Problems http://edugen.wiley.com/edugen/courses/crs1900/rc/voet9301c05/dm9ld...

1. Which peptide has greater absorbance at 280 nm?

A. Gln–Leu–Glu–Phe–Thr–Leu–Asp–Gly–Tyr
B. Ser–Val–Trp–Asp–Phe–Gly–Tyr–Trp–Ala

Answer:

Peptide B, because it contains more Trp and other aromatic residues.

2. Protein X has an absorptivity of 0.4 mL · mg–1 · cm–1 at 280 nm. What is the absorbance at 280 nm of a
2.0 mg · mL–1 solution of protein X? (Assume the light path is 1 cm.)

Answer:

Since A = cl, A = (0.4 mL · mg–1 · cm–1)(2.0 mg · mL–1)(1 cm) = 0.8

3. You are using ammonium sulfate to purify protein Q (pI = 5.0) by salting out from a solution at pH 7.0.
How should you adjust the pH of the mixture to maximize the amount of protein Q that precipitates?

Answer:

Lowering the pH from 7.0 to 5.0 would promote the precipitation of protein Q because the protein will be
least soluble when its net charge is zero (when pH = pI).
4.
(a) In what order would the amino acids Arg, His, and Leu be eluted from a carboxymethyl column at
pH 6?
(b) In what order would Glu, Lys, and Val be eluted from a diethylaminoethyl column at pH 8?

Answer:

(a) Leu, His, Arg.

(b) Lys, Val, Glu.
5. Consult Table 5-1 to complete the following: (a) On a plot of absorbance at 280 nm versus elution volume,
sketch the results of gel filtration of a mixture containing human cytochrome c and bacteriophage T7
RNA polymerase and identify each peak. (b) Sketch the results of SDS-PAGE of the same protein mixture
showing the direction of migration and identifying each band.

Answer:

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6. Explain why a certain protein has an apparent molecular mass of 90 kD when determined by gel filtration
and 60 kD when determined by SDS-PAGE in the presence or absence of 2-mercaptoethanol. Which
molecular mass determination is more accurate?

Answer:

The protein behaves like a larger protein during gel filtration, suggesting that it has an elongated shape.
The mass determined by SDS-PAGE is more accurate since the mobility of a denatured SDS-coated
protein depends only on its size.
7. Determine the subunit composition of a protein from the following information:

Molecular mass by gel filtration: 200 kD

Molecular mass by SDS-PAGE: 100 kD
Molecular mass by SDS-PAGE with 2-mercaptoethanol: 40 kD and 60 kD

Answer:

The protein contains two 60-kD polypeptides and two 40-kD polypeptides. Each 40-kD chain is disulfide
bonded to a 60-kD chain. The 100-kD units associate noncovalently to form a protein with a molecular
mass of 200 kD.
8. What fractionation procedure could be used to purify protein 1 from a mixture of three proteins whose
amino acid compositions are as follows?

1. 25% Ala, 20% Gly, 20% Ser, 10% Ile, 10% Val, 5% Asn, 5% Gln, 5% Pro
2. 30% Gln, 25% Glu, 20% Lys, 15% Ser, 10% Cys

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3. 25% Asn, 20% Gly, 20% Asp, 20% Ser, 10% Lys, 5% Tyr
All three proteins are similar in size and pI, and there is no antibody available for protein 1.

Answer:

Because protein 1 has a greater proportion of hydrophobic residues (Ala, Ile, Pro, Val) than do proteins 2
and 3, hydrophobic interaction chromatography could be used to isolate it.
9. Purification of Myoglobin (Mb). Purification tables are often used to keep track of the yield and
purification of a protein. The specific activity is a ratio of the amount of the protein of interest, in this case
Mb, obtained at a given step (μmol or enzyme units) divided by the amount (mg) of total protein. The
yield is the ratio of the amount of the protein of interest obtained at a given step (μmol or enzyme units)
divided by the original amount present in the crude extract, often converted to percent yield by
multiplying by 100. The fold purification is the ratio of the specific activity of the purified protein to that
of the crude preparation.

(a) For the purification table below, calculate the specific activity, % yield, and fold purification for
the empty cells.
(b) Which step—DEAE or affinity chromatography—causes the greatest loss of Mb?
(c) Which step causes the greater purification of Mb?
(d) If you wanted to use only one purification step, which technique would you choose?

Purification Table for Problem 9

Specific activity
mg total μmol (μmol Mb/mg total % fold
Purification step protein Mb protein) yield purification

1. Crude extract 1550 0.75 100 1

2. DEAE-cellulose 550 0.35
chromatography
3. Affinity chromatography 5.0 0.28

Answer:

(a)
Specific
mg activity(μmol
total μmol Mb/mg total
Purification step protein Mb protein) % yield Fold purification

1. Crude extract 1550 0.75 4.8 × 10–4 100 1

2. 550 0.35 6.4 × 10–4 47 1.3
DEAE-cellulose
chromato-graphy
3. Affinity 5.0 0.28 5.9 × 10–2 80 from 123-fold overall,
chromato-graphy Affinity 92-fold from
123-fold Affinity
chromato- chromatography
graphy (37

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Specific
mg activity(μmol
total μmol Mb/mg total
Purification step protein Mb protein) % yield Fold purification

overall)

(b) The DEAE chromatography step results in only a 47% yield, while the affinity chromatography
step results in an 80% yield from the step before it. The DEAE chromatography step therefore
results in the greatest loss of Mb.
(c) The DEAE chromatography step results in a 1.3-fold purification, while the affinity
chromatography step results in a 92-fold purification from the previous step. The affinity
chromatography step therefore results in the greatest purification of Mb.
(d) The affinity chromatography step is the best choice for a one-step purification of Mb in this
example.
10. Explain why the dansyl chloride treatment of a single polypeptide chain followed by its complete acid
hydrolysis yields several dansylated amino acids.

Answer:

Dansyl chloride reacts with primary amino groups, including the ε-amino group of Lys residues.
11. Identify the first residue obtained by Edman degradation of cytochrome c from (a) Drosophila, (b)
baker's yeast, and (c) wheat germ (see Table 5-5).

Answer:

(a) Gly; (b) Thr; (c) none (the N-terminal amino group is acetylated and hence unreactive with Edman's
reagent)
12. You must cleave the following peptide into smaller fragments. Which of the proteases listed in Table 5-3
would be likely to yield the most fragments? The fewest?

Answer:

Thermolysin would yield the most fragments (9) and endopeptidase V8 would yield the fewest (2).
13. Electrospray ionization mass spectrometry (ESI-MS) of proteins involves creating positively charged
ions of the protein and separating them according to their mass-to-charge ratio (m/z).

(a) What causes the different positive charges on different particles of the protein?
(b) The amino acid composition (in numbers of residues per chain) of hen egg-white lysozyme
(HEWL) is as follows:

P 2 Y 3 N 14 H 1
D 7 M 2 L 8 E 2
C 8 R 11 G 12 F 3
A 12 I 6 K 6 V 6
S 10 W 6 T 7 Q 3

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What is the maximum positive charge that can be present on a HEWL ion?
(c) The ESI-MS spectrum below was obtained for HEWL.

Using peaks 5 and 6, calculate the molecular mass of HEWL (see Sample Calculation 5-1).
[Spectrum obtained from http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm]
(d) What is the charge on the ion that makes peak 5?

Answer:

(a) The positive charges are caused by the protonation of basic side chains (H, K, and R) and the
N-terminal amino groups of the protein.
(b) There is 1 H, 6 K, 11 R, and 1 NH2 at the N-terminus. Therefore, the maximum number of
positive charges that can be obtained is 19.
(c) From Sample Calculation 5-1 we see that

Therefore

(d) Peak 5 is p1 in our calculation. From Sample Calculation 5-1,

The charge on the fifth peak in the mass spectrum is 9.00.

14. You wish to determine the sequence of a short peptide. Cleavage with trypsin yields three smaller
peptides with the sequences Leu–Glu, Gly–Tyr–Asn–Arg, and Gln–Ala–Phe–Val–Lys. Cleavage with
chymotrypsin yields three peptides with the sequences Gln–Ala–Phe, Asn–Arg–Leu–Glu, and Val–Lys–

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Gly–Tyr. What is the sequence of the intact peptide?

Answer:

Gln–Ala–Phe–Val–Lys–Gly–Tyr–Asn–Arg–Leu–Glu
15. Separate cleavage reactions of a polypeptide by CNBr and chymotrypsin yield fragments with the
following amino acid sequences. What is the sequence of the intact polypeptide?

CNBr treatment
1. Arg–Ala–Tyr–Gly–Asn
2. Leu–Phe–Met
3. Asp–Met

Chymotrypsin
4. Met–Arg–Ala–Tyr
5. Asp–Met–Leu–Phe
6. Gly–Asn

Answer:

Asp–Met–Leu–Phe–Met–Arg–Ala–Tyr–Gly–Asn
16. You wish to determine the sequence of a polypeptide that has the following amino acid composition.

1 Ala 4 Arg 2 Asn 3 Asp 4 Cys 3 Gly 1 Gln 4 Glu

1 His 1 Lys 1 Met 1 Phe 2 Pro 4 Ser 2 Tyr 1 Trp

(a) What is the maximum number of peptides you can expect if you cleave the polypeptide with
cyanogen bromide?
(b) What is the maximum number of peptides you can expect if you cleave the polypeptide with
chymotrypsin?
(c) Analysis of the intact polypeptide reveals that there are no free sulfhydryl groups. How many
disulfide bonds are likely to be present?
(d) How many different arrangements of disulfide bonds are possible?

Answer:

(a) There is one Met, so CNBr would produce two peptides.

(b) There are four possible sites for chymotrypsin to hydrolyze the peptide: following Phe, Tyr
(twice), and Trp. This would yield five peptides.
(c) Four Cys residues form two disulfide bonds.
(d) Arbitrarily choosing one Cys residue, there are three ways it can make a disulfide bond with the
remaining three Cys residues. After choosing one of them, there is only one way that the
remaining two Cys residues can form a disulfide bond. Thus there are 3 × 1 = 3 possible
arrangements of the disulfide bonds.
17. Treatment of a polypeptide with 2-mercaptoethanol yields two polypeptides:

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1. Ala–Val–Cys–Arg–Thr–Gly–Cys–Lys–Asn–-Phe–Leu
2. Tyr–Lys–Cys–Phe–Arg–His–Thr–Lys–Cys–Ser

Treatment of the intact polypeptide with trypsin yields fragments with the following amino acid
compositions:
3. (Ala, Arg, Cys2, Ser, Val)
4. (Arg, Cys2, Gly, Lys, Thr, Phe)
5. (Asn, Leu, Phe)
6. (His, Lys, Thr)
7. (Lys, Tyr)
Indicate the positions of the disulfide bonds in the intact polypeptide.

Answer:

18. You wish to sequence the light chain of a protease inhibitor from the Brassica nigra plant. Cleavage of
the light chain by trypsin and chymotrypsin yields the following fragments. What is the sequence of the
light chain?

Chymotrypsin
1. Leu–His–Lys–Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser
2. Gln–Gln–Ala–Gln–His–Leu–Arg–Ala–Cys–Gln–Gln–Trp
3. Arg–Ile–Pro–Lys–Cys–Arg–Lys–Phe

Trypsin
4. Arg
5. Ala–Cys–Gln–Gln–Trp–Leu–His–Lys
6. Cys–Arg
7. Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser
8. Phe–Gln–Gln–Ala–Gln–His–Leu–Arg
9. Ile–Pro–Lys
10. Lys

Answer:

Arg–Ile–Pro–Lys–Cys–Arg–Lys–Phe–Gln–Gln–Ala–Gln–His–Leu–Arg–Ala–Cys–Gln–Gln–Trp–
Leu–His–Lys–Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser
19. In site-directed mutagenesis experiments, Gly is often successfully substituted for Val, but Val can rarely
substitute for Gly. Explain.

Answer:

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Because the side chain of Gly is only an H atom, it often occurs in a protein at a position where no other
residue can fit. Consequently, Gly can take the place of a larger residue more easily than a larger
residue, such as Val, can take the place of Gly.
20. Below is a list of the first 10 residues of the B helix in myoglobin from different organisms.

Position 1 2 3 4 5 6 7 8 9 10
Human D I P G H G Q E V L
Chicken D I A G H G H E V L
Alligator K L P E H G H E V I
Turtle D L S A H G Q E V I
Tuna D Y T T M G G L V L
Carp D F E G T G G E V L
Based on this information, which positions (a) appear unable to tolerate substitutions, (b) can tolerate
conservative substitution, and (c) are highly variable?

Answer:

(a) Position 6 (Gly) and Position 9 (Val) appear to be invariant.

(b) Conservative substitutions occur at Position 1 (Asp and Lys, both charged), Position 10 (Ile and
Leu, similar in structure and hydrophobicity), and Position 2 (all uncharged bulky side chains).
Positions 5 and 8 appear to tolerate some substitution.
(c) The most variable positions are 3, 4, and 7, where a variety of residues appear.

Copyright © 2009 John Wiley & Sons, Inc. All rights reserved.

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