Biochemistry 3

BCH 314
Page 1

QUESTION 1 (52 marks)

a) What is the molar concentration of glucose (molecular mass = 180.16) in a
2 mg ml-1 solution? (3)

b) You are provided with a 1 mg ml -1 stock solution of albumin and are

`required to make dilutions for a standard curve. With this in mind, fill in
the missing figures in the table below. (6)

Albumin Volume Volume Dilution factor Final volume

concentration stock (l) H2O (l) (n-fold) (ml)
(g ml-1)
25 1
50 1
75 1
100 1

c) A salt-precipitated fraction of ribonuclease contained two contaminating

protein bands A and B, in addition to the ribonuclease. Further studies
showed that contaminant A has a molecular weight of about 13,000
(similar to ribonuclease) but an isoelectric point 4 pH units more acidic
than the pI of ribonuclease. Contaminant B had an isoelectric point similar
to ribonuclease but with a molecular weight of 75,000. Suggest and fully
describe an efficient electrophoretic method for the separation of the
ribonuclease from the contaminating proteins. (6)

d) Write notes on isotachophoresis (ITP) with particular reference to its

separation principles and usefulness. (8)

e) You are given a DNA sample with a concentration of 0.4 g l-1. You are
given the restriction enzyme EcoR1, which has a concentration of 4 units
l-1. You must cut 2 g of the DNA.

i) Fill in the missing volumes: (3)

Volume (l)
DNA
Buffer (10X)
Enzyme 0.5
Dist. Water
Total 20
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BCH 314
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ii) When running an agarose gel, in which direction does the DNA
migrate (from + to or to +) and why? (2)

f) Gas-liquid chromatography is a technique that uses a liquid and a gas

phase to separate compounds. Expand on this statement by describing in
detail what the basic principle behind this technique is. Concentrate on the
following:
i) Mobile and stationary phases most suited for GC. (8)
ii) The two types of capillary column systems used in GC. (8)
iii) Separation conditions employed in GC. (8)

QUESTION 2 (48 marks)

a) Biomolecules are purified using chromatography techniques that separate
them according to differences in their specific properties. Discuss this
statement in terms of the following chromatographic techniques:
i) Ion-exchange chromatography (8)
ii) Gel filtration chromatography (8)
iii) Affinity chromatography (8)

b) i) What is specific activity of an enzyme and how is it related to the

degree of purification of an enzyme?
(2)
ii) Complete the following purification table, including any missing
units: (7)
Activity Total Specific Extent of Overall
(units) protein activity purification yield (%)
(mg) ( ) (fold)
Crude 100 1000 (1.0) 100
homogenate
Ammonium 80 40
sulfate
precipitation
Ion-exchange 0 30
chromatography
iii) Briefly provide the most likely reasons for the result obtained in ion-
exchange chromatography. (3)

c) An enzyme catalyzes a reaction at a velocity of 20 mol/min when the

concentration of substrate (S) is 0.01 M. The Km for this substrate is 1 x
10-5 M. Assuming that Michaelis-Menten kinetics are followed, what will the
reaction velocity be when the concentration of S is (a) 1 x 10 -5 M and (b) 1
x 10-6 M? (7)
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d) Methanol (wood alcohol) is highly toxic because it is converted to

formaldehyde in a reaction catalyzed by the enzyme alcohol
dehydrogenase:

NAD+ + methanol NADH + H+ + formaldehyde.

Part of the medical treatment for methanol poisoning is to administer

ethanol (ethyl alcohol) in amounts large enough to cause intoxication
under normal circumstances. Explain this in terms of what you know about
enzyme mechanisms. (5)

[100]

END OF EXAMINATION