Biology 402

Spring 2001
REA/GUO

Problem Set 1: Protein Purification and Characterization

Questions:
These questions are intended to help you develop skill in extracting as much meaningful
information as possible from experimental results. Some of the questions are more
straightforward than they would at first appear; others are more complicated than they
would at first appear.

1. A protein believed to be involved in membrane transport was obtained from E. coli.

Amino acid analysis of 10 mg of the purified protein yielded 61 µg of Trp. What is
the minimum molecular weight of the protein? (Trp has an MW of 204.1).

2. A polypeptide has the sequence:

Leu-Leu-Trp-Tyr-Ser-Glu

A. Using the data in the table below and assuming a molar absorptivity for Trp and Tyr
of 5000 and 1500 L/cm.mol, respectively, estimate the extinction coefficient, ε
(units: cm2/mg) for this peptide at a wavelength, λ, of 280 nm.
B. A solution of this peptide is placed in a 1 cm thick cuvette and its absorbance is
found to be 1.3 at 280 nm. What is the concentration of the polypeptide in mg/ml?

Amino Acid MW

Leu 131.10
Trp 204.23
Tyr 181.20
Ser 105.04
Glu 147.14

Refer to the last page for a definition of terms.

3. You discover and purify a new enzyme, and obtain the following data:

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Procedure Total protein (mg) Activity (units)

Crude homogenate 20,000 4000,000

(NH4)2SO4 precipitation 5,000 3000,000
Acid precipitation 4,000 1000,000
Ion-exchange chromatography 200 800,000
Affinity chromatography 50 750,000
Gel-filtration chromatography 45 675,000

A. From the information in the table, calculate the specific activity (units/mg protein) of
the enzyme solution after each purification step.
B. Which of the purification procedures used for this enzyme is most effective - gives
the greatest increase in purity?
C. Which of the purification procedures is least effective?
D. Is there any indication from the table that the enzyme is now pure? What else could
be done to estimate the purity of the final preparation?
E. By how much has the enzyme been purified overall? Assuming that the enzyme is
now pure, what percentage of the total protein in the original crude homogenate does
the enzyme represent? What assumptions must you make in order to perform this
calculation? Hint: what are the recoveries of activity after each purification step?

4. You and your partner are characterizing three polypeptides of approximately the
same molecular weight from human plasma. Using various physical techniques you
have established that in their native states one of the polypeptides is a monomeric,
cigar-shaped molecule, the second is monomeric and approximately spherical, and
the third is a subunit of a tetramer of identical subunits. Your lab partner has
determined the amino acid compositions of the three proteins. However, when he
brings you the data, shown below, you discover that you have failed to label the
tubes containing the protein samples: you don't know which composition
corresponds to which protein. From what you know of the structures of proteins,
which composition would you assign to which protein and why?

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Number of residues per molecule

Amino acid Protein 1 Protein 2 Protein 3

Polar residues
Arg 12 4 7
Asn 9 6 5
Asp 14 5 9
Cys 7 2 6
Gln 8 7 6
Glu 11 4 6
His 4 2 4
Lys 22 6 15
Ser 20 8 21
Thr 15 5 11
Trp 2 3 3
Tyr 7 7 6

Nonpolar residues
Ala 14 28 25
Gly 9 9 8
Ile 5 16 9
Leu 3 19 7
Met 7 11 9
Phe 9 15 11
Pro 8 13 10
Val 16 29 21

5. Hemoglobin is a tetrameric protein consisting of two α and two β subunits. The

structure of the α and β subunits is remarkably similar to that of myoglobin.
However, at a number of positions, hydrophilic residues in myoglobin have been
replaced by hydrophobic residues in hemoglobin.
A. How can this observation be reconciled with the generalization that hydrophobic
residues fold into the interior of proteins?
B. What can you say about the interactions determining quaternary structure in
hemoglobin?

6. An ambitious student decided to test the proposition that many proteins stabilized by
covalent -S-S- bonds are nevertheless in minimum free energy conformations. She
treated a series of proteins containing disulfide bonds with 2-mercaptoethanol to

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reduce the -S-S- linkages, in the presence of 8M urea as a denaturant. These

reagents were removed gradually by dialysis under conditions that favor refolding
and the reformation of disulfide bonds to yield the results shown below:

Calculated recovery
of biological activity Experimental
Number of with random -S-S- bond recovery of
Protein -S-S- bonds reformation (%) activity (%)

Ribonuclease 4 0.95 100

Lysozyme 4 0.95 80
Insulin 3 6.70 7

A. With four disulfide bonds show that the expected recovery of biological activity is
0.95% if reformation of such bonds is random.
B. Do the data for ribonuclease and lysozyme support, or contradict, the student's
proposition?
C. How do you explain the data for insulin?
D. Trypsin contains 6 disulfide bonds. After subjecting trypin to the same procedure,
only 8% of its activity was recovered. What is the expected recovery of activity for
random disulfide bond formation? Do the trypsin data support, or contradict, the
student's proposition?

Beer's Law
Absorbance at wavelength λ is defined as:
Aλ = log (Io/I)
where Io = intensity of incident light and I = intensity of transmitted light.
Aλ is related to pathlength (l) and concentration c by Beer's Law:

Aλ = ελlc

where ελ is the extinction coefficient at wavelength λ for the substance being studied. If for
example, protein concentration is measured in mg/cm3 and l in cm, ελ has the dimensions cm2/mg,
since A is a dimensionless quantity.

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Answers:
1. 61 µg Trp = 0.3 µmole
If only one Trp residue/molecule, 10 mg protein = 0.3 µmole
3.33 x 10 mg = 1 µmole
MW protein = 33,300

[NB: If 2 mole Trp/mole protein,

10 mg protein = 0.15 µmole
1 µmole protein = 66.66 mg
1 mole = 66,600
In other words, 33,300 = minimum MW]

2.
A. Molar absorptivity Trp = 5000 L/cm.mol
Molar absorptivity Tyr = 1500 L/cm.mol
Peptide contains 1 Trp and 1 Tyr
Molar absorptivity peptide = 6500 L/cm.mol

Mol. wt. peptide = (131.2 + 131.2 + 204.2 + 181.2 + 105.0 + 147.1) - 5(18)

Mol. wt. = 900 - 90 = 810

g absorptivity = 6500 L/cm/810 g

= 8.02 L/cm.g
= 8.02 x 103 cm3/cm/103 mg
= 8.02 cm2/mg
B. A = εcl
1.3 = (8.02 cm2/mg)(cm)c
c = 1.3/(8.02 cm2/mg)cm
c = 0.162 mg/cm3

3.
A. Crude homogenate = 4000,000/20,000 units/mg = 200 units/mg
Ammonium sulfate ppt: 600
Acid ppt: 250
Ion-exchange: 4,000
Affinity: 15,000
Gel-filtration: 15,000
B. The greatest fold purification is after ion-exchange chromatography; namely, 16-
fold.
C. Acid precipitation results in a loss of specific activity (nearly 60% loss) and gel-
filtration does not increase the specific activity over that seen after affinity
chromatography.

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D. The finding that specific activity does not increase as a result of gel-filtration
implies that the remaining contaminants cannot be resolved on a size basis or the
enzyme is close to homogeneity. Tests of purity might include, SDS-PAGE, 2D
gel-electrophoresis, IEF, end-group analysis, laser desorption mass spectrometry,
affinity-labeling etc...
E. The overall purification is specific activity product/specific activity starting
material:
= (15,000 units/mg)/(200 units/mg) = 75-fold
If 100% of the enzyme's activity in the starting material had been recovered, then,
the enzyme would, on the basis of these data, represent 1/75 = 1.33% of this
material. However, we know that the overall recovery of enzyme activity is only
675,000 units/4000,000 units = 16.9%. If 83% of the activity has been lost
through the loss of enzyme protein, then, the actual amount of enzyme in the
starting material is: (1.33)(100/16.9)% = 7.9%. This is clearly not the case
because 7.9% of 20,000 mg = 1580 mg, which corresponds, on the basis of the
specific activity of the "purified" enzyme (15,000 units/mg), to 1580 mg x 15,000
units/mg = 23,700,000 units, i.e. nearly 6 times the activity we started with. If, on
the other hand, 83% of the activity has been lost through a loss of activity (i.e.
denaturation) without appreciable loss of enzyme protein the correction should be
applicable to the amount of pure protein.
If pure protein = 45 mg and this has a total activity of 675,000 units, but we have
only recovered 16.9% of its activity, the theoretical activity will be:
(100/16.9)(675,000) = 3,994,083 units ( i.e. the total activity we started with)
which yields a theoretical specific activity of 3,994,083 units/45 mg = 88,757
units/mg, i.e. nearly 6 times the measured final specific activity.
In short, we can only estimate that the abundance of the enzyme protein is
between 45/20,000 = 0.225% and 7.9%. 0.225% is undoubtedly too low; 7.9% is
undoubtedly too high. The former calculation assumes no loss of specific activity
while the latter assumes no loss of enzyme protein. The truth is likely to be
somewhere in between; namely losses of both specific activity and enzyme
protein.

4. Most of the polar residues in soluble proteins are on the surface of the molecule
and almost all of the nonpolar residues are folded into the interior. The ratio of
polar to nonpolar residues will determine the permissible surface area/volume
ratio and, hence, the gross shape of the molecule. For any protein molecule of a
given molecular weight, some ratio of polar to nonpolar residues will be
compatible with a spherical shape. An increase in this ratio, with no change in
molecular weight, will necessitate a greater surface area/volume ratio and lead to
a more asymmetric conformation, such as a rod or pancake shape. A decrease in
the polar/nonpolar ratio requires that the protein exist as a multimer, whose
surface/area ratio will be less than that of a monomeric sphere.

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The polar/nonpolar ratios on the three compositions are: 65:35, 30:70 and 50:50.
Therefore, the surface area/volume ratios are in the order 1 > 3 > 2, indicating that
1 is the cigar-shaped protein, 3 is the spherical protein and 2 is the tetramer
subunit.
Implicit is the assumption that the subunits are held together through hydrophobic
interactions. This is often the case.

5.
A. Hydrophobic patches occur on the outside of the hemoglobin subunits where the
α and β chains fit together. Thus, these patches are on the outside of the subunit,
but on the inside of the multimeric protein.
B. Hydrophobic interactions seem to play an important role.

6.
A. With 8 Cys residues, the first to pair has one chance in 7 of selecting the correct
partner. Of the 6 remaining Cys residues, the first to pair has 1 chance in 5 to pair
correctly. Four Cys residues remain; the first of these has 1 chance in 3 of pairing
correctly. The final pair is determined by the previous pairings. Therefore the
probability of the 3 correct choices required to produce an active enzyme is:
1/7 x 1/5 x 1/3 x 1/1 = 1/105 or 0.95%
B. These studies support the proposition because the experimental activities are
much greater than those expected from random refolding.
C. The two chains of insulin result from internal cleavages of proinsulin. Because
the noncovalent interactions that directed the initial correct folding are not all
present during the refolding process, -S-S- bond formation is random.
D. For a protein containing 12 Cys residues:
1/11 x 1/9 x 1/7 x 1/5 x 1/3 = 1/10,400 = 0.01%
An 8% recovery of enzyme activity is far in excess of the random expectation of
0.01%. Thus, the proposition is supported.
We have assumed that protein concentration is so low as to prohibit interchain
disulfide bond formation.