Biochemistry A Short Course First Edition by John L. Tymoczko – Test Bank

 

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Chapter 5   Techniques in Protein Biochemistry

 

 

Matching Questions

Use the following to answer questions 1-10:

 

Choose the correct answer from the list below. Not all of the answers will be used.

1. a) HPLC
2. b) specific activity
3. c) MALDI-TOF mass spectrum
4. d) gradient centrifugation
5. e) proteome
6. f) SDS
7. g) two-dimensional electrophoresis
8. h) Svedberg
9. i) immunoglobulin
10. j) differential centrifugation
11. k) overlap peptides
12. l) affinity chromatography

 

1.	The ratio of enzyme activity relative to total protein is called ____________.
	Ans:	b
	Section:  5.2

 

2.	The first step in protein purification from a homogenate is usually ____________.
	Ans:	j
	Section:  5.2

 

3.	____________ A type of purification that is based on the attraction of the protein for a particular chemical group.
	Ans:	l
	Section:  5.2

 

4.	____________ can be added prior to gel electrophoresis to denature the proteins.
	Ans:	f
	Section:  5.2

 

5.	Sedimentation coefficients are expressed in ____________ units.
	Ans:	h
	Section:  5.3

 

6.	Proteins with different sedimentation coefficients can be separated by ____________.
	Ans:	d
	Section:  5.3

 

7.	In order to sequence a whole protein, ____________ are used.
	Ans:	k
	Section:  5.4

 

8.	____________ The subset of gene products actually expressed by the cell.
	Ans:	e
	Section:  5.1

 

9.	____________ A protein purification technique characterized by high resolution and rapid separation.
	Ans:	j
	Section:  5.2

 

10.	____________ The separation of proteins based on charge then size.
	Ans:	g
	Section:  5.2

 

 

Fill-in-the Blank Questions

 

11.	Proteins can be separated from small molecules and ions through a semi-permeable membrane by      .
	Ans:  dialysis     Section:  5.2

 

12.	Molecular exclusion gel or gel-filtration chromatography separates molecules on the basis of     .
	Ans:  size     Section:  5.2

 

13.	In amino acid composition analysis, amino acids are visualized after separation using the chemical reagent      .
	Ans:  fluorescamine     Section:  5.4

 

14.	In the Edman procedure for peptide sequence, phenyl isothiocyanate is used to selectively remove the       residue as a PTH-derivative.
	Ans:  amino-terminal     Section:  5.4

 

15.	Antibodies used as reagents to quantify proteins or other antigens is the basis for the technique called      .
	Ans:  ELISA (enzyme-linked immunosorbent assay)     Section:  5.3

 

 

16.	allows the detection of small amounts and the size of target proteins.
	Ans:  Western blotting or immunoblotting     Section:  5.3

 

17.	Polypeptides can be fragmented into smaller peptides by cleavage with chymotrypsin, which hydrolyzes the peptide bond at the C-terminal side of      residues.
	Ans:  phenyalanine, tyrosine, and tryptophan     Section:  5.4

 

18.	gels are often used as the media for electrophoretic techniques such as SDS-PAGE and isoelectric focusing.
	Ans:  Polyacrylamide     Section:  5.4

 

19.	The      of a protein is the pH at which its net charge is zero.
	Ans:  isoelectric point     Section:  5.2

 

 

Multiple-Choice Questions

 

20.	When enzymes are purified, the assay is often based on:
	A)	light absorbance.	D)	temperature changes.
	B)	catalytic activity.	E)	mRNA levels.
	C)	pH.
	Ans:  B     Section:  5.2

 

21.	Receptor proteins are often assayed using:
	A)	binding assays.	D)	None of the above.
	B)	enzymatic activity.	E)	All of the above.
	C)	amino acid analysis.
	Ans:  A     Section:  5.3

 

22.	What is the advantage of adding SDS to gel electrophoresis?
	A)	SDS colors the proteins for visualization.
	B)	SDS reduces disulfide bonds.
	C)	SDS allows proteins to be separated on the basis of approximate mass.
	D)	None of the above.
	E)	All of the above.
	Ans:  C     Section:  5.2

 

23.	Two-dimensional electrophoresis is a combination of what two techniques?
	A)	isoelectric focusing and affinity chromatography
	B)	ion-exchange chromatography and SDS-PAGE
	C)	affinity chromatography and SDS-PAGE
	D)	isoelectric focusing and SDS-PAGE
	E)	isoelectric focusing and ion-exchange chromatography
	Ans:  D     Section:  5.2

 

 

24.	Which of the following affects the sedimentation of a particle?
	A)	mass	D)	All of the above.
	B)	shape	E)	A and B
	C)	the density of the solution
	Ans:  D     Section:  5.3

 

25.	Cyanogen bromide cleaves the peptide bond at:
	A)	the carboxyl side of Arg and Lys residues.
	B)	the carboxyl side of Met residues.
	C)	the amino terminus.
	D)	None of the above.
	E)	All of the above.
	Ans:  B     Section:  5.4

 

26.	Trypsin cleaves the peptide bond at:
	A)	the carboxyl side of Arg and Lys residues.
	B)	the carboxyl side of Met residues.
	C)	the amino terminus.
	D)	None of the above.
	E)	All of the above.
	Ans:  A     Section:  5.4

 

27.	Which of the following techniques can be used to determine the size of a target protein?
	A)	Edman degradation	D)	ELISA
	B)	affinity chromatography	E)	isoelectric focusing gel
	C)	western blot
	Ans:  C     Section:  5.3

 

28.	What types of molecules can serve as antigens?
	A)	proteins	D)	All of the above.
	B)	polysaccharides	E)	A and B
	C)	metal ions
	Ans:  E     Section:  5.3

 

29.	Affinity chromatographs:
	A)	allow high resolution and rapid separation.	D)	separate proteins based on attraction to another molecule.
	B)	separate proteins based on size.	E)	separate proteins based on charge and size.
	C)	separate proteins based on charge.
	Ans:  D     Section:  5.2

 

30.	What conditions could cause changes in the proteome of a cell?
	A)	developmental stage
	B)	environmental condition
	C)	enzymatic modification
	D)	All of the above.
	E)	None of the above.
	Ans:  D     Section:  5.1

 

31.	Which technique cannot be used for quantitative analysis?
	A)	gradient centrifugation	D)	All of the above.
	B)	ELISA	E)	None of the above.
	C)	enzyme assay
	Ans:  A     Section:  Entire Chapter

 

32.	Which of the following is true regarding gel filtration chromatography and PAGE?
	A)	In both, small proteins move most rapidly.	D)	In gel filtration, large proteins move most rapidly, but in PAGE, small proteins move most rapidly.
	B)	In both, large proteins move most rapidly.	E)	None of the above.
	C)	In PAGE, large proteins move most rapidly but in gel filtration, small proteins move most rapidly.
	Ans:  D     Section:  5.2

 

33.	Two proteins are similar in size but differ significantly in the number of acidic and basic amino acids. Which of the following techniques would be best suited to separating these two proteins?
	A)	SDS-PAGE and gel-filtration chromatography
	B)	isoelectric focusing and dialysis
	C)	immunoprecipitation and affinity chromatography
	D)	isoelectric focusing and ion-exchange chromatography
	E)	None of the above.
	Ans:  D     Section:  5.2

 

34.	Two proteins are similar in the number of acidic and basic amino acids but are different significantly in size. Which of the following techniques would be best suited to separating these two proteins?
	A)	SDS-PAGE and gel-filtration chromatography
	B)	isoelectric focusing and dialysis
	C)	immunoprecipitation and affinity chromatography
	D)	isoelectric focusing and ion-exchange chromatography
	E)	None of the above.
	Ans:  A     Section:  5.2

 

35.	Calmodulin is a calcium-binding protein expressed in eukaryotic cells. What two techniques would greatly reduce the number of steps to purify calmodulin?
	A)	SDS-PAGE and gel-filtration chromatography
	B)	isoelectric focusing and dialysis
	C)	immunoprecipitation and affinity chromatography
	D)	isoelectric focusing and ion-exchange chromatography
	E)	None of the above.
	Ans:  C    Section:  5.2

 

36.	You have isolated a protein, but by the time you have gotten it pure, you have only enough sample to do one type of analysis. Which of the following would you choose and why?
	A)	MALDI-TOF mass spectrometry to determine as much sequence data as you can.
	B)	ELIZA to identify any antigenic determinants.
	C)	Amino Acid Composition Analysis because it can be done for the whole protein.
	D)	2D gel electrophoresis to determine charge and size data of the protein.
	E)	Salting out to concentrate the protein for further study.
	Ans:  A    Section:  5.4

 

37.	You are interested in studying a powerful enzyme that is expressed in low amounts. Which of the following would you choose to determine how much is found in the tissue of interest?
	A)	MALDI-TOF mass spectrometry to determine as much sequence data as you can.
	B)	ELIZA to identify any antigenic determinants.
	C)	Amino Acid Composition Analysis because it can be done for the whole protein.
	D)	2D gel electrophoresis to determine charge and size data of the protein.
	E)	Salting out to concentrate the protein for further study.
	Ans:  B    Section:  5.4

 

Short-Answer Questions

 

38.	Why is an assay necessary for protein purification studies?
	Ans:	An assay allows researchers to accurately measure the amount of the desired protein. This is important in determining if particular purification steps are effective in isolating the protein from the other cellular material.
	Section:  5.2

 

39.	How is lactic acid dehydrogenase assayed?
	Ans:	It is assayed by the increase in NADH present. NADH has a unique absorbance at 340 nm, and the reaction can be monitored by the increase in absorbance at this wavelength.
	Section:  5.2

 

40.	How do gel-filtration and ion-exchange chromatography differ?
	Ans:	Although both are used in purification, the properties of the column material determine how the separation is accomplished. Gel filtration is based on porous beads, and molecules are separated by size. In ion-exchange chromatography, the column material is charged with either positively or negatively charged molecules. Separation is based on the protein’s charge and affinity for the column media.
	Section:  5.2

 

41.	How can a protein’s isoelectric point be used in protein purification?
	Ans:	Isoelectric focusing is an electrophoretic technique in which a gradient charge is applied. Proteins migrate through the gradient field until they reach a point at which the pH is the same as the protein’s pI.
	Section:  5.2

 

42.	What is the purpose of determining the specific activity, yield, and purification level of a protein purification protocol?
	Ans:	The measurements allow one to determine if the individual steps were effective at selectively isolating the protein while maintaining its presence and activity. In order to successfully purify protein, both the yield and purification level must remain high.
	Section:  5.2

 

43.	What type of information can be obtained from gradient centrifugation?
	Ans:	This technique can be used to determine mass and density, and to investigate molecular shape and molecular interactions.
	Section:  5.3

 

44.	Describe the Edman degradation method for protein-sequence analysis.
	Ans:	A pure protein is reacted with phenylisothiocyanate, which binds to the free amino terminus. Under mildly acidic conditions, the derivatized amino acid is liberated, and can be identified by chromatography. The steps are repeated to identify the next amino acid exposed at the amino terminus.
	Section:  5.4

 

45.	How can the amino acid sequences be used to design a DNA probe?
	Ans:	Using the amino acid sequence, the genetic code and a process known as reverse genetics, a DNA sequence can be designed. (Codon degeneracy must be considered in the design.)
	Section:  5.4

 

46.	Explain the process of immunoprecipitation.
	Ans:	Antibodies to a specific protein are chemically fixed to an inert matrix. Protein mixtures containing the protein to be purified are incubated with the antibody/matrix such that only the protein to be purified binds. The unbound proteins are washed away leaving only the purified protein attached to the antibody.
	Section:  5.4

 

47.	Explain the trend of specific activity as a protein is purified.
	Ans:	As specific activity is expressed as units of enzyme per mg of protein, a crude sample with lots of protein will have a low specific activity.  A purified sample will have a much higher ratio of units of enzyme to total protein.  Thus, as a protein is purified the specific activity is increased
	Section:  5.2

 

48.	List five possible steps in protein purification. Start with a technique used on a complex mixture of proteins such as a cell lysate through a series of steps to a pure protein.
	Ans:	homogenization, salting out, ion-exchange chromatography, gel filtration chromatography, affinity chromatography
	Section:  5.2

 

49.	Why is there a need for different digestion tools when fingerprinting a protein?
	Ans:	To generate overlapping peptides, short pieces of peptides are generated with one or two proteases and then further digested with other proteases.
	Section:  5.4

 

50.	A protein that on a SDS PAGE runs as a single 25,000 dalton band runs as a 75,000 dalton band on a native gel.  Why?
	Ans:	The protein in a SDS PAGE gel is denatured, while the native gel does not cause denaturation of the protein.  Thus, the protein before denaturing is a homotrimer of 75,000 daltons.
	Section:  5.2