BCH 214 Tutorial 2

1) A certain pentapeptide has the following sequence;

asp-arg-pro-met-lys

i) Name the basic amino acids from the sequence

ii) Name any other amino acid group that is represented in the sequence and
give an example

2) Explain why the following phrase is true; There is little twisting around the C-N
bond (peptide bond). Include illustrations in your answer.

3) Discuss non-conservative and conservative amino acids substitution. Give an

example in each case.

4) What are the different functions that proteins carry out? Give the name of a
protein for each function?

5) What is meant by the following terms? Give one example for each.
i) Diastereoisomers
ii) Enantiomers
iii) Explain the meaning of each of the following in the annotation, -D (+) -
glucopyranose: , D, (+) and pyranose.
iv) Explain why some adults experience some stomach discomfort after drinking
fresh milk, whereas children do not? What is this condition referred to as?

v) Draw the structure of the causative agent for the stomach discomfort,
mentioned in iv) above.

6) Both cellulose and -amylose consist of (14)-linked D-glucose units and can be
extensively hydrated. Despite this similarity, a person on a diet
consisting of predominantly -amylose (starch) will gain weight whereas a person
on a diet of cellulose (wood) will starve to death. Explain.

7) Describe the term amphiphilic? Give an example of an amphiphilic compound?

8) What are the roles of biological lipids?

9) Amino acids are frequently separated by paper electrophoresis.

i) What is the basis of this separation?
ii) Why is paper electrophoresis not suitable for the separation of large molecules
such as proteins?
iii) Why is it important to moisten the paper strip from below with the conducting
buffer before electrophoresis?
iv) What is cellulose acetate electrophoresis specifically used for?
10) i) Which of the two determines the gel pore size in PAGE, acrylamide or bis-
acrylamide?
ii) After casting a gel for PAGE, why is it necessary to overlay it with water before
electrophoresis?
iii) What is the function of each of the following in PAGE of proteins?
bromophenol blue and coomassie brilliant blue.
iv) Name a stain for proteins on gels following electrophoresis.
v) What is the max for (i) proteins and (ii) nucleic acids?

11) In gel electrophoresis, loading dye is added to the contents of each well because:

i) Greater accuracy is achieved

ii) The density of the dye solution helps to deliver the mixture to the bottom
of the well.
iii) Loading dye cuts the percentage of volume of the buffer
iv) Restriction enzymes are activated by the loading dye
v) None of the above is correct

12) Explain why all proteins migrate toward the anode in SDS gel
electrophoresis

13) i) What is agarose gel electrophoresis? What is it used for?

ii) Can ethidium bromide be used to stain both double and single-stranded
nucleic acids? Give a brief explanation of your answer.

14) Explain the principle and usefulness of SDS - PAGE.

15) Select from the numbers (1) to (5) the correct sequence of steps in
determining the sizes of unknown proteins.

A. A standard of known protein sizes is run along side the unknown proteins.
B. Construct a standard curve on semi - log paper.
C. Compare the sizes of known and unknown proteins on the standard curve.
D. Measure the migration distances of known proteins.
E. Read-off the molecular mass of unknown proteins from the standard
curve.
Select the correct sequence from the following:
1. A-B-C-D-E
2. A-B-D-C-E
3. A-D-B-E-C
4. D-E-C-B-A
 5. B-A-E-D-C

16) What makes it difficult to interpret the results of gel electrophoresis of a protein in
the absence of sodium dodecyl sulphate (SDS)? What does SDS, stand for and
why are gels not cooled during SDS gel
electrophoresis?

17)
Protein pI
Cytochrome c 10.7
Insulin 5.4
Ribonuclease A 7.8
Histone 10.8
Myoglobin 7.0

What would be the relative arrangement of the proteins listed in the

table above after they had been subjected to isoelectric focusing? Give an
explanation in support of your answer.

18) How do the following factors affect electrophoresis of proteins? Give a brief
explanation in each case.

(i) Pore size of the gel

(ii) Electric field

19) (i) In SDS-PAGE set-up, the protein with the highest mobility is the
one that has the (highest or lowest) ---------------------
(ii) What is the function of each of the following in PAGE of proteins?
bromophenol blue, glycerol
(iii) Name a stain for proteins on gels following electrophoresis.
(iv) What is the max for (1) proteins and (2) nucleic acids?

20) Four pure proteins were used as standard to construct a standard curve for a
molecular weight analysis using SDS-PAGE. Protein 1 (mol. wt. = 15 000) was
the smallest. Protein 2 (mol. wt. = 35 000) moved only 39% as far as protein 1.
Protein 3 (mol. wt. = 25 000) moved only 63% as far as protein 1. Protein 4 ( mol.
wt = 20 000) moved only 81% as far as protein 1.

Construct the standard curve and then determine the molecular weight of the
unknown protein that has mobility (under the same conditions) midway between
that of protein 2 and 3.
21) What is the fundamental principle of isoelectric focusing