BCH 214 Tutorial 2

1) Proteins have complex and specific structures. Give a summary of the main
features of the primary and tertiary structures of proteins.
What function do the following proteins carry out? Collagen, haemoglobulin,
immunoglobulins.
2) Differentiate between filtration and dialysis.
3) Which factors will influence the electrophoretic mobility () of a protein
molecule in an electric field assuming that the liquid medium in which the
movement occurs remains unchanged and the applied electric field is held
constant.
4) A drop of a solution containing a mixture of glycine (pI = 5.97), alanine
(pI = 6.02), glutamic acid (pI = 5.37), lysine (pI = 7.22), arginine (pI = 7.90)
and histidine (pI = 5.66) was placed in the centre of a paper strip and dried.
The paper was moistened with a buffer of pH 6 and an electric current was
applied to the ends of the strip.
(i) Which amino acid(s) moved towards the anode? Why?
(ii) Which amino acid(s) moved towards the cathode? Why?
(iii) Which remained at or near the origin? Why?
(iv) What is the basis for this separation technique?
5) Explain why the following phrase is true; There is little twisting around the C-
N bond (peptide bond). Include illustrations in your answer.
6) What are the different functions that proteins carry out? Give the name of a
protein for each function?
7) What is meant by the following terms? Give one example for each.
i) Diastereoisomers
ii) Enantiomers
8) Explain why some adults experience some stomach discomfort after drinking
fresh milk, whereas children do not? What is this condition referred to as?
Draw the structure of the causative agent for the stomach discomfort,
mentioned in iv) above.
9) Both cellulose and -amylose consist of (14)-linked D-glucose units and can
be extensively hydrated. Despite this similarity, a okoperson 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.
10) Describe the term amphiphilic? Give an example of an amphiphilic
compound?
11) What are the roles of biological lipids?
12) 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?
13) 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
14) 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.
15) Explain the principle and usefulness of SDS - PAGE.
16) 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
17) 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?

18)
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.
19) 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
20) (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?
22) What is the fundamental principle of isoelectric focusing

21) 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.

Memo
1) Primary structure
The linear sequence of amino acids.

Tertiary structure of proteins.

Secondary elements of protein structure (-helices, -sheet regions, random
coil regions) combine to give a more complex 3-dimensional shape. The
complex structure is usually the functional structure of the protein. Held in
place by:
a. Hydrophobic interactions between nonpolar R-groups
b. Electrostatic interactions between charged R-groups
c. Hydrogen bonding interactions
d. Disulfide linkages between cys residues
2) Filtration is the separation of particulate matter from liquid whereas dialysis is
the separation of dissolved molecules on the basis of their differences in size
(molecular dimensions) through a semi permeable membrane.
3) charge, size and shape of molecule
4) (i) Towards anode: glutamic and histidine - negatively charged at pH 6
ii) Towards cathode: lysine and arginine - positively charged at pH 6
iii) Near origin: glycine and alanine (slightly negatively and positively charged
resp)
5) Peptide bonds are formed when the -amino group of an amino acid residue
forms a covalent bond with the -carboxyl group of another amino acid
residue. Water is eliminated in the process.

illustration

Note: i) the C=O and the N-H bonds are nearly parallel
ii) C, O, N and H atoms are usually coplanar
iii) there is little twisting possible around C-N bond because the peptide bond
has substantial fraction of double bond character. The peptide bond can
actually be considered a resonance hybrid of the forms:

illustration
 iv) The group of atoms about the peptide bond can exist in either the trans or
cis configurations

The trans is favoured in order to minimize steric interaction between bulky R groups
on adjacent -C atoms.
6) Different functions that can be carried out by proteins:
i) Structural: e.g. collagen skin and collective tissue (tendons); keratin hair,
fingernails; silk
ii) Act as catalysts: e.g. enzymes
iii) Movement: e.g. muscle proteins (skeletal, smooth muscle for digestive
tract, cardiac tissue)
iv) Transport: e.g. O2 transport (haemoglobin in blood and myoglobin in
muscle tissue, HDL/LDL in cholesterol transport; membrane-bound
proteins for transmembrane transport)
v) Hormones: e.g. insulin, gastrin, oxytocin, vasopressin (produced by
specific organs modify function of other organs e.g. insulin involved in
glc metabolism, gastrin involved in digestion)
vi) Protection: e.g. various classes of immunoglobulins (antibodies)
7) i) Diastereoisomers: stereoisomers that are not mirror images of each other
and are not superimposable (e.g. D-glucose and D-galactose.
ii) Enantiomers: two mirror image stereoisomers that are non-superimposable
(e.g. L and D-glyceraldehyde).
8) The function of lactose in nature is to supply the newborn with an easily
available source of carbohydrate for energy. In many humans the enzyme
lactase, which catalyses the breakdown of the milk sugar lactose to galactose
and glucose, disappears from the intestinal mucosal cells after age 4 to 6,
when milk drinking usually decreases. When these people ingest fresh milk or
lactose-containing milk products, lactose passes unchanged through the first
part of gastrointestinal tract until it reaches the large intestine. The large
intestine is inhabited by millions of anaerobic bacteria that use the lactose for
energy. The action of these bacteria on the lactose causes intestinal distress,
diarrhea and excessive flatulence. This condition is referred to as lactose
intolerance. Draw the structure of lactose.
9) Inspite of the striking similarities between cellulose and -amylose(stach),
there is an important difference: the (14)-linkage in cellulose is in the -
configuration, whereas that in -amylose is in the -configuration. This
difference is readily recognized by human intestinal enzymes. Pancreatic
amylase hydrolyses glucose polymers linked -(14) but not those linked -
(1-4). Since human beings do not possess an enzyme that can hydrolyze the
(14) linkage, cellulose cannot serve as a food source.and they are usually
found in plants not on humans.

10) Amphiphilic compound is a compound that has both a polar part and a
nonpolar in its structure. Fatty acids and phospholipids
11) They serve as structural components of biological membranes.
They provide energy reserves, predominantly in the form of
triacylglycerols. Some lipids and lipid derivatives serve as vitamins and
hormones.
 12) i) Separation of amino acids is based on variations in the charge (which
leads to different migration /movement rates in an electric field) and on their
hydrophobicity.
ii) Bands tend to mix due to adsorption of proteins on paper.
iii) To drive off air bubbles
13) ii) = density of dye solution helps deliver mixture to bottom of well
14) i) It is the electrophoretic process of separating different sized and charged
fragments through an agarose gel in the presence of an electrical field. It is
maily used to separate nu cleic acids such as DNA and RNA
ii)Yes it can be used to stain for both RNA and DNA, however, the affinity of
the dye for single-stranded nucleic acids is relatively low and the fluorescenct
yield is comparatively poor compared to that of double-stranded DNA
15) During SDS gel electrophoresis, the proteins are all surrounded by SDS.
Since SDS is strongly anionic, all the proteins acquire/need the same charge
: mass ratio. Thus proteins will appear as negatively charged molecules and
will all migrate / move to the anode. As a result only differences in molecular
size form the basis of separation. This makes it possible to estimate the
molecular weight of proteins.
16) A-D-B-E-C
17) Without SDS, protein migration through a gel would be influenced by the
proteins net charge (which could be +ve or -ve) and its unique shape.
Because neither of these factors correlates with a fundamental property such
as molecular weight, meaningful comparison of different proteins based
solely upon the results of gel electrophoresis would be impossible. Sodium
dodecyl sulphate. They are already denatured by SDS
18) Since the higher the pH, the more cationic the protein, the proteins will be
focused to positions that vary from anode to cathode in the order of increasing
pI. Thus, fron anode to cathode they will take the order; insulin, myoglobin,
ribonuclease A, cytochrome c and histone.
19) i) The more open/biger porspace (less concentrated) the gel is, the easier for
proteins to move through, the more closed (concentrated) the pores are, the
more difficult for big proteins to move through.
ii)The higher the current the faster the electrophoretic mobility of proteins is
20) (i) lowest mol wt
ii) Bromophenol blue is a tracker dye, to indicate when to stop the
experiment, and glycerol is to make protein solution dense so as to sink to the
bottom of the well.
iii) Coomassie brilliant blue
iv) 280 and 260 nm resp
21) The fundamental premise of IEF is that a molecule will migrate / move so
long as it is charged. Should it become neutral, it will stop migrating in the
electric field. IEF is run in a pH gradient where the pH is low at the anode and
high at the cathode
10) i) acrylamide
ii) To keep out oxygen which inhibits polymerisation
iii) Bromophenol blue is a tracker dye, to indicate when to stop the
experiment, coomassie brilliant blue is for staining proteins.
iii) Coomassie brilliant blue
iv) 280 and 260 nm rasp

20) prot mol. wt log mol. wt mobility (%) relative mobility (Rm)
1 15,000 4.18 100 1.00
2 35,000 4.54 39 0.39
3 25,000 4.40 63 0.63
4 20,000 4.30 81 0.81

X? 51* 0.51
= Midway between 39% and 63%

Plot graph (log mol wt Vs Rm), read off log mol wt of X and convert to mol. wt.
Log mol wt of X = 4.47; mol wt of X = 29,500

2) Different functions that can be carried out by proteins:

(i) Collagen: structural - skin and connective tissue (tendons)
(ii) Haemoglobin: transport - ( O2 transport in blood)
(iii) Immunoglobulins: protection - various classes of immunoglobulins
(antibodies)
3) Mutations in a gene for a particular protein may or may not lead to a change
in the biological function of the protein. Amino acids changes within a group
often will not result in a structural change and therefore the biological function
of a protein may be unchanged. These are called conservative substitutions
e.g. Val to Leu; Ser to Thr; Gln to Asn. (The nature of the R group is the
same). Substitutions that are likely to result in structural changes are known
as non-conservative substitutions e.g. Glu to Lys (negative to positive charge);
Val to His (neutral to partial positive charge) Gly to Trp (small non polar to
large non polar) (see R groups)
iii) : denotes rotation about anomeric C-atom; D: denotes orientation of OH
group about asymmetric carbon furthest from carbonyl group; (+): denotes
rotation of plane polarized light to the right (levo); pyranose: denotes a 6-
membered ring as in pyran.