Structural Biochemistry Tutorial

Introduction
Fill the empty spaces

Large carbohydrates (polysaccharides), proteins, and nucleic acids are polymers, which are
chains of monomers
The components of lipids vary. Monomers form larger molecules by dehydration reactions
, in which water molecules are released. Polymers can disassemble by the reverse process,
hydrolysis. An immense variety of polymers can be built from a small set of monomers.

Concept maps!
Fill in the following concept maps that summarizes the chapter related on carbohydrates, lipids
and proteins

monosaccharides disaccharides polysaccharides

(CH2O)n
Glycosidic linkages

Energy Carbon,
components skeletons,
monomers Glycogen starch cellulose chitin

animals

Fats, TAG phospholipids steroids

glycerol Fatty acids Phosphate groups

Animal cell membranes

component
(cholesterol), hormones

Unsaturated saturated (no

(with C=C bond) C=C bond)

Cell membranes
Structural Biochemistry Tutorial

Proteins

Exercise 1
1. Represent a chain of a peptide constitued of 4 AA residues by representing the side
chain group with the letter R followed by a corresponding number of the AA residue.
Represent the skeleton as a zigzag. Orientate this peptide and show the peptide bonds.

2. This short peptide is derived from a polypeptide chain organized in an α helix. What
can you deduce from the interaction between residue 1 and residue 4. Name this
interaction and figure it on your diagram (Cf. question 1).

3. Residue # 3 is a cysteine residue (R: -CH2SH), what kind of bond can this AA residue
make? how can we break this bond?
Disulfide bond is realized between 2 cysteines residues, that can be broken under
denaturing conditions (heat) or by reducing agent (DTT).
Structural Biochemistry Tutorial

Exercise 2

The 5 proteins whose molecular masses and isoelectric points are given below:
a. α-antitrypsin (MW: 45000, pI: 5.4),
b. cytochrom c (PM: 13400, pI: 10.6),
c. myoglobin (PM: 17000, pI: 7.0),
d. serum albumin (PM: 69000, pI: 4.8),
e. transferrin (MW: 90,000, pI: 5.9).

They are separated by electrophoresis on polyacrylamide gel (separation according to

their size) in the presence of SDS (denaturing conditions). Give the order of their migration
from the top (the sample deposition point) to the lower part of the gel.
Answer: top to bottom : transferrin, serum albumin, α-antitrypsin, myoglobin, cytochrom c

b) If the 5 proteins are separated by isoelectrofocusing (depending on the pI), what could be
their distribution between the positive (anode, +) and negative (cathode, -) ends of the gel?
Give the highest and lowest pH values
Answer: high pH (-) cytochrom c, myoglobin, transferrin, α-antitrypsin, serum albumin low pH
(+).

Exercise 3: peptide AA sequence determination

We have a 14 AA peptide. By the action of Edman's reagent we obtain, PTH-Y; and by the
action of a carboxypeptidase, we obtain K.
After complete acid hydrolysis 6N HCl 100°C, the following AAs were found:
D; A; G; I; R; K; Y; Q; M; F;
By using CNBr which hydrolyzes specifically after Met (M - X) and Edman sequential analysis:
we obtain:

 D - I - K - Q - M;  K - F - A - M;
 K;  Y-R-G–M
By using trypsin which hydrolyzes peptide bonds after positively charged residues (K, R) and
Edman sequential analysis.
 Q - M - K;  F - A - M - K;
 G - M - D - I - K;  Y–R
Structural Biochemistry Tutorial

Exercise 4
Cation exchange column chromatography is a method for separating and purifying the different
constituents of a mixture. This cation exchange column in this case consists of resin grains
carrying sulfonated groups (-SO3-) at the surface; these fillers are found initially in the form of
sodium salts SO3- Na+.
This gives a mixture of α-amino acids containing methionine, lysine, aspartic acid and arginine.
Elution is carried out using buffer solutions with an increasing pH gradient.

1) In what ionized form must this AA mixture be in order to be separated and purified by
cation exchange column chromatography? Explain your answer and explain at what
pH should we be.
Answer: positively charged since the column consists of negatively charged resin grains.
So amino acids must be at an acidic pH and therefore low to be negatively charged

2) We give the isoelectric pH of these 4 AA: Met: 5.75 Lys: 9.74 Asp: 2.87 Arg: 10.76
At what pH should we stand respectively to elute these 4 different AAs?
Answer: we must set ourselves to a pH value ≥ pHi
3) This same AA mixture is subjected to zone electrophoresis at pH 6. Indicate by a
diagram the position of these 4 AAs after electrophoresis.
Answer:
Cathode (-) Arginine (+) Lysine (+) Methionine (-) Asp (-) Anode (+)
Structural Biochemistry Tutorial

Exercise 5:
The in-depth biochemical study of a protein has made it possible to elucidate its oligomeric
structure as being a composition of two A subunits (MW: 27 kDa each) and two B subunits
(MW: 35 kDa each) linked by weak bonds and disulfide bridges according to the diagram
below:

Before analyzing the protein by polyacylamide gel electrophoresis in the presence of SDS
(denaturing agent), three different treatments were carried out:
Treatment 1: incubation of the protein in the presence of 1% SDS and 8 M urea.
Treatment 2: incubation of the protein in the presence of 1% SDS, 8 M urea and Dithiothreitol
(DTT, redox molecule) 3.7%
Treatment 3: incubation of the protein in the presence of 1% SDS and 3.7% (w / v) dithiothreitol
(DTT) only.

1. What is the action of the following molecules: SDS, DTT and urea
2. Schematize the electrophoretic profiles corresponding to each treatment of the protein.
Justify your answer.

Answer 1: SDS is a detergent and strong ionic surfactant negatively charged, it will allow the
peptides to be negatively charged in order to be able to separate them by electrophoresis in
denaturing conditions.
The urea will induce the denaturation of the protein and therefore break the weak type bonds
between the different subunits.
DTT behaves as a reducer of any disulfide bridges (2 excess R-SH + X-S-S-X -> R-S-S-R +
X-SH HS-X). This makes it possible to separate any subunits linked by disulphide bridges and
to ensure the complete "unfolding" of the polypeptides comprising disulphide bridges
(intrachain).

Answer 2:
- Treatment 1: a band at 70 kDa (2 B subunits always linked by disulfide bridges) + 1 band at
27 kDa
- Treatment 2: one band at 27 and 35 kDa
- Treatment 3: a strip at 62 kDa (27 + 35 kDa) broken diS bridges
Structural Biochemistry Tutorial

Exercise 6: sequencing a hexapeptide

A hexapeptide is obtained by hydrolysis of a protein
1) What procedure should be followed to determine the raw formula of the hexapeptide?
The result is: Ala1, Arg1, Leu1, Phe1, Tyr1, Val1

2) Determine the sequence of the peptide, knowing that:

- Aminopeptidase releases alanine
- Hydrolysis by trypsin (cut after Lys / Arg) leads to two tripeptides, one of which consists
of alanine, arginine and tyrosine.
- Partial acid hydrolysis leads to a mixture of peptides: peptide P1, consisting of arginine
and phenylalanine; P2 peptide consisting of leucine and valine; peptide P3 consisting
of phenylalanine, leucine and arginine. Deduce the structure of the hexapeptide.

1/ Procedure to be followed to determine the formula of the hexapeptide: Total acid hydrolysis
then electrophoresis and / or ion exchange chromatography.
2 / Structure of the hexapeptide: Ala-Tyr-Arg-Phe-Leu-Val

Exercise 7: sequencing a hexapeptide

For the following AA, write their form according the pH taking into account of the pKa

1) Under which form glycine (R = -H) will be at pH= 2, pH= 7 and pH= 11 knowing that:
a. pKaCOOH =2.34
b. pKaNH2= 9.6

2) Under which form aspartic acid (R = -CH2-COOH) will be at pH= 1.4, pH= 3 and pH= 7 et pH=11
knowing that:
a. pKaCOOH =2.1
b. pKaNH2= 9.8
c. pKR=3.9
Structural Biochemistry Tutorial

1. Carbohydrates
Exercise 1
Fill in the blanks to review monosaccharides
You can recognize a monosaccharide by its multiple (a) hydroxyl groups and its one (b)
carbonyl group, whose location determines whether the sugar is a(n) (c) aldose or aldehyde
sugar(carbonyl at the end o carbon skeleton) or a(n) (d) ketose or ketone sugar (carbonyl
within carbon skeleton).In aqueous solutions, most five- and six-carbon sugars form (e) rings
. The names for most sugars end in (f) -ose.

Aldose= aldehyde function always carried by C1

Ketose = ketone function always carried by C2

Exercise 2
Number the carbons in the following 2 oses. What is the name of those oses according to their
heterocycles.

Circle the atoms that will be removed by a dehydration reaction. Then draw the resulting
disaccharide with its 1-2 glycosidic linkage. What is the name of that bond ?

Exercise 3
1- Represent in linear structure two aldopentoses from the D serie, which are
enantiomers (Fischer representation)

or

2- Then represent in cyclic formula one of these sugars which you will name according to
its isomeric α form (Haworth representation).
1) Write formula as Fisher (aldehyde function top)
2) D serie
3) reaction C1 with C4
4) -OH in C1 down like α form
5) OH fischer right => down and OH left in Fisher => up
Structural Biochemistry Tutorial

3- What are the main substituents which can react with the hydroxyl group carried by the
pseudo-aldehyde of an ose ?

Exercise 5
Permethylation followed by acid hydrolysis of a non-reducing diholoside gives, after
chromatography, a single molecule identified as a 2,3,4,6 tetramethylated derivative of a
hexose.
The acid hydrolysis of this diholoside followed by oxidation with nitric acid (HNO3) gives a
diacid inactive on polarized light. Give the formula (s) for this diholoside.

Exercise 6
What are the D-aldoses whose reduction leads to polyalcohols identical to those which one
obtains
by reduction of D-fructose? Indicate their linear structure.
Exercise 7:
A carbohydrate analysis provided the following information:
- compound of molar mass: 342 g.mol-1
- non-reducing compound
- hydrolyzable
Methylation followed by acid hydrolysis identifies two different methylated oses:
- ose n ° 1 methylated in 2, 3, 4 and 6
- ose n ° 2 methylated in 1, 3, 4 and 6
Ose # 1 is derived by synthesis of Kiliani-Fisher from D-arabinose. We also know that ose # 1
is a
epimer of D-mannose.
Ose # 2 is a D isomer and has one less asymmetric carbon than ose # 1.
5-1- Write the formula of the 2 oses
5-2- Is the above information sufficient to write the expanded formula for this diholoside? Justify
your answer.
Structural Biochemistry Tutorial

B. Lipids

Exercise 1
What do you get when a glycerol molecule is esterified by:
1-1- three molecules of C17H33COOH.
1-2- the following three fatty acids:
 C15H31COOH
 C17H35COOH
 C17H31COOH
Write the formulas for the products obtained and name them.

Reminder: an FA (simple): Cn H2n O2

insaturated FA: Cn H2n -2i O2

Oleic acid

Trioleyl-glycerol

Palmitic acid C16:0

glycerol
Stearic acid C18/0
Linoleic acid C18:2 Δ 9,12

Generally, glycerol has a simple FA on C1 and insaturated FA on C2

Palmitic acid

linoleic acid
stearic acid

Name: 1-palmityl-2-linoleyl-3stearyl glycerol

Exercise 2
Among the following properties attributed to stearic acid, two are inaccurate, which ones?
a- has 18 carbon atoms.
True

b- fixes the iodine

True : definition of lipid

c- is insoluble in water
True : definition of lipid

d- gives with soda an amphiphilic compound soluble in water.

e- absorbs UV.
Structural Biochemistry Tutorial

For d and e: those 2 properties are specific to the double bonds (fluorescent molecules after
UV)

Exercise 3
We propose to analyze a mixture of glycerides in solution in water. By shaking this mixture in
the presence of benzene (no longer used as a carcinogen), an enrichment of the phase is
caused aqueous in compound A and of the organic phase in compound B.

Reminder: Glycerides = Glycerol + GA  Simple lipids: Triacylglycerol

Si + P and radical  complex lipids: glycerophospholipids

1- What conclusions can be drawn from this experience?

Compound A is more soluble in the aqueous phase
Compound B has better solubility in organic solvent: B is hydrophobic
 A is more polar than B because it stays in water.

2- Compound A is subjected to mild alkaline hydrolysis. We get 3 products: A1, A2 and g.

Analysis of the compounds A1 and A2 indicates the following chemical compositions:
A1 = C16O2H32 and A2 = C18O2H32
Compound A3 is polar and by acid hydrolysis gives a tri-alcohol A4 and an acid.
Name and formula of A.

Mild alkaline hydrolysis: hydrolysis of ester bonds (= saponification), release of FA in soaps

A  A1 + A2 + A3
A1 = C16O2H32: Palmitic acid C16: 0
A2 = C18O2H32: Linoleic acid C18: 2 9.12
A3  A4 + acid
Trialcool A4: glycerol (since we know that we are working on a glyceride)
Acid: H3PO4: It is therefore a complex lipid: glycerophospholipid
A is therefore a phosphatidic acid: 1-palmityl-2-linoleyl-phosphatidyl-glycerol

3- B is also subjected to alkaline hydrolysis. We obtain 4 products: B1, B2, B3 and B4. B4 is
identical to A4. B1, B2 and B3 have the same carbon content: 18 atoms per molecule. Through
gas chromatography, three peaks are obtained where B1 comes out first, B3 comes out last.
Name (s) and formulas of B.

Alkaline hydrolysis: B = B1 + B2 + B3 + B4
B4 is the same as A4: glycerol
B1, B2, B3= FA in C18

 B is a Triacylglycerol: Simple lipid

CPG gas chromatography

Separative method.
The separation of analytes is based on the difference in affinity of these compounds for the
mobile phase and for the stationary phase. The mixture to be analyzed is vaporized and then
Structural Biochemistry Tutorial

transported through a column (stationary phase). Elution is done using an inert gas (mobile
phase).

 separation according to:

- polarity
- molar mass
- temperature…

FA: + the number of C is low, + the retention time decreases

+ the number of double bond (DB) is high, + the retention time increases
So as the 3 FAs have the same number of Cs, they are distinguished by the number of DB:
B1 has the least DB and B3 has the most DB. B2 therefore has an intermediate DB number.
Different possibilities:

C1 C2 C3 name of FA
C18:0 C18:1 Δ9 C18:2 Δ9,12 1- stearyl -2-oleyl -3-linoleyl -glycerol
C18:0 C18:1 Δ 9 C18:3 Δ 9,12,15 1- stearyl -2-oleyl -3-linolenyl -glycerol
C18:0 C18:2 Δ 9,12 C18:3 Δ 9,12,15 1- stearyl -2-linoleyl -3-linolenyl -glycerol
C18:1 Δ 9 C18:2 Δ 9,12 C18:3 Δ 9,12,15 1- oleyl -2-linoleyl -3-linolenyl -glycerol

Exercise 4
A partition chromatography on silica gel (using an organic solvent) is subjected to mixture of
two lipids A and B:
A: CH2O-CO-R B: CH2O-CO-R

CHO-CO-R CHO-CO-R

CH2O-CO-R CH2O-PO2-O-CH2-CH2-N+ -(CH3)3

Which of these two lipids will migrate the furthest?

Partition chromato:
Thin layer chromatography based on the difference in solubility of analytes with solvents.
Stationary phase: paper containing a polar solvent (water)
Mobile phase: liquid organic solvent
 Compounds + soluble in the stationary aqueous phase move slowly: migrate a bit
 Compounds + soluble in the mobile organic phase move rapidly: migrate far

A: Triacylglycerol
B: Glycerophospholipid = Phosphatidyl-choline

B: the more polar (phosphate and N+), will be the more polar in the aqueous phase (=
stationary)

B will migrate little, the least far

A: the less polar, will have more affinity for the organic solvent (= mobile phase) and will be
entrained with this solvent A will migrate the furthest
Structural Biochemistry Tutorial

Exercise 5
A lipid is isolated from the nervous tissue. Three components are identified:
1- One is an amino alcohol unsaturated with 18 carbons.
Rq: in general, nervous tissue = sphingolipids
Sphingosine: unsaturated amino alcohol at 18C
2 -OH functions in C1 and C3
1 amine function in C2 (amide bond with FA)

Fixation of FA
1 DL trans between C4 and C5

2- The second is soluble in chloroform and benzene, but insoluble in water. It becomes soluble
in water after treatment with alcoholic potash KOH.
soluble in organic solvents, but insoluble in water: LIPID!
treatment with alcoholic potash: saponification = a soap is obtained so it is a FA (no details on
its nature)

3- The third is active on polarized light. It reduces the fehling liquor. With the acid nitric, it gives
an inactive compound on polarized light.
What is the structure of this lipid?
Reaction with other carbohydrates
Reduces Fehling solution
Inactive on polarised light after strong oxidation: symmetry axe in the molecule

Mucic acid from

D-galactose