Download FST 123 - Enzymology Homework IS `13

FST 123 - Enzymology
Homework I
S '13
Name____________
1. Briefly define the following terms:
a. Hydrophobic effect
b. Quaternary structure:
c. Domain
d. Primary structure
e. Ramachandran diagram
f. Salt Bridge
g. Torsion angle
h. Tertiary structure
2. If a protein is composed of 225 amino acids:
a. What is its approximate molecular weight? What is its approximate volume?
b. Assuming it is a sphere, what is its radius?
3. The course website contains a link to a Kinemage file depicting the structures of four
proteins. Download them, view them using Mage or King
(http://kinemage.biochem.duke.edu/software/index.php), and classify them according to
Chothia’s four categories.
4. A buffer was made by dissolving 18.92 g of lactic acid and (pKa = 3.86) 32.71 grams of
sodium lactate in 1L or water. (Mr.: Lactic acid, 90.08; NaLactate, 112.8).
a. What is the pH of this buffer?
b. What is the concentration of the buffer?
c. What is the ionic strength of the buffer?
5. Protein A contains 30 carboxyl groups and 18 amino groups; protein B contains 15 carboxyl
groups and 20 amino groups. Assume that the carboxyl groups have pKa's of 4.0 and that the
amino groups have pKa's of 9.0.
a. Neglecting second-order effects, estimate the pI of each protein.
b. Which of the two proteins would be more likely to adsorb to a DEAE-cellulose
chromatography column in buffer of low ionic strength at pH 8.2? What about a CMcellulose column at pH 6.2?
c. From the following table, select the acids that would be the best choices for making
the two buffers described in part b.
acid
formic
acetic
benzoic
Ka
acid
carbonic
4.3 x 10-4
1
1.7 x 10-5
MES
2
6.4 x 10-5
Phosphoric
1MES = 2-[N-morpholino]ethanesulfonic acid
2the second ionization is given for phosphoric acid.
3Tris-Cl = tris(hydroxymethyl)aminomethane•HCl.
Ka
4.3 x 10-7
6.3 x 10-7
6.2 x 10-8
acid
hypochlorous
Tris-Cl3
hypoiodous
Ka
3.0 x 10-8
7.1 x 10-9
2.3 x 10-11
6. The following information is known about four proteins:
Protein
A
B
C
D
pI
5.2
9.1
6.2
5.8
Monomer Mr
13,100
14,200
66,700
50,000
subunits
1
1
2 identical, 66.7 k each
4 identical,
50,000 each
a. Sketch below, the elution profile expected if this mixture is run on a Sephadex G-100
gel filtration column run in 50 mM phosphate buffer, pH 6. Label the peaks.
b. On the following page, sketch the gel you would expect to obtain from running these
proteins on an SDS polyacrylamide electrophoresis gel at pH 8.0, (after staining and
destaining).
c. What predictions can you make about the results of a native PAGE at pH 7.6 (State
any assumptions you might need to make about the % acrylamide in the gel.)
d. Sketch the elution profile of these proteins from a carboxymethyl cellulose ion
exchange chromatography column, run at pH 6.25 (with a salt gradient, if necessary).
Label the peaks.
G-100 Column
SDS Gel
CMC column
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7. List two distinctly different criteria used to assess the purity of an enzyme and briefly discuss
their relative merits. Note that sometimes you think a protein is pure, but you have really just
been unable to separate the impurities, so you don’t know they are there. It’s always a good
idea to use more than one technique.
8. Complete the following purification table:
Step
Crude extr
total vol
(mL)
500
Protein
(mg/mL)
2.0
Activity
(U/mL)
total
protein
(mg)
total
activity
sp Act
(U/mg)
Purification
(fold)
Recovery
(%)
6.2
20% Ammon.
18
6.7
4
sulfate ppt
20% Ammon.
499
1.6
5
sulfate super.
40% Ammon.
10
2.1
169
sulfate ppt
40% Ammon.
550
1.5
1.4
sulfate super.
(40% ammonium sulfate super. means the supernatant solution obtained from the 20% ammonium sulfate
supernatant by adding enough ammonium sulfate to bring the concentration to 40% of saturation, then centrifuging
it.)
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