a protein is least soluble. The graph below shows the solubility of a protein as a function of pH in solutions of different ionic strengths. ✓ 4



(a) Why is the protein least soluble at its pI (indicated by the red arrow in the figure)?

(b) Why does increasing the salt concentration increase the protein’s solubility?

21.  Protein sequencing 1. Determine the sequence of hexapeptide on the basis of the following data. Note: When the sequence is not known, a comma separates the amino acids. (Table 5.3)

Amino acid composition: (2R,A,S,V,Y)

Amino-terminal analysis of the hexapeptide: A

Trypsin digestion: (R,A,V) and (R,S,Y)

Carboxypeptidase A digestion: no digestion

Chymotrypsin digestion: (A,R,V,Y) and (R,S)

22.  Protein sequencing 2. Determine the sequence of a peptide consisting of 14 amino acids on the basis of the following data.

Amino acid composition: (4S,2L,F,G,I,K,M,T,W,Y)

Amino-terminal analysis: S

Carboxypeptidase A digestion: L

Trypsin digestion: (3S,2L,F,I,M,T,W) (G,K,S,Y)

Chymotrypsin digestion: (F,I,S) (G,K,L) (L,S) (M,T) (S,W) (S,Y)

Amino-terminal analysis of (F,I,S) peptide: S

Cyanogen bromide treatment: (2S,F,G,I,K,L,M*,T,Y) (2S,L,W)

M*, methionine detected as homoserine

23.  Dialysis. Suppose that you precipitate a protein with 1 M (NH₄)₂SO₄, and you wish to reduce the concentration of the (NH₄)₂SO₄. You take 1 ml of your sample and dialyze it in 1000 ml of buffer. At the end of dialysis, what is the concentration of (NH₄)₂SO₄ in your sample? How could you further lower the (NH₄)₂SO₄ concentration?

24.  Sedimenting spheres. What is the dependence of the sedimentation coefficient s of a spherical protein on its mass? How much more rapidly does an 80-kDa protein sediment than does a 40-kDa protein?