Chapter 3 Exploring Proteins and Proteomes
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.
a) HPLC
b) specific activity
c) MALDI-TOF mass spectrum
d) zonal centrifugation
e) nascent
f) SDS
g) epitope
h) Svedberg
i) immunoglobulin
j) centrifugation
k) overlap peptides
l) affinity chromatography
1 The ratio of enzyme activity relative to total protein is called ____________.
Ans b
:
Section: 3.1
2 The first step in protein purification from a homogenate is usually
____________.
Ans j
:
Section: 3.1
3 ____________ A type of purification that is based on the attraction of the
protein for a particular chemical group.
Ans l
:
Section: 3.1
4 ____________ can be added prior to gel electrophoresis to denature the
proteins.
Ans f
:
Section: 3.1
5 Sedimentation coefficients are described as ____________ units.
Ans h
:
Section: 3.1
6 Proteins with different sedimentation coefficients can be separated by
____________.
Ans d
:
Section: 3.1
7 In order to sequence a whole protein, ____________ are used.
Ans k
:
Section: 3.2
8 ____________ The name used to describe the original, uncleaved protein.
Ans e
:
Section: 3.2
9 ____________ Another name for an antibody.
Ans j
:
Section: 3.3
10 ____________ Another name for an antigenic determinant.
Ans g
:
Section: 3.3
Fill in the Blank Questions
11 Proteins can be separated from small molecules and ions through a semi-
permeable membrane by __________________.
Ans: dialysis Section: 3.2
12 Exclusion gel or gel-filtration chromatography separates molecules on the
basis of __________________.
Ans: size Section: 3.1
13 __________________ is a chemical reagent that is often used to detect the
presence of amino acids.
Ans: Ninhydrin Section: 3.2
14 In the Edman procedure for peptide sequence, phenyl isothiocyanate is used
to selectively remove the __________________ residue as a PTH-derivative.
Ans: N-terminal Section: 3.2
15 Disulfide bonds in peptides and proteins are readily oxidized to cysteic acid
residues by treatment with __________________.
Ans: performic acid Section: 3.2
16 MALDI-TOF is the abbreviation for __________________.
Ans: Matrix-Assisted Laser Desorption and Ionization Time of Flight mass
spectrometer Section: 3.5
17 Automated peptide synthesis involves the activation of the carboxyl group of
the incoming amino acid by __________________ and then reaction with
the amino group of the growing peptide chain.
Ans: dicyclohexylcarbodiimide Section: 3.4
18 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: 3.2
19 __________________ gels are often used as the media for electrophoretic
techniques such as SDS-PAGE and isoelectric focusing.
Ans: Polyacrylamide Section: 3.2
20 The mobility of proteins in SDS-PAGE is inversely proportional to the
_____________.
Ans: logarithm of their mass Section: 3.1
Multiple Choice Questions
21 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: 3.1
22 Proteins that are not catalysts are often assayed using
A) antibody binding assays. D) None of the above.
B) catalytic activity. E) All of the above.
C) amino acid analysis.
Ans: A Section: Introduction
23 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: 3.1
24 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: 3.1
25 Which of the following affect 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: 3.1
26 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: 3.2
27 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: 3.2
28 Which of the following techniques can be used to determine the site of a
disulfide bond?
A) Edman degradation D) MALDI-TOF
B) affinity chromatography E) SDS-PAGE
C) diagonal electrophoresis
Ans: C Section: 3.2
29 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: 3.3
30 An ELISA can be used for
A) quantitative analysis. D) All of the above.
B) size analysis. E) None of the above.
C) absorbance measurements.
Ans: A Section: 3.3
31 A technique used to identify proteins after gel electrophoresis, which
employs antibodies in the detection process.
A) None of these. D) Southern Blot
B) Southwestern Blot E) Northern Blot
C) Western Blot
Ans: C Section: 3.3
32 A technique that can be used to study protein location in cells.
A) NMR spectroscopy D) Western blotting
B) fluorescent microscopy E) ion-exchange chromatography
C) X-ray crystallography
Ans: B Section: 3.3
33 The use of synthetic peptides includes
A) use as antigens for making antibodies.
B) drugs.
C) “hooks” used in purification.
D) All of the above.
E) a and c.
Ans: D Section: 3.3
34 Which technique cannot be used for quantitative analysis?
A) X-ray crystallography D) All of the above.
B) ELISA E) None of the above.
C) absorbance spectroscopy
Ans: A Section: Entire Chapter
35 Techniques that can be used to obtain information about protein shape are
A) X-ray crystallography. D) a and b.
B) NMR spectroscopy. E) a, b, and c.
C) SDS-PAGE.
Ans: D Section: 3.5
Short-Answer Questions
36 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: 3.1
37 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: 3.1
38 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: 3.1
39 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: 3.1
40 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: 3.1
41 What type of information can be obtained from ultracentrifugation?
Ans This technique can be used to determine mass and density, and to
: investigate molecular shape and molecular interactions.
Section: 3.1
42 What is peptide-mass fingerprinting?
Ans This is a technique in which proteins are analyzed by a method requiring
: two-dimensional electrophoresis, followed by enzymatic cleavage and
identification of the mass-by-mass spectrometry. The resulting
fingerprint can be used for identification by comparison to a database.
Section: 3.1
43 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: 3.2
44 How can the amino acid sequences be used to design a DNA probe?
Ans Using the amino acid sequence and the genetic code, a DNA sequence
: can be designed. (Codon degeneracy must be considered in the design.)
Section: 3.2
45 What is one advantage of using the recombinant DNA methods to determine
protein sequences?
Ans Large proteins can be difficult to sequence by traditional methods
: because only short peptides can be sequenced. Long sections of DNA
can be cloned and sequenced, and the genetic code used to determine
the amino acid sequence.
Section: 3.2
46 What is an epitope?
Ans An epitope is the specific group or part of a molecule that the antibody
: recognizes.
Section: 3.3
47 Why are monoclonal antibodies more useful than polyclonal antibodies?
Ans Monoclonal antibodies are more specific, consisting of one type of
: antibody with a specific binding site and affinity. Polyclonal antibodies
contain several different antibodies with slightly different binding
affinities and specificities. Monoclonal antibodies are more suitable for
examining protein structure and studying mechanisms.
Section: 3.3
48 How are monoclonal antibodies made?
Ans They are made by fusion of an immortal myeloma cell line with a short-
: lived, antibody-producing cell.
Section: 3.3
49 Briefly describe how an ELISA works.
Ans In ELISA, the antigen of interest is complexed with a specific antibody
: under appropriate assay conditions, and excess antibody is removed.
The antibody is complexed to an enzyme, which can be measured
quantitatively using an appropriate substrate and assay tool, often by a
colorimetric or radioactive product formation.
Section: 3.3
50 What are some of the advantages of NMR spectroscopy compared to X-ray
crystallography?
Ans While both are expensive and time-consuming techniques, NMR does
: not require a crystal and allows protein structure in solution to be
determined.
Section: 3.5
