RHODES UNIVERSITY

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

BIOCHEMISTRY 202
Paper 2

PRACTICAL EXAMINATION: NOVEMBER 2019

Internal Examiners: Prof H Hoppe MARKS: 100
Prof B Pletschke DURATION: 3 hours
SUB-MINIMUM: 40%

GENERAL INSTRUCTIONS TO CANDIDATES

1. There are 6 questions. Answer ALL questions.

2. Time management is very important. The value of the mark for each question should be
used as a rough guide to the amount of time allocated to answer the question (100 marks
in 180 minutes).

3. It is in your best interest to write legibly.

4. At the end of the examination, place all answer books and graph paper inside answer
book 1.

5. Graph paper (4 cartesian sheets) and a calculator are required.

6. The Oxford concise English dictionary may be used during this examination.

PLEASE DO NOT TURN OVER THIS PAGE UNTIL TOLD TO DO SO

Biochemistry 202 Paper 2 (Practical) Page 1 of 5
Question 1

a) At a wavelength of 572 nm, the molecule resorufin has an extinction coefficient (ε572) of
7000 M-1.cm-1. You place a solution of resorufin in a 1 cm cuvette, read the absorbance at
572 nm and obtain A572 = 0.7.
i) What is the concentration of resorufin in the solution in mM? (3)
ii) Resorufin has a molecular weight of 235 Da. What is its concentration in the solution in
mg/ml? (2)
b) The enzymes malate dehydrogenase and fumarase catalyse the following reactions:

i) You have a sample with an unknown concentration of malate. To determine the

concentration of malate, which two molecules or proteins would you add to the sample? (2)
ii) If you wanted to determine the concentration of fumarate in a sample, which additional (1)
molecule or protein would you add?
iii) At what wavelength would you read the absorbance? (1)

c) If malate dehydrogenase produces 4 µmol of oxaloacetate in 2 minutes and the reaction

contained 2 mg of enzyme, what is the enzyme’s specific activity? (1)
d) i) What is the protein concentration of a sample that has an absorbance at 280 nm of 0.3? (1)
ii) What wavelength is used to determine the concentration of nucleic acids? (1)
e) Which reagent is commonly used to determine the concentration of proteins? (1)
[13]
Question 2

a) You carry out an SDS-PAGE experiment with a molecular weight marker (size standards) in
one lane and an unknown protein X in another. After staining the gel and measuring the
migration distances of the standards, you obtain the values shown in the table below:
Molecular weight Migration distance
standard (kDa) (mm)
140 10
90 33
65 51
40 76
30 92
The migration distance of protein X is 62 mm
The migration distance of the dye front is 120 mm

Use the graph paper provided to draw a standard curve and determine the molecular weight
of protein X. Provide the molecular weight of X in kDa. (Hand in the graph paper with your
answer book. You may use the answer book to do your calculations) (5)

Biochemistry 202 Paper 2 (Practical) Page 2 of 5

b) You run three protein samples (A, B and C) on an SDS-PAGE gel. Each sample contains one
of the following proteins:
BSA (66 kDa), Actin (43 kDa) or Globin (14 kDa).
After staining the gel with Coomassie Blue, you obtain the following result:

(3)
Which protein was present in samples A, B and C, respectively?
c) Why is TEMED added to acrylamide/bisacrylamide solutions to form an SDS-PAGE gel? (1)
d) Why is β-mercaptoethanol added to the SDS-PAGE sample buffer? (1)
e) Give two reasons why bromophenol blue is added to the sample buffer. (2)
[12]
Question 3

Research suggests that the protein p15 (molecular weight 15 kDa) is only present in virulent TB bacteria
(virulent bacteria cause disease, while non-virulent bacteria don’t). It further suggests that humans infected
with virulent TB bacteria contain antibodies to p15 in their bloodstream.
a) Describe an experiment you could perform to confirm that p15 is only present in virulent TB
bacteria.
(For this experiment, you have been supplied with a sample of virulent bacteria and one of
non-virulent bacteria, as well as mouse anti-p15 antibodies.) (8)
b) Describe a diagnostic method you could perform to determine if a human patient is infected
with virulent TB bacteria.
(You have been supplied with a 96-well plate containing purified p15 protein attached to the
wells, as well as a patient blood sample). (5)
[13]
Question 4

With reference to the purification table below, answer the following questions:
Steps Total protein Total activity Specific Yield (%) Fold purification
(mg) (U) activity
(U/mg)
Crude extract 330 8500 A 100.00 1.00
Ammonium 90 7700 85.50 B 3.34
sulphate pellet
Cation exchange 20 6250 312.50 73.50 C
chromatography
Size exclusion 4 D 750.00 35.20 29.29
chromatography
a) Calculate the values that are missing in the table above:
i) A (1)
ii) B (1)
iii) C (1)
iv) D (1)

Biochemistry 202 Paper 2 (Practical) Page 3 of 5

b) Why does the Total Activity decrease but the Specific Activity increase during the purification? (4)
c) Instead of ammonium sulphate precipitation, name two other methods that you could use to
concentrate your target protein as a second step.
Very briefly describe these methods and the principles on which they are based. (4)
[12]
Question 5

a) In your ion-exchange chromatography practical, you used a carboxy-methyl (CM) cellulose

column. This is a cation exchange resin, which is negatively charged and so binds positively
charged molecules. Based on what you did during this practical:
i) Explain the mechanism involved in the separation of:
Blue dextran. (2)
DNP glycine. (3)
Cytochrome C (include the principle of cytochrome C elution). (5)

ii) If a diethylaminoethyl exchange resin had been used instead of the carboxy-methyl resin,
what would have been the theoretical elution profile of the three compounds, if a 0.1 M
buffer at pH 6 was used? Explain your answer. (3)

b) Explain the principle of molecular exclusion chromatography. (4)

c) The relative molecular mass (Mr) of a myosin was investigated by gel filtration
chromatography using a Sephacryl S300 column and aldose, catalase, ferritin, thyroglobulin
and blue dextran as standards. The following results were obtained:
Mol. wt. (Da) Elution volume (ml)
Aldose 158 000 22.5
Catalase 210 000 21.4
Ferritin 444 000 18.4
Thyroglobulin 669 000 16.4
Blue Dextran 2 000 000 13.6
Myosin unknown 19.2

Calculate the molecular weight of myosin by constructing an appropriate graph. (8)

[25]
Question 6

a) Banana tyrosinase catalyses the following reaction:

DOPA (dihydroxyphenylalanine) → DOPA-CHROME (Red Colour)

You are provided with the following reagents and equipment: 0.1 M phosphate buffer (pH 7.2),
2.5 mg/ml DOPA substrate in 0.1 M phosphate buffer (pH 7.2), banana extract,
spectrophotometer and cuvettes, autopipettes and graph paper.

Describe how you would determine the kinetic parameters Vmax and Km.
In your answer, also discuss the types of plots you would need to draw to accurately determine
your kinetic parameters. (15)

Biochemistry 202 Paper 2 (Practical) Page 4 of 5

b) The kinetics of an enzyme are measured as a function of substrate concentration in the presence
and absence of 2 mM inhibitor:

Velocity (µmoles/min)
[S] (uM) No inhibitor Inhibitor
3 10.4 4.1
5 14.5 6.4
10 22.5 11.3
30 33.8 22.6
90 40.5 33.8

i) What are the values of Vmax and Km in the absence of the inhibitor? (4)
ii) What are the values of Vmax and Km in the presence of the inhibitor? (4)
iii) What type of inhibition is displayed by the enzyme? (2)

[25]

END OF THE EXAMINATION PAPER

Biochemistry 202 Paper 2 (Practical) Page 5 of 5