Exercises

BIOCHEMISTRY
Unit 1 and 2

1
What you need to know:
- Structure and functions of water.
- How to calculate pH of a strong acid, base and a weak acid.
- Buffer, function of buffer.

2
pH calculation:
pH =  log[H+]

For a basic solution:

pOH =  log[OH]  pH = 14 – pOH

Henderson-Hasselbalch equation:
[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝐻𝐴]
where: A–: conjugated base or deprotonated form
HA: acid or protonated form
3
1. Solubility of ethanol in water
Explain why ethanol (CH3CH2OH) is more soluble in water than is ethane
(CH3CH3).

Ethanol Ethane

4
2. Calculate the pH of a Molar Concentrations
What is the pH of a solution containing 0.12 mol/L of NH4Cl and 0.03 mol/L
of NaOH (pKa of NH4+/NH3 is 9.25)?

Acid Conjugated base

NH4+ + OH-  NH3 + H2O

0,12 0,03

=> [NH3] = [OH-] = 0.03 (mol/L)

 Remaining of [NH4+]: 0.12 – 0.03 = 0.09 (mol/L)
[𝐴− ]
 𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝐻𝐴]
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3. Identifying the Conjugate Base
Which is the conjugate base in each of the pairs below?
(a) RCOOH, RCOO−
(b) RNH2, RNH3+
(c) H2PO4−, H3PO4
(d) HPO42- , H2PO4-
(e) PO43- , HPO42-
(f) H2CO3, HCO3-
(g) CO32−, HCO3−

6
4. Use of Molar Concentrations to Calculate pH
What is the pH of a solution that contains 0.20 M sodium acetate and 0.60 M
acetic acid (pKa = 4.76)?

CH3COOH CH3COO- + H+
protonated form deprotonated form

CH3COONa CH3COO- + Na+

[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝐻𝐴]

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5. Acidity of Gastric HCl
In a hospital laboratory, a 10.0 mL sample of gastric juice, obtained several hours
after a meal, was titrated with 0.1 M NaOH to neutrality; 7.2 mL of NaOH was
required. The patient’s stomach contained no ingested food or drink; thus assume
that no buffers were present. What was the pH of the gastric juice?

HCl + NaOH  NaCl + H2O

n HCl = nNaOH = CM (NaOH) . V NaOH = 0.1 x 7.2.10-3 = 0.72. 10-3 mol
=> n HCl = 0.72. 10-3 mol
=> CM (HCl) = (0.72. 10-3) / (10. 10-3) = 0.072 M
HCl  H+ + Cl-
[H+] = [HCl] = 0.072 M
 pH = - log [H+]
8
6. Preparation of an Acetate Buffer
Calculate the concentrations of acetic acid (pKa = 4.76) and sodium acetate
necessary to prepare a 0.2 M buffer solution at pH 5.0.

[CH3COO- ]
pH = pKa + log
[CH3COOH]

[CH3COO- ]
log = pH - pKa
[CH3COOH]
[CH3COO- ]
[CH3COO- ] and [CH3COOH]
[CH3COOH]
9
7. Calculation of the pH of a Strong Acid or Base
(a) Write out the acid dissociation reaction for hydrochloric acid. (b) Calculate
the pH of a solution of 6.0 × 10–5 M HCl. (c) Write out the acid dissociation
reaction for sodium hydroxide. (d) Calculate the pH of a solution of 5.0 × 10–3
M NaOH
HCl  H+ + Cl
pH =  log[H+]

NaOH  Na+ + OH

pOH =  log[OH-]
pH = 14 – pOH

10
8. pH and Drug Absorption
Aspirin is a weak acid with a pKa of 3.5.

It is absorbed into the blood through the cells lining the stomach and the small
intestine. Absorption requires passage through the plasma membrane, the rate
of which is determined by the polarity of the molecule: charged and highly
polar molecules pass slowly, whereas neutral hydrophobic ones pass rapidly.
The pH of the stomach contents is about 1.5, and the pH of the contents of the
small intestine is about 6. Is more aspirin absorbed into the bloodstream from
the stomach or from the small intestine? Clearly justify your choice.
11
 8. pH and Drug Absorption
[𝐴− ]
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝐻𝐴]
Pass rapidly
[𝐴− ]
[𝐻𝐴]
acid/protonated form (HA)

[HA]

Pass slowly
–

Conjugated base/deprotonated form (A– ) 12

Part I: Amino acids
What you need to know:
- Structure and properties of the amino acids
- Amino acid classification
- Optical properties of the amino acids
- Isoelectric point (pI)

13
1. Separation of amino acid by paper electrophoresis
A mixture of aspartic acid (pI = 2.77), cysteine (pI = 5.07), and lysine (pI = 9.74)
is spotted in the center of the paper, the two ends of the paper are dipped in a
buffer of pH 6.5, high voltage is applied. Determine the migration of these
amino acids in an electric field.

14
pI application
Paper electrophoresis

15
https://khonggiansinhhoc.com/dien-di-la-gi/
1. Separation of amino acid by paper electrophoresis
 pH = pI, amino acid is uncharged  immobilized.
 pH < pI, amino acid has positive charge (+)  amino acid moves to the Cathode (-).
 pH > pI, amino acid has negative charge (-)  amino acid moves to the Anode (+).

pI value of some amino acids:

Aspartate (Asp): 2,77
Cysteine (Cys): 5,07
Lysine (Lys): 9,74

What are the movements of these amino

acid above?
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1. Separation of amino acid by paper electrophoresis
Anode (+) pH 6.5 Cathode (-)

Lys +
pI=9,74
pI=2,77
- Asp

- - H +

- Cys
pI=5,07
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2. Relationship between the Titration Curve
and the Acid-Base Properties of Glycine.
A 100 mL solution of 0.1 M glycine at
pH 1.72 was titrated with 2 M NaOH solution.
The pH was monitored and the results were
plotted as shown in the following graph.
The key points in the titration are designated
I to V.
For each of the statements (a) to (o), identify
the appropriate key point in the titration and
justify your choice.

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2. The titration curve of Glycine

Net charge: +1 0 –1

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 Net charge: +1 0 –1
a) Glycine is present predominantly as
the species +H3NCH2COOH pK2

HOOC-CH2-NH2 + H+  HOOC-CH2-NH3+ pI

pK1

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 Net charge: +1 0 –1
b) The average net charge of glycine is
+ 1/2. pK2

HOOC-CH2-NH2 + H+  HOOC-CH2-NH3+ pI

HOOC-CH2-NH3+  -OOC-CH2-NH3+ + H+
pK1

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 Net charge: +1 0 –1
c) Half of the amino groups are ionized.
pK2

-OOC-CH -NH + + OH-  -OOC-CH -NH + H O

2 3 2 2 2
pI

pK1

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 Net charge: +1 0 –1
d) The pH is equal to the pKa
of the carboxyl group. pK2

HOOC-CH2-NH3+  -OOC-CH2-NH3+ + H+ pI

[HOOC-CH2-NH3+]
pH = pKa – log pK1
[-OOC-CH2-NH3+]

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 Net charge: +1 0 –1
e) The pH is equal to the pKa
pK2
of the protonated amino group.

-OOC-CH -NH + + OH-  -OOC-CH -NH + H O pI

2 3 2 2 2

[-OOC-CH2-NH3+]
pH = pKa – log pK1
[-OOC-CH2-NH2]

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 Net charge: +1 0 –1
f) Glycine has its maximum buffering
capacity. pK2

pK1±1 and pK2±1 pI

pK1

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 Net charge: +1 0 –1
g) The average net charge of glycine is zero.
pK2

pI

pK1

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 Net charge: +1 0 –1
h) The carboxyl group has been completely
titrated (first equivalence point). pK2

HOOC-CH2-NH3+ + OH-  -OOC-CH2-NH3+ + H2O pI

pK1

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 Net charge: +1 0 –1
i) Glycine is completely titrated (second
equivalence point). pK2

-OOC-CH -NH + +
2 3 OH-  -OOC-CH2-NH2 + H2O pI

pK1

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 Net charge: +1 0 –1
j) The predominant species is
+H NCH COO pK2
3 2
.
pI

pK1

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 Net charge: +1 0 –1
k) The average net charge of glycine is 1.
pK2
-OOC-CH -NH + + OH-  -OOC-CH -NH + H O
2 3 2 2 2
pI

pK1

30
 Net charge: +1 0 –1
l) Glycine is present predominantly as a
50:50 mixture of pK2
+H NCH COOH and +H NCH COO
3 2 3 2

pI
Midpoint
-COO-  -COOH
pK1

31
 Net charge: +1 0 –1
m) This is the isoelectric point.
pK2

pI

pK1

32
 Net charge: +1 0 –1
n) This is the end of the titration.
pK2

H2N-CH2-COO-
pI

pK1

33
 Net charge: +1 0 –1
o) These are the worst pH regions for
buffering power. pK2

pI

pK1

34
3. Absolute Configuration of Citrulline The citrulline isolated from
watermelons has the structure shown below. Is it a D- or L-amino acid?
Explain.

35
How to identify the chiral form of an amino
acid (D, L form)
• Draw the molecule as a Fischer projection with the carboxylic acid
group on top and side chain on the bottom. (The amino group will not
be at the top or bottom.)
• If the amine group is located on the right side of the carbon chain, the
compound is D. If the amine group is on the left side, the molecule is L.
• If you wish to draw the enantiomer of a given amino acid, simply draw
its mirror image.

36
 R

Citrulline 37
4. pKa of Amino acid The amino acid histidine has three ionizable groups,
with pKa values of 1.8, 6.0, and 9.2.
(a) Which pKa corresponds to the histidine side chain?
(b) (b) In a solution at pH 5.4, what percentage of the histidine side chains
will carry a positive charge?.

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 Acid conjugated base

Net charge: +2 +1 0 –1

[conjugated base]
pH = pKa + log
[acid]
5.4

 [acid] = 4 [conjugated base]

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Part II: Protein
What you need to know:
• Know the definitions of primary, secondary, tertiary, and quaternary
structures or protein
• Understand the differences between globular and fibrous proteins
• Understand the type of bonds that stabilize these structures
• List the roles proteins play in the human body.
• Understand that certain changes in protein sequence can change the
structure and function of protein.

40
5. Peptides and proteins Lys (Mr = 146) residues make up 10.5% of the
weight of ribonuclease. The ribonuclease molecule contains 10 Lys residues.
Calculate the molecular weight of ribonuclease.

Mr = 18 Mr of Lys residue = 146 – 18 = 128

10 Lys residues = 10.5% of the weight of ribonuclease

1 Lys residue = 1.05%

Mr of ribonuclease =? 41
6. Protein structure Name factors (bonds or other forces) that contribute to
stabilizing the native structure of a protein, and describe one condition or
reagent that interferes with each type of stabilizing force.

42
6. Protein structure

SDS, Urea

Heat, SDS

Mercaptoethanol, dithiothreitol

Changes in pH

43
7. Protein denaturation Why do crab/shrimp/lobster turn red when cooked?

Astaxanthin-crustacyanin Heat
Astaxanthin (red)
complex

https://pubs.rsc.org/en/Content/ArticleLanding/2015/CP/C4CP06124A#!divAbstract
44
8. Subunit Composition of a Protein A protein has a molecular mass of 400 kDa
when measured by gel filtration. When subjected to gel electrophoresis in the
presence of sodium dodecyl sulfate (SDS), the protein gives three bands with
molecular masses of 180, 160, and 60 kDa. When electrophoresis is carried out
in the presence of SDS and dithiothreitol (DTT), three bands are again formed,
this time with molecular masses of 160, 90, and 60 kDa. Determine the subunit
composition of the protein.

45
8. Subunit Composition of a Protein

• What is the role of SDS?

• What is the role of DTT?
>> How many subunits in the protein?
46
Part III: Enzyme
What you need to know:
- What are enzymes?
- Functions of the enzyme.
- Mechanism of the enzyme activity.
- Factors that effect the activity of the enzyme.

47
How do enzymes work?
Many enzymes can bind to more than one substrate molecules
• Substrates bind to the enzyme
• The enzyme brings molecules close together so that they can react
with one another
• The bonds inside the substrate are stretched slightly out of position,
which weakens the bonds
• The reaction takes place and the product is released from the
enzyme.

https://www.youtube.com/watch?v=yk14dOOvwMk
48
8. Enzyme actvity why do apple slides turn brown?

• https://www.youtube.com/watch?v=5GR4gNn0Qrc
49
Brown color

50
9. Protection of an Enzyme against Denaturation by Heat
When enzyme solutions are heated, there is a progressive loss of catalytic
activity over time due to denaturation of the enzyme.
A solution of the enzyme hexokinase incubated at 45oC lost 50% of its
activity in 12 min,
but when incubated at 45oC in the presence of a very large concentration of
one of its substrates, it lost only 3% of its activity in 12 min.
Suggest why thermal denaturation of hexokinase was retarded in the
presence of one of its substrates.

51
9. Protection of an Enzyme against Denaturation by Heat

Which form is more stable?

52
10. Clinical Application of Differential Enzyme Inhibition Human blood serum
contains class of enzymes known as acid phosphatases, which hydrolyze
biological phosphate esters under slightly acidic conditions (pH 5.0):

Acid phosphatases are produced by erythrocytes, the liver, kidney, spleen, and
prostate gland. The enzyme of the prostate gland is clinically important because
its increased activity in the blood can be an indication of prostate cancer. The
phosphatase from the prostate gland is strongly inhibited by tartrate ion, but
acid phosphatases from other tissues are not. How can this information be
used to develop a specific procedure for measuring the activity of the acid
phosphatase of the prostate gland in human blood serum? 53
10. Clinical Application of Differential Enzyme Inhibition

Tartrate ion

Phosphatase of prostate gland

Phosphatase of other tissues

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9. Enzyme inhibitor: How is ciprofloxacin used to treat the bacterial infections?

Ciprofloxacin
(Fluoroquinolone)

Inhibits DNA gyrase and topoisomerase

IV  prevents DNA replication  kill
bacteria.

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https://www.youtube.com/watch?v=IkKZ_gxAOXI
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