Biochemistry 4th Edition by Donald Voet - Test Bank

Biochemistry 4th Edition by Donald Voet – Test Bank

 

Instant Download – Complete Test Bank With Answers

 

 

Sample Questions Are Posted Below

 

Chapter 5: Proteins: Primary Structure

 

 

 

Matching

 

A)	electrophoresis
B)	hydrophobic interaction
C)	enzyme-linked immunosorbent assay
D)	three-dimensional shape
E)	N-terminal amino acid
F)	negative charge
G)	nucleases
H)	chromophore
I)	foaming
J)	high level expression
K)	2-mercaptoethanol
L)	positive charge
M)	cation exchange
N)	pI
O)	chymotrypsin
P)	C-terminal amino acid
Q	Sodium dodecyl sulfate

 

 

1. One of the reasons the primary structure is important for a protein is that it determines the ______ the molecule adopts in aqueous solutions.

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

2. If the cDNA for a protein has been cloned, it may be possible to obtain large quantities of the protein by _________________ in bacteria.

 

Ans:  J

Level of Difficulty:  Easy

Section: 5.2.A

Learning objective: Protein Purification and Analysis

 

3. To help prevent denaturation of proteins in solution, steps are taken to avoid _________ and adsorption to surfaces.

 

Ans:  I

Level of Difficulty:  Moderate

Section: 5.2.A

Learning objective: Protein Purification and Analysis

 

 

4. Molecules that contain a(n) ______ are capable of absorbing light.

 

Ans:  H

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

5. If antibodies to the protein being assayed are available, a(n) ______ can be carried out.

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

6. In general, proteins are least soluble in water when the pH is close to the ______.

 

Ans:  N

Level of Difficulty:  Moderate

Section:  5.2.B

Learning objective: Protein Purification and Analysis

 

 

7. ______ chromatography is a method of fractionating a protein mixture according to differences in polarity.

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

8. In order for DEAE to act as an anion exchanger, it must have a ______.

 

Ans:  L

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

9. In ______ chromatography, a protein mixture must be applied to the column at a low pH so that the proteins will have a net positive charge and bind to the column.

 

Ans:  M

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

10. In SDS-PAGE, disulfide-linked polypeptides can be separated after reacting the protein first with ______.

 

Ans:  K

Level of Difficulty:  Easy

Section:  5.2.D

Learning objective: Protein Purification and Analysis

 

 

11. Either dansyl chloride or Edman’s reagent can be used to identify the ______ of a protein.

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.3.A

Learning objective: Protein Sequencing

 

 

12. The endoprotease ______ cleaves polypeptides on the C-terminal side of certain bulky hydrophobic amino acid residues.

 

Ans:  O

Level of Difficulty:  Moderate

Section:  5.3.B

Learning objective: Protein Sequencing

 

Multiple Choice

 

13. Proteins are synthesized in vivo by the translation of
14. A)
15. B)
16. C)
17. D)
18. E)

 

Ans:  E

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

14. Since there are 20 standard amino acids, the number of possible linear polypeptides of length N can be expressed as:
15. A) n × 20
16. B) 20ⁿ
17. C) 20 × 10ⁿ
18. D) n²⁰
19. E) n × 10²⁰

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

15. Natural proteins most commonly contain linear polypeptides between 100 and 1000 residues in length. One of the reasons polypeptides outside this range may be disfavored is that
16. A) larger polypeptides would likely be insoluble.
17. B) smaller polypeptides do not form stable folded structures.
18. C) smaller polypeptides typically assemble into prion-like aggregates.
19. D) amide linkages are not strong enough to keep larger polypeptides intact.
20. E) ribosomes are unable to synthesize larger polypeptides.

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

16. The vast majority of polypeptides contain between ______ amino acid residues.
17. A) 10 and 50
18. B) 50 and 100
19. C) 100 and 1000
20. D) 1000 and 2000
21. E) 2000 and 34,000

 

Ans:  C

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

17. Which of the following has the most dramatic influence on the characteristics of an individual protein?
18. A) the amino-acid sequence
19. B) the amino-acid composition
20. C) the location of its encoding gene within the genome
21. D) the stereochemistry at the a-carbon
22. E) the sequence of tRNA molecules involved in its translation

 

Ans:  A

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

18. Which statement about insulin is correct?
19. A) Insulin is composed of two polypeptides, the A chain and the B chain.
20. B) Insulin contains an intrachain disulfide bond.
21. C) Insulin contains interchain disulfide bonds.
22. D) The A chain and the B chain of insulin are encoded by a single gene.
23. E) All of the above are correct.

 

Ans:  E

Level of Difficulty:  Easy

Section:  5.1

Learning objective: Polypeptide Diversity

 

19. A fast and common method for determining the protein concentration in column effluent is
20. A) tandem mass spectrometry.
21. B) salting in with ammonium sulfate.
22. C) drying a portion and weighing the solid.
23. D) measuring light absorption at 280 nm.
24. E) Edman degradation.

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

20. The salting in of proteins can be explained by:
21. A) salt counter-ions reducing electrostatic attractions between protein molecules.
22. B) salt ions reducing the polarity of the solution.
23. C) salt ions increasing the hydrophobic interactions.
24. D) releasing hydrophobic proteins from nonpolar tissue environments.
25. E) hydration of the salt ions reducing solubility of proteins.

 

Ans:  A

Level of Difficulty:  Moderate

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

21. The quantitation of proteins due to their absorbance at ~280 nm (UV region) is due to the large absorbtivity of the ________ amino acids.
22. A) anionic
23. B) dansylated
24. C) cleaved
25. D) polar
26. E) aromatic

 

Ans:  E

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

22. Which of the following ‘assays’ would be most specific for a particular protein?
23. A) Bradford assay
24. B) UV absorptivity
25. C) radioimmunoassay
26. D) molar absorptivity
27. E) amino acid analysis

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

23. An enzyme-linked immunosorbent assay requires
24. A) a radioactive substrate.
25. B) a radioactive standard for binding to the antibody.
26. C) aromatic amino acids.
27. D) an antibody that binds the protein of interest.
28. E) a catalytic antibody.

 

Ans:  D

Level of Difficulty:  Moderate

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

24. ELISA is an example of a(n):
25. A) enzyme assay.
26. B) biological assay.
27. C) binding assay.
28. D) immunological assay.
29. E) none of the above

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

25. You are purifying a nuclease by affinity chromatography. To determine which fractions contain the protein of interest, you test samples of all fractions for their ability to break down DNA.  This is an example of
26. A) a binding assay.
27. B) a biological assay.
28. C) an enzyme assay.
29. D) an immunological assay.
30. E) none of the above

 

Ans:  C

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

26. A radioimmunoassay requires
27. A) an enzyme-linked antibody.
28. B) a coupled enzymatic reaction.
29. C) a radiolabeled antibody.
30. D) a catalytic antibody.
31. E) a radiolabeled standard protein that is used to compete for binding to the antibody.

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

 

27. Five graduate students prepare extracts from 5 different tissues. Each student measures the total amount of alcohol dehydrogenase and the total amount of protein in his or her extract.  Which extract has the highest specific activity?

 

	Total protein (mg)	Total alcohol dehydrogenase activity (units)
A	300	60,000
B	200	80,000
C	3000	96,000
D	5000	100,000
E	1000	200,000

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.A

Learning objective: Protein Purification and Analysis

 

28. Which physical characteristic is not commonly used in protein separation?
29. A) solubility
30. B) stereochemistry
31. C) size
32. D) charge
33. E) polarity

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.B

Learning objective: Protein Purification and Analysis

 

 

29. Adding additional salt to a protein solution can cause:
30. A) an increase in solubility called ‘salting in’.
31. B) a decrease in solubility called ‘salting out’.
32. C) protein precipitation from solution.
33. D) all of the above
34. E) none of the above

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.2.B

Learning objective: Protein Purification and Analysis

 

 

30. A first step in purifying a protein that was initially associated with fatty substances would be
31. A) Coomassie Brilliant Blue dye staining.
32. B) analytical ultracentrifugation.
33. C)
34. D) Western blotting.
35. E) hydrophobic interaction chromatography.

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

31. The acronym HPLC stands for
32. A) hydrophobic protein liquid chromatography.
33. B) high performance liquid chromatography.
34. C) hydrophilic partition liquid chromatography.
35. D) high priced liquid chromatography.
36. E) hydrostatic process liquid chromatography.

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

32. A technique that can be used to separate proteins based primarily on the presence of non-polar residues on their surface is called
33. A) ion-exchange chromatography.
34. B) gel filtration chromatography.
35. C) affinity chromatography.
36. D) gel electrophoresis.
37. E) hydrophobic interaction chromatography.

 

Ans:  E

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

33. A technique that can be used to separate proteins based primarily on their pI is called
34. A) ion-exchange chromatography.
35. B) gel filtration chromatography.
36. C) affinity chromatography.
37. D) isoelectric focusing.
38. E) hydrophobic interaction chromatography.

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

34. Which of the following amino acids would be last to elute at pH 8.0 from an anion-exchange column?
35. A) lysine
36. B) alanine
37. C) glutamic acid
38. D) asparagine
39. E) glycine

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

35. Which of the following amino acids would be first to elute at pH 8.0 from an anion-exchange column?
36. A) lysine
37. B) alanine
38. C) glutamic acid
39. D) asparagine
40. E) glycine

 

Ans:  A

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

36. The pK₁, pK₂, and pK_R of the amino acid lysine are 2.2, 9.1, and 10.5, respectively. The pK₁, pK₂, and pK_R of the amino acid arginine are 1.8, 9.0, and 12.5, respectively.  A student at SDSU wants to use ion exchange chromatography to separate lysine from arginine.  What pH is likely to work best for this separation?
37. A) 5
38. B) 5
39. C) 5
40. D) 5
41. E) 5

 

Ans:  E

Level of Difficulty:  Difficult

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

37. The pK₁, pK₂, and pK_R of the amino acid histdine are 1.8, 9.3, and 6.0, respectively. The pK₁, pK₂, and pK_R of the amino acid arginine are 1.8, 9.0, and 12.5, respectively.  You have a mixture of histidine and arginine, how would you try to separate these two amino acids?
38. A) anion exchange chromatography at pH 2
39. B) anion exchange chromatography at pH 4
40. C) cation exchange chromatography at pH 2
41. D) cation exchange chromatography at pH 4
42. E) cation exchange chromatography at pH 9

 

Ans:  E

Level of Difficulty:  Difficult

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

38. What can be done to increase the rate at which a protein of interest moves down an ion-exchange chromatography column?
39. A) reduce the ion concentration in the eluant
40. B) add a small amount of a non-ionic detergents to the eluant
41. C) change the pH of the eluant
42. D) add a protease inhibitor to the eluant
43. E) reduce the temperature of the eluant

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

39. Hydrophobic interaction chromatography can be used to separate proteins based on differences in
40. A) ionic charge.
41. B)
42. C)
43. D)
44. E) binding specificity.

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

40. You are trying to separate five proteins, which are listed below, by gel filtration chromatography. Which of the proteins will elute first from the column?
41. A) cytochrome c (12 kDa)
42. B) RNA polymerase (99 kDa)
43. C) glutamine synthetase (621 kDa)
44. D) interferon-g (34 kDa)
45. E) hemoglobin (62 kDa)

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.2.C

Learning objective: Protein Purification and Analysis

 

 

41. SDS-PAGE separates proteins primarily due to differences in
42. A) isoelectric point.
43. B)
44. C)
45. D)
46. E) amino acid sequence.

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.D

Learning objective: Protein Purification and Analysis

 

42. Which of these techniques is used to separate proteins mainly based on mass?
43. A) polyacrylamide gel electrophoresis (in the absence of SDS)
44. B) SDS-PAGE
45. C) isoelectric focusing
46. D) immunoblotting
47. E) Western blotting

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.2.D

Learning objective: Protein Purification and Analysis

 

43. Which of these techniques uses antibodies to detect very small amounts of specific proteins following separation by SDS-PAGE.
44. A) immunoblotting
45. B) silverstaining
46. C) Coomassie Brilliant Blue staining
47. D) ELISA
48. E) RIA

 

Ans:  A

Level of Difficulty:  Easy

Section:  5.2.D

Learning objective: Protein Purification and Analysis

 

 

44. Disulfide bonds can be cleaved using
45. A)
46. B) dansyl chloride.
47. C) 2-mercaptoethanol (b-ME).
48. D)
49. E)

 

Ans:  C

Level of Difficulty:  Easy

Section:  5.3.A

Learning objective: Protein Sequencing

 

 

45. Which of these reagents is commonly used to determine the number of polypeptides in a protein?
46. A) iodoacetate
47. B) dansyl chloride
48. C) 2-mercaptoethanol (b-ME)
49. D) cyanogen bromide
50. E) DEAE

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.3.A

Learning objective: Protein Sequencing

 

46. Enzymes that hydrolyze the internal peptide bonds (not the peptide bonds of the terminal amino acids) of a protein are classified as
47. A)
48. B)
49. C)
50. D)
51. E)

 

Ans:  C

Level of Difficulty:  Easy

Section:  5.3.B

Learning objective: Protein Sequencing

 

 

47. Which of the following substances cannot be used to cleave peptide bonds in polypeptides?
48. A) trypsin
49. B) cyanogen bromide
50. C) endopeptidases
51. D) 2-mercaptoethanol
52. E) pepsin

 

Ans:  D

Level of Difficulty:  Moderate

Section:  5.3.B

Learning objective: Protein Sequencing

 

 

48. Which of these are commonly used to cleave peptide bonds in polypeptides?
49. A) 2-mercaptoethanol (b-ME)
50. B) dansyl chloride
51. C) iodoacetate
52. D) sodium dodecyl sulfate
53. E) trypsin

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.3.B

Learning objective: Protein Sequencing

 

49. The peptide Leu─Cys─Arg─Ser─Gln─Met is subjected to Edman degradation. In the first cycle the peptide first reacts with phenylisothiocyanate under basic conditions.  The product of this reaction is incubated with anhydrous trifluoroacetic acid and subsequently with an aqueous acid.  What are the products generated in the first cycle.

 

1. A) PTH─Leu, PTH─Cys, PTH─Arg, PTH─Ser, PTH─Gln, and PTH─Met
2. B) PTH─Leu─Cys─Arg─Ser─Gln─Met
3. C) PTH─Met and Leu─Cys─Arg─Ser─Gln─Met
4. D) PTH─Leu─Cys and PTH─Arg─Ser─Gln─Met
5. E) PTH─Leu and Cys─Arg─Ser─Gln─Met

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.3.C

Learning objective: Protein Sequencing

 

 

50. Edman degradation can be used to
51. A) identify the N-terminal amino acid of a polypeptide.
52. B) identify the C-terminal amino acid of a polypeptide.
53. C) separate the subunits of a multi-subunit protein.
54. D) cleave a protein at specific sites.
55. E) cleave disulfide bonds within a protein so that the individual polypeptides can be separated.

 

Ans:  A

Level of Difficulty:  Easy

Section:  5.3.C

Learning objective: Protein Sequencing

 

 

51. Although a protein’s primary sequence can be inferred from the nucleotide sequence, modifications such as ______ can be determined most easily by tandem mass spectrometry followed by protein database searching.
52. A) phosphorylation
53. B) disulfide crosslinks
54. C) glycosylation
55. D) acetylation
56. E) all of the above

 

Ans:  E

Level of Difficulty:  Easy

Section:  5.3.D

Learning objective: Protein Sequencing

 

52. The positive charge on proteins in electrospray ionization mass spectrometry is the result of
53. A) protons fired at the gas-phase protein molecules.
54. B) protonated side chains of Asp and Glu residues.
55. C) protonated side chains of Arg and Lys residues.
56. D) a high pH.
57. E) electrons fired at the gas-phase protein molecules.

 

Ans:  C

Level of Difficulty:  Moderate

Section:  5.3.D

Learning objective: Protein Sequencing

 

 

53. ______________ has emerged as a technique for protein sequencing.
54. A) NMR spectroscopy
55. B) Mass spectrometry
56. C) Gel electrophoresis
57. D) Phylogenetic analysis
58. E) Limited proteolysis

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.3.D

Learning objective: Protein Sequencing

 

 

54. Protein sequences are customarily ‘reconstructed’ from sequenced fragments because
55. A) protein purification invariably results in the fragmentation of the protein of interest.
56. B) proteins are naturally and inevitably cleaved by proteolytic enzymes.
57. C) proteins are composed of multiple subunits.
58. D) large polypeptides cannot be directly sequenced.
59. E) all of the above

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.3.E

Learning objective: Protein Sequencing

 

55. You have purified a new peptide hormone. To determine its amino acid sequence you have digested the polypeptide with trypsin and in a separate reaction you have cleaved the polypeptide with cyanogen bromide.

 

Cleavage with trypsin yielded 5 peptides that were sequenced by Edman degradation as shown in the following.

 

1. Ser─Leu
2. Asp─Val─Arg
3. Val─Met─Glu─Lys
4. Ser─Gln─Met─His─Lys
5. Ile─Phe─Met─Leu─Cys─Arg

 

Cleavage with cyanogen bromide yielded 4 peptides that were sequenced by Edman degradation:

 

1. His─Lys─Ser─Leu
2. Asp─Val─Arg─Val─Met
3. Glu─Lys─Ile─Phe─Met
4. Leu─Cys─Arg─Ser─Gln─Met

 

Determine the identity of the N-terminal amino acid after reconstructing the intact protein.

 

1. A) Asp
2. B) Ser
3. C) His
4. D) Glu
5. E) Ile

 

Ans:  A

Level of Difficulty:  Difficult

Section:  5.3.E

Learning objective: Protein Sequencing

 

 

56. In two homologous proteins, which residue is most likely to replace a Glu residue as a conservative substitution?
57. A) Asp
58. B) Trp
59. C) Met
60. D) Ile
61. E) Lys

 

Ans:  A

Level of Difficulty:  Easy

Section:  5.4.A

Learning objective: Protein Evolution

 

57. A phylogenetic tree depicts ___________ of proteins.
58. A) folding patterns
59. B) hypervariable residues
60. C) invariable residues
61. D) evolutionary relationships
62. E) gene sequences

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.4.A

Learning objective: Protein Evolution

 

 

58. A protein that has had few changes in its amino acid sequence over evolutionary history is labeled
59. A) a fibrinopeptide.
60. B) evolutionarily conserved.
61. C)
62. D) a product of pseudogenes.
63. E)

 

Ans:  B

Level of Difficulty:  Easy

Section:  5.4.B

Learning objective: Protein Evolution

 

 

59. Paralogous genes are
60. A) genes that do not encode protein.
61. B) genes of slowly evolving proteins.
62. C) relics of genes that are not expressed.
63. D) genes of rapidly evolving proteins.
64. E) the results of gene duplication.

 

Ans:  E

Level of Difficulty:  Moderate

Section:  5.4.B

Learning objective: Protein Evolution

 

60. A fast way for nature to generate new proteins is:
61. A) generation of pseudogenes.
62. B) mutation by neutral drift.
63. C) shuffling protein domains or motifs.
64. D) hypervariable positions.
65. E) liberal substitution.

 

Ans:  C

Level of Difficulty:  Easy

Section:  5.4.B

Learning objective: Protein Evolution

 

 

61. ___________ is an example of a very slowly evolving protein.
62. A) Histone H4
63. B) Hemoglobin
64. C) Cytochrome c
65. D) Fibrinopeptides
66. E) none of the above

 

Ans:  A

Level of Difficulty:  Moderate

Section:  5.4.B

Learning objective: Protein Evolution

 

 

62. Proteins are often constructed from multiple segments of 40-200 amino acid residues, commonly called
63. A)
64. B) hypervariable residues.
65. C) protolytic fragments.
66. D)
67. E)

 

Ans:  D

Level of Difficulty:  Easy

Section:  5.4.B

Learning objective: Protein Evolution

 

SHORT ANSWER

 

63. Proteins can vary in size from approximately 40 to 34,000 amino acids.
64. Why is there a lower limit to the size of proteins?
65. Why is there an upper limit to the size of polypeptides?

 

Ans:  a.  Polypeptides shorter than 40 residues are unable to maintain a stable fold (probably because there are not enough functional groups to help stabilize the folded structure).

1. The longer the polypeptide, the longer the corresponding mRNA and gene, resulting in an increase in mistakes made during transcription and translation. Beyond a certain size every protein encoded for by a very large gene will include debilitating mistakes.

Level of Difficulty:  Moderate

Section:  5.1

Learning objective: Polypeptide Diversity

 

 

64. We are able to purify proteins because they differ from each other in various physical or chemical properties. List 5 physicochemical properties of proteins that can be used as basis for their separation.  Give a method of separation based on each of these properties (match the method with the property).

 

Ans:  1. solubility — salting out; 2. ionic charge — ion exchange chromatography, electrophoresis (in the absence of SDS), or isoelectric focusing; 3. polarity — hydrophobic interaction chromatography; 4.  size (mass) — gel filtration chromatography (size exclusion chromatography) or SDS-PAGE 5. ability to bind to specific molecules — affinity chromatography

Level of Difficulty:  Moderate

Section:  5.2A

Learning objective: Protein Purification and Analysis

 

65. One technique commonly used in protein purification is chromatography.
66. Explain briefly the general principle of column chromatography
67. Name four types of chromatography and indicate for each of these types the basis for separation (match types of chromatography with the properties that form the basis for separation).

 

Ans:  a.  In chromatography there are two phases, the stationary phase and the mobile phase.  The stationary phase is the column material and the mobile phase is the buffer that runs through the column.  Proteins or molecules are separated based on their relative affinities for these two phases.  A molecule with very high affinity for the stationary phase will spend most of its time associated with the column material and will move very slowly down the column.  A molecule with high affinity for the mobile phase will spend most of its time suspended in the buffer and will move quickly through the column.  Molecules with high affinity for the stationary phase will elute late from the column.  Molecules with a high affinity for the mobile phase will elute early.

1. 1. ion-exchange chromatography — ionic charge; 2.  gel filtration chromatography — size (mass); 3.

hydrophobic interaction chromatography — polarity; 4.  affinity chromatography — ability to bind to specific molecules.

Level of Difficulty:  Moderate

Section:  5.2C

Learning objective: Protein Purification and Analysis

 

 

66. Ion exchange chromatography is a commonly used method for separation of biomolecules.
67. What are the two types of ion exchange chromatography?
68. In what order would Arg, Val, and Glu elute from a carboxymethyl column at pH 6.0. Carboxymethyl is negatively charged at pH 6.0.
69. You are trying to purify a protein using ion exchange chromatography. Unfortunately, your protein remains bound to the ion exchange column or it is eluting very slowly.  What are the two changes you can make, to try to elute your protein more quickly from this column?

 

Ans:  a.  cation exchange chromatography and anion exchange chromatography.

6. First Glu, then Val, followed last by Arg, because at pH 6.0 Glu is has an electric charge of -1, Val is neutral, and Arg has an electric charge of +1.
7. To make your protein move more quickly through the column you need to reduce the interactions with the column material. This can be done by changing the pH (changing the charge on your protein) or by increasing the ion concentration in the eluant (ions will compete with your protein for binding to the column material).

Level of Difficulty:  Moderate

Section:  5.2C

Learning objective: Protein Purification and Analysis

 

67. A variety of chromatographic techniques are available for protein purification.
68. Explain briefly the principle of hydrophobic interaction chromatography.
69. Name three changes that can be made to the eluant that can be used to speed up elution of the protein of interest from a hydrophobic interaction chromatography column.

 

Ans:  a.  The column material is substituted with nonpolar groups.  Proteins with nonpolar regions on their surface will be driven to interact with the column material as a result of the hydrophobic affect.

1. To weaken the interaction between the column material and the nonpolar regions on your protein you can include detergents (they will shield nonpolar regions on proteins from interacting with water or other nonpolar groups), change the pH (will change the polarity of your protein), or reduce the ion concentration of the eluant (this will make it less polar).

Level of Difficulty:  Moderate

Section:  5.2C

Learning objective: Protein Purification and Analysis

 

 

68. You are interested in receptor protein-tyrosine kinases and you are purifying a novel phosphotyrosine-binding protein (NPBP-1). In order to characterize this protein you need to separate it from three other proteins (X, Y, and Z) that are still present in your partially purified material.  The proteins in your preparation have the following properties:

 

	binds to phosphotyrosine	Size (kDa)	pI
protein X	yes	85	7.2
protein Y	yes	29	2.9
protein Z	no	31	7.4
NPBP-1	yes	28	7.5

 

1. What type of separation technique can be used to separate protein NPBP-1 from protein X?
2. What type of separation technique can be used to separate protein NPBP-1 from protein Y?
3. What type of separation technique can be used to separate protein NPBP-1 from protein Z?

 

Ans:  a. On a size (mass) basis:  gel permeation chromatography, SDS-PAGE, or ultracentrifugation.

1. On a charge basis: ion exchange chromatography or isoelectric focusing
2. On a binding behavior basis: phosphotyrosine affinity chromatography

Level of Difficulty:  Moderate

Section:  5.2C

Learning objective: Protein Purification and Analysis

 

69. You have purified the receptor for a hormone by affinity chromatography. During gel filtration chromatography under native conditions the receptor elutes between pyruvate decarboxylase (250 kDa) and glutamine synthetase (620 kDa).  During SDS-PAGE, in the absence of reducing agents, the receptor migrates as a single band of approximately 230 kDa.  When SDS-PAGE is carried out in the presence of 2-mercaptoethanol the receptor migrates as two bands of approximately 95 and 135 kDa.  Explain this result.

 

Ans:  The receptor is a heterotetramer composed of two a subunits of 95 kDa and two b subunits of 135 kDa.  Heterodimers of one a subunit and one b subunit are stabilized by disulfide bonds.  Two heterodimers are held together by hydrophobic interactions.  In the absence of detergents during gel filtration all subunits stay together and we observe a 460 kDa protein.  During SDS-PAGE (in the absence of reducing agents) SDS interferes with the hydrophobic interactions that hold the two ab dimers together and we observe a 230 kDa protein.  Finally, when we add 2-mercaptoethanol during SDS-PAGE the disulfide bonds are broken and we observe 2 polypeptides of 135 and 95 kDa.

 

Level of Difficulty:  Moderate

Section:  5.2D

Learning objective: Protein Purification and Analysis