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Biochemistry 3

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Question 1 (37 marks)
a) A sugar solution (0.5 ml) was assayed by the Nelson-Somogyi method
and yielded an A520 of 0.2 in a 1 cm light path. If the slope of the standard
curve for this sugar (i.e. the K-value) was 5 x 10 -3 absorbance units g-1,
calculate its concentration in g ml-1. (5)

b) What volume of 0.5 mol. dm-3 Tris-HCl must be added to 45 cm3 of water
to bring the final concentration of Tris-HCl to 0.05 mol dm -3? Assume that
all volumes are strictly additive. (5)

c) Most pure proteins are insoluble in pure distilled water but dissolve in
dilute salt concentrations. However, the addition of high concentrations of
neutral salts to an aqueous solution of protein causes it to precipitate.
Name this phenomenon and suggest a molecular explanation for the
observation that high concentrations of added salts decrease the solubility
of proteins. (6)

d) A protein has a molecular weight of 160,000. On a reducing SDS-PAGE

gel it migrates as two protein bands corresponding to molecular weights of
80,000 and 40,000 respectively. Explain this observation. (3)

e) Capillary gel electrophoresis (CGE) forms part of a family of related

techniques that comprise capillary electrophoresis (CE). Give a brief
description of this technique with particular reference to its separation
characteristics and applications. (15)

f) Give an explanation at a molecular level of how ethidium bromide interacts

with DNA. (3)

Question 2 (28 marks)

a) Dextran blue is a high molecular weight carbohydrate (mol. wt. 2 x 10 6)
which is often run in Sephadex columns as a marker protein. Explain why
this is necessary when protein partition coefficient (K av) values are to be
determined. Give a reason for obtaining a K av greater than a value of 1 for
a solute following chromatography on a molecular exclusion gel. (7)

b) You wish to purify an ATP-binding enzyme from a crude extract that

contains several contaminating proteins. In order to purify the protein
rapidly and to the highest purity, you must consider some sophisticated
strategies, among them affinity chromatography. Explain how affinity
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chromatography may be applied to this separation and explain the
physical basis of the separation. (9)

c) Concanavalin A, a protein that binds carbohydrates, can be purified by

affinity chromatography on Sephadex. The column distribution coefficient
for concanavalin A is greater than 1.0. Explain how this will affect a
molecular weight determination of concanavalin A by gel filtration
chromatography? (4)

d) Write short descriptions that explain clearly the following (concerning

1) Resolution (2)
2) Efficiency (2)
3) Selectivity (4)

Question 3 (35 marks)

a) As a protein is purified, both the amount of total protein and the activity of
the partially purified protein fraction decrease. Why, then, does the specific
activity of the purified protein increase? (5)

b) All the -galactosidase from Erwinia chrysanthemi (10 mg) is dissolved in

buffer (10 ml). A sample of this solution is found to have an activity of 10 3
units ml-1 after a 100-fold dilution. What is the activity of -galactosidase in
units mg-1 Erwinia? (5)

c) Methanol (wood alcohol) is highly toxic because it is converted to

formaldehyde in a reaction catalyzed by the enzyme alcohol

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. (4)

d) A major concern in protein purification is stability. What precautions must

be taken in order to minimize protein degradation and decrease the
chances of denaturation? (5)
e) 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
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the reaction velocity be when the concentration of S is (a) 1 x 10 -5 M and
(b) 1 x 10-6 M? (7)

f) i) Micrococcus lysodeikticus cells (1.9 ml) were mixed with muramidase

(100 l) and the resulting reaction monitored at 600 nm in a 1 cm cuvette.
The linear rate of decrease in absorbance was 0.24 per minute. If one unit
of enzyme activity is defined as a change in absorbance (at 600 nm) of
0.001 per second, calculate the units per ml of activity. (6)
ii) If 75 ml of the enzyme preparation in (i) above was prepared from 15 g
of egg white, what would be the specific activity of the enzyme. (3)