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FCH 530 Homework 3

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1. You are asked by your advisor to purify a protein from Escherichia coli cells. You
know that your protein is intracellular (in the cytosol) and that it has a pI of 10. Describes
the steps and techniques you might use to purify this protein.

Given the following data:

Ammonium 0 10 20 30 40 50 60 70 80 90
Sulfate (%

Sample A280 1000 900 600 300 100 75 50 40 15 10

Activity assay 200 200 200 200 200 200 200 190 160 30

What is our best option for salting out this protein?

Activity of the protein remains in solution up to 60-70% ammonium sulfate. So the first
step would be to bring the protein solution to 70% ammonium sulfate to precipitate out
the proteins and bring the sample A280 to 40. The protein could then be precipitated out
at 90% ammonium sulfate. Once this is done, the protein would need to be resuspended
and the ammonium sulfate should be removed by dialysis.

Because we know that the protein has a pI of 10, we know it is a basic protein. In order
to purify this protein, we should use ion exchange chromatography with a cation
exhanger like CM cellulose. Cation exchangers interact with basic proteins (pI 10).
Anion exchangers interact with acidic protein.
2. Describe the steps to make a monoclonal antibody.

3. A mixture of the following amino acids (Ala, Ser, Phe, Leu, Arg, Asp, and His) is
subjected to paper electrophoresis at pH 3.9. Which will go toward the anode? Which
will go toward the cathode?
At pH 3.9, what are the charges of the various amino acids?
Ala =0, Ser =0, Phe =0, Leu =0 but will have a net positive charge (some carboxyl groups
will be –COOH but all amino groups will be in NH3+ state) thus these will move
slightly towards cathode and not separate. His and Arg have pIs closer to 7.6 and 10.8
and would move towards the cathode, separating from the other amino acids. Asp with a
pI close to 3.0 will move towards the anode.

Amino acids with identical charges often separate slightly during paper electrophoresis;
for example Gly separates from Leu. Can you suggest an explanation?
Although identically charged, a large molecule will move more slowly than a smaller one
with the same charge during electrophoresis because the charge-to-mass ratio is smaller
and accordingly the force causing migration is smaller per unit mass.
Assume you have a mixture of Ala, Val, Glu, Lys, and Thr at pH 6.0. Draw the pattern
that will be obtained by ninhydrin staining of the amino acids following paper
electrophoresis. Indicate the anode, cathode, origin and any unresolved amino acids.

A comparison of pI’s indicates that Glu, with a net negative charge, will move toward the
anode; Lys, with a net positive charge, will move toward the cathode. At pH =6, Val,
Ala, and Thr are near their isoelectric points. Though Thr might be expected to move
away from Val and Ala, it does not do so in practice. (see below)

4. What chromatographic method would be suitable for separating the following pairs of
(a) A-F-K, A-A-K
These two differ in the central residue. Since Phe is less polar than Ala, paper
chromatography can effectively separate these tripeptides.

(b) lysozyme, ribonuclease A

Since the pI’s of lysozyme (pI=11.0) and ribonuclease (pI = 7.8) differ so much, cation
exchange chromatography at a pH between these pI’s would separate these proteins. For
example, CM-cellulose chromatography at pH 8.0.

(c) hemoglobin, myoglobin

Hemoglobe is 64.5 kD and myoglobin is 16.9 kD and differ significantly in molecular
mass. Thus gel filtration chromatography on a gel with a fractionation range will
work. Sephadexes G-50, G-100, G-200, or Bio-Gels P-10, P-30, P-100, or Sepharose 6b
(see Table 6-3) will work.

5. What is the order of elution of the following proteins from a Sephadex G-50
column: catalase, -chymotrypsin, concanavalin B, lipase, and myoglobin?
This resin will fractionate based on size from 1 to 30 kD with the largest eluting first.
According to the masses in Table 6-5, the order would be:

Catalase (222 kD)>concavalin B (42.5 kd)>-chymotrypsin (21.6 kD)>myoglobin

(16.9 kD)>lipase (6.7 kD)
6. Explain why the molecular mass of fibrinogen is significantly overestimated when
measured using a calibrated gel filtration column (Fig 6-10 in your book) but can
be determined with reasonable accuracy from its electrophoretic mobility on an
SDS-polyacrylamide gel (see Table 6-5).
Table 6-5 indicates that fibrinogen with a f/f0 =2.336, is a highly asymmetric
molecule. In its native state it will be more likely to penetrate a given gel pore than a
spherical molecule of the same molecular mass. Under gel filtration it will migrate
more rapidly than this equivalent spherical molecule and hence appear to have a
molecular mass lower than it really has. In contrast, SDS-PAGE denatures fibrinogen
so it will migrate at the same rate as almost any other protein of its molecular mass