UNIVERSITY OF ZULULAND

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

SEMESTER 1:
BIOMOLECULES AND ENZYMOLOGY (SBCH 211)
WATER and PROTEINS
INTRODUCTION
All animals, plants and microorganisms are composed of small units known as cells.
Different tissues contain different types of cells which are distinguished not only by their
different structures but their different metabolic activities. Biochemistry is the study of the
molecular basis of life:
a) Chemical constituents of a living cell
b) The chemical reactions taking place in the cell and
c) The energies resulting from the chemical reactions.
In this module, we shall look at the first aspect i.e. we shall consider the interplay between
the three dimensional structure of biomolecules and their functions.

There are two types of biomolecules namely micro and macro molecules. Macromolecules
by nature have molecular mass in excess of 10-4 and they are more abundant than the micro
molecules.
Macromolecules include proteins, carbohydrates, lipids, nucleic acids; water is often added to
this list.

Course outcome:
Students should be able to generally describe and explain the biomolecules in terms of
1. Their structure
2. Their chemical and physical properties
3. Their functions
4. Their structure and function relationship

Students should be able to generally describe and explain enzymes in terms of

5. Their names
6. The rate of catalysis
7. Factors that control enzymes actions
Queries
In case of any problem, students should feel free to contact the lecturer.

Study materials
Prescribed text books:
1. Garrett R.H. and Grisham C.M. Biochemistry
 2. Devlin, T.M..Textbook of Biochemistry with clinical correlations
3. Harvey A.R and Champe P.C. Lippincott’s illustrated Biochemistry
4. Any textbook of Biochemistry

Assessments
Criteria
1. A firm basis in understanding the course’s outcomes is essential
2. The learner should be able to critically analyse and apply the course’s outcome to
relevant case studies
3. The learner should be equipped with the necessary internet skills
4. The capacity and ability to assess and coordinate teamwork within a group is to be
established
 Functional Groups

In organic chemistry, functional groups (Table 1) are specific groups of atoms within
molecules that are responsible for the characteristic chemical reactions of those molecules.
The same functional group will undergo the same or similar chemical reaction(s) regardless
of the size of the molecule it is a part of.
Polarity of Organic Compounds

Principles of Polarity:

The greater the electronegativity difference between atoms in a bond, the more polar the
bond. Partial negative charges are found on the most electronegative atoms, the others are
partially positive. In general, the presence of an oxygen atom is more polar than a nitrogen
atom because oxygen is more electronegative than nitrogen.

The combination of carbons (C) and hydrogens (H) as in hydrocarbons or in the hydrocarbon
portion of a molecule with a functional group is always NON-POLAR.

Summary of Polarity
Polarity Ranking of the Functional Groups:

Amide > Acid > Alcohol > Ketone ~ Aldehyde > Amine > Ester > Ether > Alkane

1. WATER

Although not an organic biomolecule, water is the most ubiquitous component in the living
cells and accounts for 60-95% of their weights.
Water is required:
a) As a solvent – e.g. dissolving waste products for excretion
b) As a medium- enzymatic reactions require aqueous medium
c) For transporting substances
d) For regulating body temperature
e) In the production of body fluids- e.g. digestive enzymes
f) In maintaining the structure of macromolecules

These functions are attributed to the unique physical and chemical properties of water.

1. Water is a polar molecule- water molecule is V-shaped and is an electric dipole.

The chemical bonds in the water molecule are covalent bonds since the hydrogen atoms
combine with the oxygen atom in shared electron pairs. It is the unique distribution of the
electrons in the resulting chemical bond that causes the hydrogen atoms to bond with the
oxygen atom at a bond angle of 104.5o. This makes the water molecule electrically
asymmetric and producing an electric dipole (Figure 1).

0
H H
Figure 1: molecular structure of water

The oxygen atom exerts a relatively strong pull on the shared electron pair causing the
hydrogen atoms to become electropositive regions and the oxygen atom to become an
electronegative region. Because the positive and negative regions are not evenly distributed
around a centre point, permanent dipole moments are created. Thus the water molecule is
termed a polar molecule.
The polar nature of the water molecule causes it to become electrostatically attractive to other
water molecules as well as other ions in solution and contact surfaces with electrostatic sites.
The electropositive hydrogen atoms on the water molecule will be attracted to the
electronegative oxygen atoms of adjacent water molecules. This “bridging” phenomenon is
called hydrogen bonding.

NB: The polarity of the molecules determines the forces of attraction between the molecules
in the liquid state. The greater the forces of attraction the higher the boiling point or the
greater the polarity the higher the boiling point.

2. Water is highly cohesive- water molecules can be attracted to each other by

electrostatic forces (hydrogen bonds). At any time, in liquid water at room
temperature, each water molecule is believed to be hydrogen bonded to 3.4 other
water molecules.

Hydrogen bonds are not unique to water (See examples given). It is a common system in
biological molecules. They occur intermolecularly and intramolecularly.
A. C=O...H-N the type found in proteins and nucleic acids
B. C-OH...O=C a weak bond found in proteins
C. N-H...N the type found in DNA
Although the energy of hydrogen bond is rather small (4.5 ≈ 5 Kcal/mol) the presence of
several bonds acting cooperatively constitute a considerable stabilizing force.

3. Excellent solvent- the excellent solvent properties of water are a reflection of the
dipolar character of water. The attraction between the ionic components of the
molecule and the water dipoles is sufficient to overcome the attraction between the
ions themselves- water makes solvation shells around ions, e.g. when crystalline NaCl
is exposed to water the dipolar water is attracted to the Na+ and Cl- ions and pulls
them away to form hydrated Na+ and Cl- ions in solution (Figure 2).

Non-ionic polar compounds, e.g. sugars, simple alcohols, are soluble because of the
functional polar groups, e.g. OH, C=O, which readily hydrogen bonds with water molecule
dispersing the compounds among the water molecule.

Amphiphatic compounds- a compound with a hydrophilic polar head, and a hydrophobic

tail, e.g. salt of a fatty acid (potassium laurate) form micelles- aggregate of molecules with
polar carboxylic ions on the exterior and non-polar hydrocarbon chains in the interior, which
creates an internal hydrophobic environment.

Figure 2: Hydration of NaCl

4. Because of the hydrogen bonding, water differs remarkably in its properties from
other related liquids. Hydrogen bonding is responsible for most of the unusual
properties of water (high freezing and boiling points, high heat capacity, high
heats of fusion and evaporation, solvency, and high surface tension).
Dielectric constant (D)
Ions will exist if the solvent in which they are formed prevents their natural association- i.e. a
proper solvent will very well support the existence of oppositely charged particles by
minimizing the forces of attraction between them. In biological systems, charges are
separated by water, other molecules, or parts of molecules. Thus, the cellular medium shields
charges from each other.

The capacity of a system to insulate oppositely charged particles from mutual attraction is
reflected by the property called dielectric constant (D).
The relationship of D and the force of attraction (F) between two particles carrying opposite
charges (Q-, Q+) and are separated by a distance r is given by

F = Q- Q+
Dr2 (Coulomb’s law)

Note then that F will be reduced in a medium with large D.

Dielectric constant is high for a polar solvent and low for non-polar organic solvents (See
Table 3). Water has a large D and very capable of supporting the existence of an ionic
environment.

Table 3: Dielectric constants of some solvents

Solvent Dielectric constant (D)
water 80.4
methanol 33.6
Ethanol 24.3
Ammonia 17.3
Acetic acid 6.5
chloroform 4.81
Ethyl ether 4.34
Benzene 2.28
Carbon tetrachloride 2.24

ACID-BASE CONCEPT

A most useful definition of acids and bases in biochemistry is that proposed by Brönsted-
Lowry. An acid is a proton donor. A base is a proton acceptor.
Acid H+ + Base
e.g. CH3COOH H+ + CH3COO-
NH4+ H+ + NH3
HCl H+ + Cl-

The species formed by the ionization of an acid is its conjugate base. Conversely,
protonation of a base yields its conjugate acid.
There are two types of acids: a strong acid- has little affinity for its protons will therefore,
completely dissociate in water e.g. HCl, H2SO4.
A weak acid- has high affinity for its protons and will not readily dissociate in water e.g.
CH3COOH, H2CO3.

The selective dissociation of weak acids in water is of importance in biochemistry. Water is

not inert in its service as a solvent for biochemical reactions. In addition to its abilities as a
solvent, water is chemically reactive. It is a weak electrolyte that dissociates only slightly to
form hydronium (H3O+) and hydroxide (OH-) ions. For simplicity, we refer to the hydronium
ions as hydrogen ions (H+) and we write

H2O H+ + OH- (2H2O H3O+ + OH-)

The equilibrium constant, Keq of this dissociation is given by

Keq = [H+][OH-]
[H2O]

Because the concentration of H2O (55.5 M) is little changed by ionization we can write

Kw = [H+][OH-]

Where Kw is the ionic product or ionization constant of water. At 25oC, Kw = 1x10-14. Note
that the concentration of H+ and OH- are reciprocally related.
In pure water, [H+] = [OH-] = 1x10-7M.
pH
The pH measures how acidic or basic a substance is. The pH scale ranges from 0 to 14. The
pH of a solution is a measure of its hydrogen ion concentration [H+]. The pH scale is
logarithmic and as a result, each whole pH value below 7 is x10 more acidic than the next
higher value, eg. pH 4 is x10 more acidic than pH 5 and x100 more acidic than pH 6. The
same holds true for pH values above 7, each of which is x10 more basic than the next lower
value, e.g. pH 10 is x10 more basic than pH 9 and x100 more basic than pH 8.

The pH is defined as, pH = log 1/H = -log [H+], simply pH = -log [H+],

Also [OH-] can be expressed as pOH:

Kw = [H+][OH-]
1x10-14 = [H+][OH-]
-log (1x10-14) = -log [H+] –log [OH-]
14 = pH + pOH
... pH + pOH = 14

If given pH or pOH, to calc. [H+] or [OH-] we use

[H+] = 10-pH, [OH-] = 10-pOH

Effect of Acids and Bases on Water Equilibrium:

If an acid (H+) is added to the water, the equilibrium shifts to the left and the [OH-] decreases.

Water Equilibrium: H2O H+ + OH-

In any acid solution: [H+] > [OH-], [H+] > 10‐7 M, [OH‐] < 10‐7M

If a base (OH-) is added to water, the equilibrium shifts to left and the [H+] decreases.

In any basic solution: [H+] < [OH-], [H+] < 10‐7 M, [OH‐] > 10‐7M

NB: At 25oC, regardless of the increase in [H+] and [OH-], Kw = 10-14.

BOTH H+ and OH- ions are ALWAYS PRESENT in any solution. A solution is acidic if
the H+ ions are in excess. A solution is basic, if the OH- ions are in excess.

IONIZATION OF WEAK ACIDS

HA H+ + A-
The equilibrium constant, Ka (acid dissociation constant) for this ionization is
Ka = [H+][A-]
[HA]
The pKa of an acid is defined as, pKa = log 1/Ka = -log Ka, simply pKa = -log Ka

The pKa of an acid is the pH at which a weak acid is half dissociated.

Henderson-Hasselbalch equation defines the relationship between the pH and

concentrations of the conjugate acid and base.
pH= pKa + log[A-] or pH = pKa + log [proton acceptor]
[HA] [proton donor]

Thus, the pH of a solution can be calc. if the molar proportion of A-, HA and the pKa are
known.
Note that the equation only works for weak acids and bases.

Some facts about the equation

1. If pH = pKa, then [A-] = 1, log 1 = 0
[HA]

2. As the pH increases or decreases by 1 unit relative to the pKa, the ratio of the dissociated
form [A-] to the associated form [HA] of the acid changes by factor 10.
That is, if the pH of a solution is 6 and pKa is 7, the ratio of [A-] = 0.1
[HA]
If the pH of a solution is 5, the ratio will be 0.01
If the pH of a solution is 7, the ratio will be 1

3. If the pH< pKa, the ratio <1

If the pH= pKa, the ratio = 0
If the pH> pKa, the ratio > 1

Percentage ionization
A weak acid is 50% unprotonated (dissociated) at pH equals pKa (pH = pka)
90% unprotonated (dissociated) at 1 pH unit above its pKa (pH=pKa + 1)
99% unprotonated (dissociated) at 2 pH units above its pKa (pH=pKa + 2)

pKa of any dissociable group is a characteristic property of a molecule.

Note that protonation of bases also is dependent on the pH. Despite the differences between
acids and bases, pKa can also be used to quantify the relative strength of bases. Notice that
while the pKa values for acids are generally less than 7, pKa values for bases are usually
greater than 7. For instance, ethanolamine has a pKa of 9.5 and is thus half protonated when
the pH is 9.5.

An acid-base conjugate pair has an important property: it resists changes in the pH of the
solution i.e. it acts as a buffer.

Consider the addition of OH- to a solution of acetic acid (HA)

HA + OH- A- + H2O

A plot of the dependence of the pH of the solution on the amount of OH- added is called a
titration curve (Figure 4).

Equivalents hydroxide
Figure 4: Titration curve showing how neutralization of an acid by a base.
Upon addition of 0.5 equivalents of base, 1/2 of the acid will be converted to the conjugate
acid, such that [A–] = [HA], and pH = pKa. This condition will allow the largest
concentration of both species, and therefore the maximum buffering effect. The flatter
midsection of the curve corresponds to the buffering region with midpoint of that region
corresponding to the pKa value. The shape of this curve applies to any weak monoprotic
acid.

MONOPROTIC/DI-/POLYPROTIC ACIDS
Some acids have 1 dissociable proton. Such are called monoprotic acids. Those with 2 are
diprotic and those with 3 are triprotic. In general, any acid with more than one dissociable
proton is referred to as polyprotic acid. Such acids will therefore, have more than one pka
value. The number of pKa values will correspond to the number of dissociable protons e.g.
phosphoric acid (H3PO4) has 3 pKa values. See Table 2.

O O O O
|| || || ||
HO-P-OH pKa1= 2.12 HO-P-O- pKa2 = 7.21 HO-P-O- pKa3 = 12.3 -O-P-O-
| | | |
OH OH O- O-

H3PO4 H2PO4- + H+ HPO42- + H+ PO43- + H+

Note that H3PO4 is the conjugate acid of H2PO4-, which is the conjugate base of H3PO4 and
also the conjugate acid of HPO42-. In turn, is HPO42- is a conjugate base of H2PO4- and a
conjugate acid of PO43-. Thus any conjugate pair could be used in a buffering system-
depending on the pH of choice.

Table 2: Some biological acids and their pKa values

Acid pKa1 pKa2 pKa3
Formic HCOOH 3.75
Acetic H3CCOOH 4.76
Pyruvic H3CCOCOOH 2.50
Lactic H3CCHOHCOOH 3.86
Succinic HOOC(CH2)2COOH 4.21 5.63
Malic HOOCCH2CHOHCOOH 3.40 5.26
Carbonic H2CO3 6.35 10.3
Citric HOOCCH2COHCOOHCH2COOH 3.09 4.75 5.41
Phosphoric H3PO4 2.12 7.21 12.3

BUFFERS
Control of pH is an essential property of biological systems. The physiological pH of human

blood is 7.4. An increase or decrease of this pH is not compatible with life. The control of pH

in natural biochemical systems is accomplished by buffers.

A buffer is a solution that resists large changes in pH on the addition of small amounts of

another acid or a base.

e.g. acid added: CH3COO- + H+ CH3COOH

 base added: CH3COOH CH3COO- + H+
OH-
H2O
Most commonly the buffer solution consists of a mixture of a weak acid and its conjugate
base e.g. mixture of CH3COOH/NaCH3COO- .

Two factors determine the effectiveness or capacity of a buffer solution:

1. The molar concentration of the buffer components- the buffer capacity is directly
proportional to the concentration of the buffer components. The buffer concentration may be
defined as the sum of the concentration of the weak acid and its conjugate base. Thus, a 0.1
M of acetic acid buffer could contain 0.05 M acetic acid (CH3COOH) and 0.05 M sodium
acetate (NaCH3COO-) in 1L of water.
It could also contain 0.065 M CH3COOH and 0.035 M NaCH3COO- in 1L of water.

2. The ratio of the conjugate base to the concentration of the weak acid- the pH of an
equimolar (50/50) solution of a conjugate acid-base pair equals to its pKa value. Since such a
solution has maximum buffering effectiveness, it follows that this is also the pH at which the
solution buffers best. The values of log1/9 = (-0.95) and log 9/1 = (0.95) correspond roughly
to -1 and +1. Hence, the range of pH where the buffering is effective corresponds to pKa
± 1 and is maximum at the pKa.

IONIC STRENGTH (interaction of macroions in solution)

Large polyelectrolytes (compounds that carry multiple of only one kind of charge- cationic or
anionic, e.g. DNA) or polyampholytes (compounds with many acidic and basic groups) are
together classified as macroions. Depending on the pH of the solution they may carry
substantial net charge, and the electrostatic forces of attraction or repulsion between such
particles play a major role in determining their behaviour in solutions, e.g. negatively charged
DNA molecules in water will repel each other. Thus nucleic acids tend to remain apart in
solution. A protein will be in solution at a pH above or below their isoelectric point (pI)-
they carry net + or – charges. At pI, proteins carry no net charge (i.e + = -), but the
negatively charged portion will be attracted to the positively charged portion i.e self
association at pI.
If positively charged molecules are in solution with negatively charged molecules, the
electrostatic attraction between the molecules makes them tend to attract or associate with
each other, e.g. DNA associates with proteins (histones) to form chromatin found in
chromosomes. Such associations will be influenced or modified by the presence of small ions
(counterions), e.g. Na+, Cl-, Mg2+. Each macroion will collect about its self a counterion
atmosphere enriched in oppositely charged small ions (Figure 5). Macroions are therefore
screened from each other. The amount of counterions in solution is measured as the ionic
strength (I).This phenomenon is the Debey-Hückel theory which is quantitatively expressed
as
r=K
I½
Where r = effective radius of the counterion atmosphere, K = constant, depends on dielectric
constant of the medium and temperature, I = ionic strength, which is a function of
concentration.
Debye-Huckel theory attempts to measure the radius across which macroions can interact
with each other
The larger 'r' is, the longer the distance two macroions can affect each other. Thus, as the
ionic strength (concentration) increases, macroions must be closer together before they can
interact with each other, due to the shielding or shell of the counterions.

Figure 5: The influence of small ions on macroion interactions

The ionic strength (μ), I = ½∑CiZi2
Where the sum is taken over all small ions in solution. For each ion type C i = molar
concentration, Zi = stoichiometric charge.

Note that the ionic strength is a solution property related to both the concentration of the ions
in solution and the ionic nature i.e. the charge of the ions. Cellular activity is a function of
both e.g. for a 1:1 electrolyte like NaCl we have ZNa+ = +1 and ZCl- = -1 and since CNa+ =
CNaCl, we found that INaCl = CNaCl. Thus, for a 1:1 electrolyte, ionic strength equals salt
molarity. This is obviously not the true of divalent, trivalent etc ions. For such
electrolytes I > C.

For cellular studies, ionic strength of 0.15 M (physiological ionic strength) is used. Ionic
strength of solutions is important in: Plasmolysis and Salting in or salting out.

Plasmolysis
When whole cells are placed in a solution that does not contain an ionic concentration at least
approximately equivalent to the ionic concentration of the intracellular fluid (protoplasm),
one of the two things (both undesirable) will occur. When the ionic strength of the
extracellular fluid is much less than that of the intracellular, water will enter the cell, causing
it to swell and ultimately burst. Alternatively, if the ionic strength of the extracellular fluid is
much greater than that of the intracellular fluid, water will leave the cell, causing it to shrink
and collapse.

Tutorials
1. Rank the following compounds by decreasing polarity. i.e. more polar – most non-polar.
Methanol; ethanoic acid, hexane, ethanamide, aminomethane, formaldehyde [Hint: functional
group determines the polarity of a compound]

2. Calculate the [H+] for a solution with: (a) pH = 3.4; (b) pH = 8.2

3. What is the pH of: (a) 100mM NaOH; (b) 0.1 M H2SO4, 25mM Ca(OH)2?

4. Differentiate between monoprotic and polyprotic acids.

5. Answer these statements with TRUE or FALSE; if false explain why.

a) Henderson-Hasselbalch equation is described by:
a) pH = log Ka + log [salt]
[acid]

b) –pH = log Ka + log[salt]

[acid]
c) pH = pKa + log[acid]
[salt]

d) pKa = pH – log[salt]
[acid]

e) pH = pKa + log[proton acceptor]

[proton donor]

b) Water
i. Possesses a high heat capacity
ii. Dissociates completely into H+ and OH-
iii. Readily forms hydrogen bonds with polar and charged molecules
iv. Displays high surface tension
v. Serves as an excellent solvent for all substances.

6. At what pH will 90% of a weak acid be protonated; at what pH will 90% be unprotonated,
99% unprotonated? Use the Henderson-Hasselbalch equation to show your answers.

7. What would be the pH and concentration of the resulting buffer solution when:
a) 7.58 g Na2CO3 and 2.40 g NaHCO3 are dissolved in 500 ml of water? (pKa1 = 6.1;
pKa2 =10.3).
b) 3.48 g of K2HPO4 and 2.72 g KH2PO4 are dissolved in 250 ml of water? (pKa1 = 2.12;
pKa2 =7.2; 12.3).
8. A Biochemistry student wanted to prepare a phosphate buffer (0.1M, pH 7.4) and was
supplied with the following conjugate acid-base pairs: H2CO3/HCO3-, pKa = 6.1; H2PO4-
/HPO4-2, pKa= 7.2; HPO4-2/PO4-3, pKa= 12.3.
Choose the correct conjugate acid-base pair to prepare the buffer and calculate the
concentration of each component.

9. A 0.2 M acetate buffer solution contains 0.1M acetic acid and 0.1M sodium acetate.
Calculate the pH after addition of 4 ml of 0.025M HCl to 10 ml of the buffer.
(pKa = 4.76).

10. What is the difference between heat of fusion and specific heat capacity?
11. What is the chemical explanation for the high heat of fusion of water (80 Kcal mol-1)?
What is the biological significance of the large specific heat capacity of water (1.0 cal g-1
degree-1)?
 2. PROTEINS
Proteins exhibit functional versatility:
1. Enzymes- proteins with catalytic activities e.g. amylase, trypsin, Ribonuclease

2. Transport proteins- carry small molecules and ions e.g haemoglobin, transferrin

3. Nutrient & storage proteins- stored for growth of the embryo e.g gliadin (wheat),
ovalbumin (egg), casein (milk), ferritin (of animal tissue store iron)

4. Contractile & motile proteins- help cell to contract, change shape and move about, e.g.
actin & myosin (muscle), tubulin (flagella & cilia).

5. Structural proteins- give strength, protection, support to biological structures, e.g. collagen
(skin), keratin, fibrion (silk).

6. Defence proteins- defend and protect organisms against invasion by other species e.g.
antibodies (immunoglobulins), fibrinogen & thrombin (loss of blood), venoms (snakes,
insects), toxic plant and microbial proteins.

7. Regulatory proteins- control physiological activities, e.g. hormones

8. Stress protein- mediate physiological responses to stress e.g. Heat shock proteins

9. Other proteins- functions are too exotic for classification, e.g. monellin- has intense sweet
taste, “antifreeze protein” in Antartic fish.

All proteins are polymers made from α-amino acids (monomers)

H
2 1
R Cα COOH

NH2

General structure of α-amino acid

Table 1: Amino acids commonly found in proteins

Essential Non-essential
Isoleucine (Ile, I) Alanine (Ala, A)
Leucine (Leu, K) Glycine (Gly, G)
Lysine (Lys, S) Cysteine (Cys, C)
Methionine (Met, M) Aspartate (Asp, D)
Phenylalanine (Phe, F) Glutamate (Glu, E)
Threonine (Thr, T) Serine (Ser, S)
Tryptophan (Trp, W) Tyrosine (Tyr, Y)
Valine (Val, V) Asparagine (Asn, N)
Arginine (Arg, R) Glutamine (Gln, Q)
Histidine (His, H) Proline (Pro, P)

There are 20 amino acids commonly found in proteins (Table 1). The different amino acids
are distinguished by their different side chains (R-groups).
R-groups vary in structure, size, and in their tendency to interact with water. Thus amino
acids are classified on the basis of polarity of their R-groups in water (pH 7):

1. Non-polar or hydrophobic- R- group is hydrocarbon in nature and thus hydrophobic

Amino acids with aliphatic side chain (R-grp)

Glycine (Gly) R≡H
Alanine (Ala) R ≡ -CH3

CH3 More
Valine (Val) R ≡ -CH hydrophobic
CH3
CH3
Leucine (Leu) R ≡ -CH2CH Note- the more
hydrophobic
CH3 amino acids
prefer an
Isoleucine (Ile) R ≡ -CHCH2CH3 environment
within a protein
CH3 molecule

Proline (Pro, P) shares many properties with this group, although it is a cyclic amino acid
(imino acid)
 CH 2
H 2C CH2

H 2N C COO-
H

2. Polar but uncharged R-groups- R-group is more soluble in water because they contain
functional groups that can hydrogen bond with water:

Amino acids with –OH or S-containing side chain (R-grp)

Serine (Ser, S) R ≡ -CH2OH

Cysteine (Cys, C) R ≡ -CH2SH

Threonine (Thr, T) R ≡ -CHCH3

OH
Methionine (Met, M) R ≡ -CH2-CH2-S-CH3

These are more hydrophilic- because of their

weakly polar R grps- than the aliphatic.
Met is fairly hydrophobic.

NOTE-1. Cys R grp can ionize at high pH

SH S-

CH2 CH 2
pKa = 8.3 +NH + H+
+NH C COO - 3 C COO -
3

H H

2. Oxidation can occur between pairs of Cys R grps to form a disulfide bond- important in
structural proteins also in 3D
 H H
+
+NH
3 C COO- NH3 C COO-

CH 2 CH2

SH S
oxidation +
reduction S + 2H + 2e-
SH
CH2 CH 2
+NH + NH COO-
3 C COO- 3 C

H H

Cysteine Cystine (derived amino acid)

Aromatic amino acids

R≡
OH

NH

CH2 CH2 CH2

Phenylalanine Tyrosine Tryptophan

(Phe, F) (Tyr, Y) (Trp, W)

NOTE- Phe, Val, Leu, Ile are the most hydrophobic amino acids. Tyr and Trp have
hydrophilic properties.

Tyr can ionize at high pH

OH O-

pKa = 10.1 H+
+
CH2 CH2
+NH COO- +
NH3 C COO -
3 C

H H

Because of the conjugated ring they (Tyr and Trp) strongly absorb light at 280 nm (used in
the analytical detection of protein)
A280= ԐCL (Beer-Lambert’s law)
Where, Ԑ ≡ molar absorbance coefficient
C≡ concentration
L ≡ length of light path (in cm)
A ≡ absorbance

A= log10Io/I; Io ≡ intensity of incident light

I ≡ intensity of transmitted light

3. Basic amino acids (diamino-monocarboxylic acids)

R≡
NH2

C NH2+

NH3+ NH

N
NH+ (CH2)3 (CH2)2

CH2 CH2 CH2

Histidine (His, H) Lysine (Lys, K) Arginine (Arg, R)

These amino acids carry basic groups in their side chains. They are strongly polar and are
therefore usually found in the exterior of proteins.

4. Acidic amino acids (monoamino-dicarboxylic acids) and their amides (diamino-

dicarboxylic acids).
R≡
O
O-
O
O-
C
C CH2

CH2 CH2

Aspartate (Asp, D) Glutamate (Glu, E)

 O
NH2
O C
NH2
C CH2

CH2 CH2

Asparagine (Asn, N) Glutamine (Gln, Q)

Acidic amino acids exist as negatively charged molecules at physiological pH as aspartate

and glutamate. They are very polar.Their amides (Asn, Gln) are polar uncharged amino acids.

NOTE
 All amino acids, except glycine, exhibit stereoisomerism.
 Almost all biologically active amino acids are in the L-forms (There is a limited
biological use of D-amino acids, e.g. in cell walls and some antibiotics).

COOH COOH

NH 2 H 2N C H
H C

R R

D-isomer L-isomer

Modified amino acids

Modified forms of some amino acids are found in certain proteins, e.g.
-Collagen contains 4-hydroxyproline and 5-hydroxylysine
H H

C CH3 +H N-CH -CH-CH -CH -C-COO-

HO 3 2 2 2

3HC COO-
H2C OH NH3+

+NH
2

4-OH proline 5-OH lysine

-Some enzymes contain phosphoserine

-N-formylmethionine initiates protein synthesis in E. coli
-Oncogenes (cancer cells) contain phosphotyrosine
-Myosin contains N-methyl lysine
Acid-Base behaviour of amino acids

Amino acids are amphoteric compounds i.e. they contain both acidic and basic groups.

As proton donor (acid)

H3+N CHRCOO- H+ + H2NCHRCOO-

As proton acceptor (base)

H+ + H3+NCHRCOO- +
NH3CHRCOOH

Their ionization state of amino acids depends on the pH-depending on the nature (pH) of the
solution in which they are, they are capable of bearing a net charge. Thus, as a base is added
to an amino acid, the two groups will be titrated.
H H H
OH- OH-
H3+N C COOH + C COO- H2N C COO-
H+ H3 N H+
R pKa1 R pKa2 R

Predominant form Predominant form Predominant form

at pH 1 at pH 7 at pH 11
net charge (+1) (0) (-1)
Zwitterion

At physiological pH, amino acids are predominantly dipolar ions (Zwitterions).

NOTE- amino acids have at least 2 pKa values- that of the α-COOH and that of the α-+NH3
group.

The pKa values of some amino acids (25oC)

AA α-COOH α-NH3+ side chain (R)
Ala 2.3 9.6
Gly 2.4 9.8
Phe 1.8 9.1
Ser 2.1 9.2
Val 2.3 9.6
Asp 2.0 10.0 3.9
Glu 2.2 9.7 4.3
His 1.8 9.2 6.0
Cys 1.8 10.8 8.3
Tyr 2.2 9.1 10.9
Lys 2.2 9.2 10.8
Arg 1.8 9.0 12.5
Isoelectrict point/pH or isotonic point (pI)

The pH at which the average net charge is zero is called the Isoelectric point/pH (pI).
pI value of an amino acid is calculated as:

pI = pKa1 + pKa2
2

NOTE
Amino acid is: +ve if pH<pI
± (neutral) if pH=pI
-ve if pH>pI

The pI value is a constant for a compound at a particular ionic strength.

Because the net charge on zwitterion molecule is zero (at pI), it will not migrate (move) in an
electrical field.

Reactions of Amino Acids

Amino acids react with a variety of reagents that may be used to investigate the function of
specific side chains.

a) Reaction with alcohols

O O
+ OHR 2 R 1-C + H 2O
R 1C
OH OR2
Acid alcohol ester

b) Amino acids could be

(i) decarboxylated to their respective amines

H3N+ CH2 + CO2

H3N+ CH COOH decarboxylase
R R

(ii) deaminated or transaminated to yield keto acids

O
H3N + CH COOH R C COOH NH3
+
R
c) Amino groups of amino acids react with ketones or aldehydes to form Schiff base (-
HC=N-). Schiff base is important in enzymatic reactions.

d) Amino group of amino acids can react with ninhydrin to give a blue (purple) colour
(absorbs light at 540 nm).

This is used in the qualitative and quantitative test for amino acids. The reaction is also +ve
for primary amines and ammonia, but CO2 is not liberated. Imino acids give a yellow colour
(absorbs light at 440 nm).

e) Amino groups of amino acids also reacts with 1-Flouro-2,4-dinitrobenzene (FDNB)-

Sanger reagent- in a mild alkaline solution to give a yellow 2,4-dinitrophenyl derivative.
H

R C COO-
F
HN
H NO2
C COO- + NO 2
R

H 2N
HF
NO2
1 Flouro-2,4-dinitrobenzene NO 2
2,4-dinitrophenyl amino acid (yellow)

This is used in the quantitative determination of amino acids and in the identification of the
terminal amino acid of a polypeptide.

f) The α-carboxyl group of an amino acid can react with the α-amino group o another amino
acid to form a peptide bond (also called an amide bond).
 H O H O H O H O

H3N+ C C O- + H3N+ C C O- H3N+ C C N C C O-

+ H2O
R2 R1 H R2
R1

Peptide bond
The equilibrium of the reaction lies on the side of hydrolysis rather than synthesis.

The peptide unit is rigid and planar

O H
1.3 2
Å C Å
1.5 C R
R C N 1.46 Å

H H

A C-N single bond= 1.49 Å; A C=N double bond = 1.27Å.

The C- bond of a peptide = 1.32 Å, therefore it has partial double bond character.
O O-
C N C N+

H H

The C- and N-termini could be linked to give a cyclic oligopeptide.

Many proteins have the N-termini blocked, e.g. by

O R O
O R O
C N C C
N C

O- H CH 3 H
N-formyl group N-acetyl group
And a few have the C-termini modified to amine
R O
N C C
NH2
H H
C-terminal amide

Reactions of the peptide bond

1. Peptide hydrolysis can be catalysed in several ways:
a) non-enzymatic:- by heating in a strong mineral acid (6M HCl) or base

b) by reacting with cyanogen bromide (N≡C-Br).

N≡C-Br specifically cleaves the peptide bond to the carboxyl side of Met in any polypeptide.

c. Proteolytic enzymes catalyse the hydrolysis of specific bonds in a peptide, e.g.

R1
O R2 O

N C C N C C
N-terminal C-terminal
H
H H

(i) Trypsin R1= Lys, Arg

(ii) Chymotrypsin R1= Tyr, Phe, Leu, Ile, Val, Trp, (His at high pH).
(iii) Pepsin R1= Phe, Leu, many others
(iv) Thrombin R1= Arg
(v) Papain R1= Arg, Lys, Phe-x (C=O side of residue next to Phe)
(vi) Bromelain R1= Lys, Ala, Tyr, Gly
(vii) Carboxypeptidase A R1= C-terminal amino acid.
2. Peptides react with biuret reagent to give a pink colour. Amino acids do not give a
positive result. This reagent is used to identify and estimate proteins in solution.

C O
C O
HN
NH

R1 CH
R3 CH
Cu2+
C O
C O

HN
NH
R2 CH R4 CH

Charges carried by polypeptide

COOH

CH 2
CH 3
CH 2 H H
O
CH N HC
C N COOH
H 3N + C H 2C N C
HC

O Gly H Ala O
Glu (CH 2) 4

H 3N +
Lys
pKa1 2.2 2.4 2.3 2.2
pKa2 9.7 9.8 9.6 9.2
pKa3 4.3 - - 10.8

NOTE- every amino acid, peptide and protein has an isoelectric point (pI). The pH at which it
lies depends on the relative numbers and kinds of acidic and basic groups in the molecule.

Protein is +ve if pH<pI

-ve if pH>pI

Isoelectric point (pI) is the pH at which a protein carries no net charge. Below the isoelectric
point proteins carry a net positive charge, above it a net negative charge. Due to a
predominance of weakly acid residues in almost all proteins, they are nearly all negatively
charged at neutral pH. The isoelectric point is of significance in protein purification because
it is the pH at which solubility is often minimal and at which mobility in an electro focusing
system is zero (and therefore the point at which the protein will accumulate).

PROTEINS
Proteins are not just polypeptides. They are polypeptides of defined sequence. Every protein
has a defined order (number & sequence) of amino acid residues referred to as the (1)
primary structure (1o) of the protein.
2. Secondary structure (2o) - refers to the specific geometrical arrangement of the
polypeptide chain along one axis. It consists of (i) α-helix, (ii) parallel or anti-parallel β-
pleated sheets.

(i). An α-helix is stabilised by H-bonds- since peptide bond is rigid and planar, the
polypeptide chain has only 2 degrees of freedom (Φ, φ). And since the same values recur
over long stretches of chain- i.e each residue turn to form the same Ramachandran angle. As
a result polypeptide fall into a natural right-handed α-helix with the –NH group of each
amino acid residue hydrogen bonded to the –C=O of the 4th following residue.

(ii). β-pleated sheets depend on intermolecular (interchain) H-bonding. They are suitable for
creating fiber-like molecules.

3. Tertiary structure (3o) - is the complete functional form assumed by the polypeptide
chain. This is the result of the overall effect of many intramolecular forces.
Due to the α-helical nature of the polypeptide chain, and the interplay of forces:
(a) electrostatic interactions
(b) hydrogen bonding
(c) hydrophobic interaction of non-polar side chains
(d) dipole-dipole interaction
(e) disulfide linkage- the only covalent bond in the 3o structure-
among the various R-groups, the chain folds into a 3-D structure referred to as the tertiary
structure.

4. Quaternary structure (4o) – concerns interactions by which 2 or more polypeptide chains

associate in a specific manner to form biologically active protein. Such proteins are said to be
oligomeric- each subunit has its own tertiary structure. The forces available for creating 4o
structure are the same as those that create 2o and 3o structures.

Classification of proteins
In addition to classifying proteins according to their functions, they could be classified based
on (1) gross structure as:

a) Fibrous proteins- are insoluble in water. They are composed of polypeptides which are
often cross-linked by various types of bonding to yield tough and water insoluble structural
proteins, e.g. collagen, silk, hair.

b) Globular proteins- are water soluble and spheroidal, compact structures. They are
biologically active proteins in living systems, e.g. enzymes, antibodies.

(2). Chemical composition as:

a) Simple proteins- contain only amino acids, e.g. albumin
b) Conjugate proteins- consist of amino acids and prosthetic group e.g. nucleoprotein
lipoprotein
phosphoprotein
metalloproteins
glycoproteins

Properties of proteins- All the properties of polypeptides

Proteins are very labile to extremes of pH, temperature, organic solvents, ions of heavy
metals and certain solutes. Under these conditions proteins loose their 4o, 3o, and 2o structures
thus their biological activities.
NOTE that 1o structure is not hydrolyzed (peptide bond is not hydrolyzed) in these
conditions.
This change in the 3D structure of a protein molecule without the hydrolysis of peptide bond
is known as denaturation of proteins.
-enzymes loose activity, globular proteins become insoluble and random coiled, e.g. egg
coagulate on heating.
How denaturation occurs at levels of protein structure

1. Primary structure is not disrupted by denaturation

2. In secondary structure denaturation, proteins lose all regular repeating patterns such as
alpha-helices and beta-pleated sheets, and adopt a random coil configuration.

3. Tertiary structure denaturation involves the disruption of:

 Covalent interactions between amino acid side-chains (such as disulfide

bridges between cysteine groups)
 Noncovalent dipole-dipole interactions between polar amino acid side-chains
(and the surrounding solvent)
 Van der Waals (induced dipole) interactions between nonpolar amino acid
side-chains.

4. In quaternary structure denaturation, protein sub-units are dissociated and/or the spatial
arrangement of protein subunits is disrupted.

Loss of function

Most biological proteins lose their biological function when denatured. For example,
enzymes lose their activity, because the substrates can no longer bind to the active site.

Reversibility and irreversibility

In some proteins denaturation is reversible (the proteins can regain their native state when the
denaturing influence is removed).

 Heat dehydrates proteins, disrupt bonds (notably H-bonds) - with gentle heating and
slow cooling the process is reversible.
 8 M urea or guanidine-HCl- disrupts non-covalent interaction
 β-mercaptoethanol- reduce disulfide bonds to yield cysteinyl residues
 Ammonium sulphate- used for salting in-salting out of proteins. It disrupts protein-
water, protein-protein interactions.
  Heavy metal ions- because proteins carry +ve or –ve charges, they can form insoluble
salts with metal cations or anions.
 pH- extreme pHs change net charge of proteins and thus disrupt the electrostatic
interactions. Proteins are least soluble in any solvent when pH=pI (at pI, net charge =
0; ... electrostatic repulsion is minimal between protein molecules).

Salting in or salting out- (Effects of ionic strength or pH on protein solubility)

As the ionic strength increases, macroion (i.e. protein) solubility at first increases and this is
referred to as salting in. But beyond a certain point, the solubility begins to decrease and this
is referred to as salting out (Figure 1).

Figure 1: Effect of ionic strength on solubility of macroion (protein)

At low ionic strength, the ionisable groups of the protein become surrounded by counterions
which prevent interaction between the ionisable groups. Thus, the protein-protein interactions
are decreased and the solubility is increased (salting in). Alternatively, at high ionic strength,
so much water becomes bound by the counterions that not enough remains to properly
hydrate the protein. Thus, protein-protein interactions exceed protein-water interactions, and
the solubility decreases (salting out).
Note- because of the different amino acids composition and consequent different structures of
proteins, different proteins have different behaviours in solution.

At a pH<pI, protein is +vely charged and electrostatic repulsion will keep the protein in
solution. Similarly, -vely charged proteins (at pH>pI) will be kept in solution. Thus, protein
will be least soluble if pH=pI and protein molecules will aggregate and precipitate (Figure
2).
Figure 2: Effect of ionic strength on solubility of a protein

If the ionic strength is increased, the counterions atmosphere shrinks about the charged
regions, and the attractive interaction between +ve or –ve groups are effectively screened.
Thus, solubility increases, even at isoelectric point (pI)- salting in.
Raising the salt concentration to very high levels brings in another effect- salting out.

AMINO ACID ANALYSIS OF PROTEINS

1. Determination of the amino acid composition of a protein
 Hydrolysis of the protein to its constituent amino acids
 Separation of the amino acids in the mixture
 Quantification of the individual amino acids

The protein is hydrolyzed to constituent amino acids by heating it at 110oC in 6M HCl for 24
hrs (or 18-36 hrs), in sealed tube under vacuum to avoid oxidation of the oxygen sensitive
groups. This treatment cleaves the peptide bonds, and releases the individual amino acids,
which can be separated by cation-exchange chromatography- the separation is based on (1)
charge- since it is negatively charged, acidic amino acids will pass first while basic ones are
retained. The pH of the eluting buffer is then increased to facilitate separation. (2) The
hydrophobic nature of the resin used will hold up more hydrophobic amino acids.

NOTE-side chain amides of Asn and Gln are hydrolyzed to Asp and Glu and free NH3; they
are included within Asp and Glu content in the analysis.
The separated amino acids could be detected and quantified by reacting them with ninhydrin,
dansyl chloride or similar flourophoric reagents.

2. Determination of the amino acid sequence of a protein

Amino acids are commonly sequenced by:

N-termini
a) Sanger Reagent (FBNB)
The reagent reacts with the N-terminal amino group of a polypeptide. If the polypeptide is
hydrolyzed and separated (paper chromatography, gel electrophoresis) then the N-terminal
residue would be identified as DNP derivative.
NO 2 NO2 NO2

H 2N

R2 CH
NO 2 NO2 NO2 C O
F NH NH
O-
+
R1 CH H 2O R1 CH
NH 2 +
C O C O +
R1 CH H2N
O-
O HF HN
C
R2
R3 CH
CH
HN
C O C O
R2 CH
HN O-
C O
HN R3 CH

R3 CH C O

C O

b) Dansyl chloride
This compound also reacts with the N-terminal amino group at high pH. It has an advantage
that it is intensely fluorescent and therefore could be used for low concentrations (1 nM).
c) Edman’s reagent (Edman degradation)
This reagent has the advantage over the other 2 reagents in that the polypeptide is not
destroyed by hydrolysis. Only the N-terminal amino acid is cleaved. Thus when used
sequentially the complete sequence of the protein can be elucidated.
O O
H
CH C N CH C N C S
N C S + H 2N O O
H Step 1 C N CH C
R1 R2 HN CH
Phenylisothiocyanate N-terminus of peptide chain R1 H R2
Phenylthiocarbomyl derivative of chain
2H+
Step 2
H+
N C S H O
N C S
NH Step 3 H3+N CH C
O C
CH HN+ C O
R2
R1 CH
Peptide chain shortened by 1 unit
Phenylthiohydantoin (PTH) derivative of R1
R1

The compound phenylisothiocyanate reacts with the N-terminal amino group to yield a
phenylthiocarbomyl derivative of the peptide (step 1). This derivative is then treated with a
strong anhydrous acid, which results in cleavage of the peptide bond between residues 1 and
2 (step 2). The derivative of the N-terminal residue then rearranges to yield a
phenylthiohyndantoin (PTH) derivative of the amino acid (3). NOTE- the N-terminal amino
acid has been marked, and the rest of the polypeptide has not been destroyed- it has only been
reduced by one residue. The whole process can be repeated over and over again for the whole
polypeptide chain to be read.

C-termini
a) Hydrazinolysis
The reaction leads to the cleavage, with modification of every residue except the C-terminal
one, which can be identified.
 O O
CH COO -
H 3+N CH C NH CH C NH
Rn-1 Rn
R1
C-terminal
H2N-NH 2
hydrazine

O
HN CH C H 3+N CH COO-
NHNH 2
R1 + + Rn
O
C-terminal residue
NH CH C
NHNH2
Rn-1

b) Lithium borohydrate
This compound reduces the COOH to an alcohol (α-amino alcohol). If the peptide chain is
then hydrolyzed, all the other amino acids will be present as free amino acids and the amino
alcohol will be identified.

-------NH CH COOH -------NH CH CH2OH

LiBH4
R R

c) Carboxypeptidase
This proteolytic enzyme catalysis the sequential hydrolysis of a polypeptide chain starting
from the C-terminal.
O O
NH CH COO-
HN CH C NH CH C
R n-1 Rn
R n-2
C-terminal
H 2O

O
O
HN CH C NH CH C + H 3+N CH COO -
O-
Rn-1 Rn
Rn-2

peptide shortened by 1 residue C-terminal residue

How to sequence a protein

It is difficult to sequence longer polypeptides (>50 amino acid residues long). It is therefore
proper to cleave polypeptides at specific sites to get smaller fragments (≤50 residues),
determine the amino acid composition and sequence of each polypeptide, and then by
overlapping, the sequence of the whole protein could be obtained.
To break disulfide bonds, and thus separate 2 chains (e.g insulin) the protein is treated with
performic acid or β-mercaptoethanol.
O O
CH C N CH C
N O
CH 2 CH2
H HC O H
OH
S SO 3-
performic acid
S SO3-

CH2 CH 2 O
O
CH C N CH C
N

H H

Specific cleavage of the protein can be achieved by chemical or enzymatic methods.

Chemical cleavage
Chemical cleavage side
Cyanogen bromide carboxyl side of Met
o-Iodobenzoate carboxyl side of Try
Hydroxylamine Asn-Gly bond
2-Nitro-5-thiocyanobenzoate amino side of Cys

Enzymatic cleavage
Enzyme cleavage side
Trypsin carboxyl side of lys or Arg residue
Clostripain carboxyl side of Arg residue
Staphylococcal protease carboxyl side of Asp or Glu res
SEQUENCING A PROTEIN

Suppose the amino acid analysis gives the following composition:

(Ala, Arg, Cys, 2Glx, Gly, Ile, 2Lys, 2Met, Ser, Trp, Tyr, Val)
Glx indicates both Glu & Gln and Asx indicates Asp & Asn are present. The ( ) indicates a
composition of unknown sequence.

Suppose the N-terminal has been determined to be Ala, and the C-terminal is Gly.
:. H3+N-Ala-(Arg, Cys, 2Glx, Ile, 2Ile, 2Lys, 2Met, Ser, Trp, Tyr, Val)-Gly-COO-
Suppose we treated the peptide with trypsin and obtain 4 fragments and Edman degradation
gives the following sequence of each fragment: (Trypsin cleaves to give Arg/Lys as C-
terminal residues)

T1: H3+N-Ala-Gln-Glu-Lys-COO-
T2: H3+N-Tyr-Met-Ser-Met-Ile-Arg-COO-
T3: H3+N-Val-Cys-Lys-COO-
T4: H3+N-Trp-Gly-COO-

Since T1 has the same N-terminal as original, it means T1 is N-terminal fragment. And since
T4 has no Arg/Lys as C-terminal, it follows that T4 is the C-terminal fragment.
Suppose we then treat the whole protein with CNBr and obtain 3 fragments and Edman
degradation gives the following sequence (CNBr cleaves Met as C-terminal):

CB1: H3+N-Ala-Gln—Glu-Lys-Tyr-Met-COO-
CB2: H3+N-Ser-Met-COO-
CB3: H3+N-Ile-Arg-Val-Cys-Lys-Trp-Gly-COO-

The sequences of the protein can be established by overlapping:

T1: H3+N-Ala-Gln-Glu-Lys-COO-
CB1: H3+N-Ala-Gln-Glu-Lys-Tyr-Met
T2 : Tyr-Met-Ser-Met-Ile-Arg
CB2: Ser-Met
CB3: Ile-Arg-Val-Cys-Lys-Trp-Gly-COO-
T3 : Val-Cys-Lys
T4 : Trp-Gly-COO-
H3+N-Ala-Gln-Glu-Lys-Tyr-Met- Ser-Met-Ile-Arg-Val-Cys-Lys-Trp-Gly-COO-
 Tutorials

1. Rank the following amino acids by increasing polarity. i.e. more non-polar – most polar.
ser ; glu ; asp ; lys ; ala ; gln

2. Match the levels of protein structures in the left column with the appropriate descriptions
in the right column.

a) Primary (1) Association of protein subunits

b) Secondary (2) Linear amino acid sequence
c) Tertiary (3) Spatial arrangement of amino acids that are near
each other in linear sequence
d) Quarternary (4) Necessary for the catalytic activity of an enzyme

3. A hexapeptide has Asp as the N-terminal amino acid, Glu as the C-terminal amino acid and
has the sequence: Asp-Lys-Ala-Glu-Arg-Glu.

a) Write the structure of the hexapeptide.

b) What net electrical charge will the hexapeptide carry at pH 7.0 and 12.5. Give brief
reasons for your answer (consult books/notes for pKa values of the amino acids).
c) Indicate what are the products of the reaction of the peptide and 6 M HCl, 100oC, 21
hours?
d) Products of c (above) plus ninhydrin.
e) Peptide plus dansyl chloride followed by 6 M HCl, 100oC, 21 hrs.
f) Peptide plus trypsin.
g) Staphylococcal protease

4. Write down the reaction of alanine treated with a deaminating reagent

5. Ethanol (70%) rather 90% alcohol is used as a disinfectant. Explain?

6. Poly-L-leucine in an organic solvent such as dioxane is α-helix, whereas poly-L-isoleucine

is not. Why do these amino acids with the same number and kinds of atoms have different
helix-forming tendencies?

7. A solution of a protein whose sequence includes three tryptophan residues, no tyrosine

residues, and no phenylalanine residues has an absorbance of 0.1 at 280 nm in a cell with a
path length of 1cm (= 3400 M-1 cm-1). Estimate the concentration (M) of the protein.

8. Answer these statements with TRUE or FALSE; if false explain why.

a) In a globular protein, these amino acids would probably be positioned near the surface
i. Leucine
ii. Aspartate
 iii. Phenyl alanine
iv. Tyrosine
v. Arginine
vi. Valine

b) The peptide bond:

i. Results from condensation of any R-group or side chain of different amino acids with
each other
ii. Results from the condensation reaction between the alpha-carboxyl group of one
amino acid with the alpha-amino group of a second amino acid
iii. Results from the condensation reaction between the alpha-carboxyl group of one
amino acid with the alpha-carboxyl group of a second amino acid
iv. Results from the condensation reaction between the alpha-carboxyl group of one
amino acid and ammonia
v. Results from the condensation reaction between the alpha-carboxyl group of one
amino acid and ammonium ion

c) The following statements are about amino acids

i. The most hydrophobic amino acids have side chains with either a negative or positive
charge at physiological pH
ii. The most hydrophobic amino acids have side chains that are polar but uncharged at
physiological pH
iii. The most hydrophilic amino acids have no oxygen or nitrogen in their side chains
iv. The most hydrophilic amino acids have side chains that are polar but uncharged at
physiological pH
v. The most hydrophobic amino acids have side chains that are unpolar and uncharged at
physiological pH

d) The sequencing of a peptide can involve any of the following steps

i. Hydrolysis of the peptide.

ii. Partial hydrolysis by proteases.
iii. Using Sanger's reagent to identify N-terminal amino acids.
iv. Identify the C-terminal amino acid by using a carboxypeptidase.
v. The use of paper chromatography to separate amino acids.
vi. Use Edman degradation to analyze the peptide.
9. An oligopeptide was analyzed. Given the data below, choose the correct sequence from the
options given below.
 Amino acid analysis revealed the composition Asp, Asn, 2Glu, Gly, Lys, 2Met, Phe,2Pro
 Carboxypeptidase digestion gave the results shown in this plot

 Sanger N-terminal analysis afforded the DNP derivative of glutamic acid

 Treatment of the peptide with cyanogen bromide gave three fragments. Sanger N-
terminal analysis of these three fragments gave the DNP derivatives of Glu, Pro, and
Asp.
 Cleavage of the oligopeptide with trypsin gave two fragments. Sanger analysis of
these two fragments both gave the DNP derivative of Glu
 Cleavage of the oligopeptide with chymotrypsin gave two fragments. Sanger analysis
of these two fragments gave DNP derivatives of Glu and Lys.

A) Glu-Pro-Met-Pro-Gly-Lys-Phe-Met-Asp-Asn-Glu
B) Glu-Met-Pro-Pro-Phe-Lys-Gly-Asp-Met-Asn-Glu
C) Gly-Asp-Met-Asn-Glu-Pro-Pro-Met-Phe-Lys-Glu
D) Glu-Met-Pro-Pro-Phe-Lys-Glu-Asn-Met-Asp-Gly
E) Glu-Pro-Met-Pro-Lys-Phe-Glu-Asn-Met-Asp-Gly
 pKa Values of Common Amino Acids
 -COOH  -NH + pK
Amino Acid 3 a R group pKa
pKa
Alanine 2.4 9.7
Arginine 2.2 9.0 12.5
Asparagine 2.0 8.8
Aspartic acid 2.1 9.8 3.9
Cysteine 1.7 10.8 8.3
Glutamic acid 2.2 9.7 4.3
Glutamine 2.2 9.1
Glycine 2.3 9.6
Histidine 1.8 9.2 6.0
Isoleucine 2.4 9.7
Leucine 2.4 9.6
Lysine 2.2 9.0 10.5
Methionine 2.3 9.2
Phenylalanine 1.8 9.1
Proline 2.1 10.6
Serine 2.2 9.2 ~ 13.00
Threonine 2.6 10.4 ~ 13.00
Tryptophan 2.4 9.4
Tyrosine 2.2 9.1 10.1
Valine 2.3 9.6

NOTE: THIS IS JUST BRIEF INFORMATION. DO NOT ONLY RELY ON THIS

INFORMATION, FIND MORE OTHER STUDY MATERIALS