Unit 4 Amino Acids

4
R
+
H3 N CH C O UNIT
–
O

AMINO ACIDS

Structure
4.1 Introduction Zwitterionic Structure
Expected Learning Outcomes Isoelectric Point
4.2 Amino Acids: The Building Titration Curve
Blocks of Proteins 4.5 Separation of Amino Acids
Structure and Representation of Paper Chromatography
Amino Acids
Paper Electrophoresis
Configuration of Amino Acids
4.6 Summary
Classification of Amino Acids
4.7 Terminal questions
4.4 Physical Properties Amino
Acids 4.8 Answers

4.1 INTRODUCTION
In the units of the first block of this course you were introduced to the important
features of a typical cell present in all living organisms. You also studied about
carbohydrates, the important energy providing biomolecules and their
significance in the functioning of the living system. In the second block you will
be introduced to another important and complex class of biomolecules, known
as proteins. These molecules are central to all biological process and
constitute half the dry mass of a cell. These are made up of α-amino acids as
the monomers and are dealt in the present unit of this block.

We would give you a broad view of how amino acid molecules join into
polypeptide chains, which in turn form the primary, secondary, tertiary and the
quaternary structures of proteins. Thus, you will study the structure,
classification and properties of the amino acids. You would also study how
these properties help us in the separation of amino acids by paper
chromatography and paper electrophoresis. The next unit deals with peptides
formed by the combination of amino acids.

Expected Learning Outcomes

After studying this unit, you should be able to:

 describe the structure of amino acids as the monomeric units of proteins, 81

Block 2 Amino Acids, Peptides and Proteins

 classify amino acids on the basis of their structure,

 draw and explain the zwitterionic structure of amino acids,

 define isoelectric point and explain the titration curve for amino acids,
and

 describe the process of separation of amino acids by paper

chromatography and paper electrophoresis.

4.2 AMINO ACIDS: THE BUILDING BLOCKS OF

PROTEINS
Proteins are significant biomolecules that are essential for performing a
Amino acids are the number of functions in the cells. Similar to polysaccharides, proteins are also
main components of polymeric in nature. The monomeric units of proteins are amino acids. When
protein molecules and proteins are completely hydrolysed, the twenty L- α-amino acids (L notation for
free amino acids are
rarely distributed in amino acids will be explained later) are produced. Let us try to recollect the
nature. basic structure of amino acids and learn to represent these in the form of
The dietary proteins symbols.
are hydrolysed during
digestion to release 4.2.1 Structure and Representation of Amino Acids
amino acids which
are then reassembled The amino acids have a common structural motif. You would note that amino
into proteins. acids have an amino group at one end and a carboxyl group at the other end,
both of which are attached to the α-carbon atom i.e. the carbon atom next to
the carboxyl group. Those amino acids which have their amino group attached
to this α-carbon are known as the α-amino acids. The α-carbon also carries a
hydrogen atom. The fourth group on this carbon is known as the side chain
and is denoted as R. The simplest of α-amino acids i.e., glycine has a
hydrogen atom as the side chain. In other α-amino acids, this side chain
consists of long carbon chain and more complex chemical groups. All the
α-amino acids differ from each other in the nature of their side chains only.
Thus, the general structure of an α-amino acid can be represented as given in
Fig. 4.1 (a). The structure of glycine as an example is given in Fig. 4.1 (b).

R-group or
side chain
R H
carbon hydrogen carbon hydrogen
+ +
H 3N CH C O H 3N CH C O
– –
amino group O amino group O
carboxyl group carboxyl group

(a) (b)

Fig. 4.1: a) General structure of an amino acid; b) Structure of glycine.

The biologically significant amino acids are principally α-amino acids.

However, depending upon the position of amino group on the carbon chain
attached to the carboxyl carbon, these are called ,  and  amino acids.
The amino acids which have amino group attached to the carbon atom
82 adjacent to the carboxyl group are - amino acids or 2-amino acids. In amino
Unit 4 Amino Acids

acids with longer alkyl chains, the amino group can be placed in different
Position isomers:
positions with respect to carboxyl groups giving rise to,  etc. amino acids. are the isomers that
For example, amino group in amino butanoic acid can be placed in three differ in the position of
positions viz.,,  and  which gives rise to three position isomers of this a functional
group/multiple
amino acid. The structures of the three isomers are given below. bond/substituent in
the same carbon
  chain.

4 3 2 1 4 3 2 1 4 3 2 1
H3C CH2 CH COOH H3C CH CH2 COOH H2C CH2 CH2 COOH
NH2 NH2 H2N

- Aminobutanoic acid - Aminobutanoic acid - Aminobutanoic acid

2- Aminobutanoic acid 3- Aminobutanoic acid 4- Aminobutanoic acid

You can observe from the general structure of amino acids that these differ
from one another with respect to the R groups i.e., the side chains bonded to
the α-carbon atom. Most living organisms contain twenty α-amino acids which
constitute the vast variety of proteins. These amino acids are listed along with
their names, structures and representation in symbol forms in Table 4.1. You
may note the different groups under which the amino acids are categorised.
You will be able to appreciate this when you study about the classification of
amino acids in subsection 4.2.3. You would also observe that the symbol
representation is using three and one letter. Let us now understand the
representation of amino acids to have a better clarity.

Representation of amino acids

The amino acid is commonly represented by a three letter symbol or one

letter symbol. The three letter symbol is derived from the trivial name of the
amino acid and includes the first three letters of this name. The amino acids
asparagine, glutamine, isoleucine and tryptophan are the exceptions in this
representation. Thus, the three letter symbols are written as one capital letter
followed by two lowercase letters. For example, glycine is represented as Gly
and not as GLY or gly. This applies even when we have to write the symbol in
the middle of a sentence.

In addition to the above, one letter symbol is also in use. The one letter
symbol for eleven of the twenty coded amino acids is the first letter of their full
name and for the rest nine distinct letters have been assigned so as to avoid
any confusion. It is important to know that the symbols or we may say
abbreviated forms are commonly used in representing long sequences of
peptides or proteins where it would be difficult to write the full sequence of
constituent amino acids with their full names.

If we observe the arrangement of atoms or groups around the α-carbon in

amino acids as observed in the case of carbohydrates, we would be able to
appreciate the stereochemistry of amino acids. You would recall from courses
on stereochemistry that the spatial arrangement of atoms or groups in a
molecule is called the configuration of the molecule. Let us describe the
configuration at the α-carbon atom in amino acids after you have attempted
SAQ 1.
83
Block 2 Amino Acids, Peptides and Proteins

Table 4.1: Structures of amino acids with three and one letter names
Nonpolar R Group
CH3 CH3
CH3 CH3 CH CH2
H CH3 CH3 CH CH2 CH3 CH
H2N C COOH H2 N C COOH H2N C COOH H2N C COOH H2N C COOH
H H H H H

Glycine (Gly, G) Alanine (Ala, A) *Valine (Val, V) *Leucine (Leu, L) *Isoleucine (IIe, I)
CH 3
NH
S
CH2
CH 2
H2C CH2
CH2 CH 2 CH2
HN C COOH
H2N C COOH H 2N C COOH H2N C COOH
H H H H
Proline (Pro, P) *Phenylalanine (Phe, F) *Methionine (Met, M) *Tryptophan (Trp, W)

Polar, Uncharged R Group

OH SH CH3
CH2 CH2 CH OH

H 2N C COOH H2N C COOH H2N C COOH

H H H
Serine (Ser, S) Cysteine (Cys, C) *Threonine (Thr, T)

O NH 2 OH
C
O NH2
C CH 2
CH2 CH 2 CH2
H2N C COOH H2N C COOH H2N C COOH
H
H H
Asparagine (Asn, N) Glutamine (Gln, Q) Tyrosine (Tyr, Y)

Polar (Negative Charge) Group Polar (Positive Charge) R Group

NH 2
NH 2
O OH C NH
CH 2
C HN NH
O OH CH 2
CH2 N CH 2
C
CH 2
CH2 CH 2
CH 2 CH2
CH 2 CH 2
H2 N C COOH H 2N C COOH H2N C COOH
H2N C COOH H 2N C COOH
H H H
H H

Aspartic acid (Asp, D) Glutamic acid (Glu, E) *Histidine (his, H) *Arginine (Arg, R) *Lysine (Lys, K)
* These are essential amino acids
84
Unit 4 Amino Acids

SAQ 1
Fill in the blanks with appropriate words in the following sentences:

a) The monomeric units of proteins are _________.

b) The amino acids which have amino group attached to the carbon atom
adjacent to the carboxyl group are called________________.

c) Gln is the three letter abbreviation for ------------------ .

4.2.2 Configuration of Amino Acids

You would have observed that all the amino acids discussed above, with the
exception of gycine, have a chiral or an asymmetric carbon atom, i.e., they
have a carbon atom bonded to four different atoms or groups tetrahedrally. In
case of amino acids under discussion, it is the α-carbon which is a chiral
centre. Therefore, except glycine, all the amino acids containing chiral carbon
exhibit optical activity and are designated as (+) and (-) isomers respectively.
As in the case of carbohydrates, the configuration at the chiral carbon is
designated by the letters D and L. You may recall from Unit 2 that these
notations are used after a comparison with the configuration of
glyceraldehyde. The D and L glyceraldehydes are the mirror images as shown
below.

CHO CHO

HO H H OH

CH2OH CH2OH

L-Glyceraldehyde, (-) Rotation D- Glyceraldehyde, (+) Rotation

In the case of α-amino acids, the configuration at the α-carbon is compared to

the configuration of D and L glyceraldehydes, as shown below:

CHO COOH COOH COOH

HO C H H2 N C H H2 N C H H2 N C H

CH2OH R CH3 CH2OH

L-Glyceraldehyde L-Amino acid L-Alanine L-Serine

CHO COOH COOH COOH

H C OH H C NH2 H C H2N H C H2 N

CH2OH R CH3 CH2OH

D-Glyceradehyde D-Amino acid D-Alanine D-Serine
85
Block 2 Amino Acids, Peptides and Proteins

As depicted above, in L-amino acids the –NH2 group is on the left and in the
D form the –NH2 group in on the right side. All amino acids found in proteins
have the L-configuration and are designated either as L (+) or L (-), depending
on whether they rotate the plane polarised light to the right or the left. Although
D-amino acids do occur in nature, they are never present in proteins (in
comparison to this fact you have read that generally carbohydrates occur in
D-configuration).

The amino acids are grouped into different types according to the nature of
their side chains. Before studying the way amino acids are classified try to
answer the following question.

SAQ 2
Complete the following sentence with appropriate answer.
a) Glycine is not optically active because …………………………
b) In L amino acids the amino group is on the ………….
c) All the amino acids present in proteins have ……….configuration.
d) The configuration of amino acids is compared to the configuration
of........... to designate D or L.

4.2.3 Classification of Amino Acids

It was mentioned earlier and from Table 4.1, you can observe that the side
chains present in amino acids vary in their nature and polarity. These two
factors form the basis of the classification of amino acids. We can understand
the classification by either looking at the nature of R group or considering the
polarity of R group. Let us see how.

(A) Classification of amino acids based on the nature of R groups

 Aliphatic amino acids: The aliphatic amino acids have aliphatic chain as
R group. Five amino acids; glycine, alanine, valine, leucine and isoleucine
are included in this group.

 Hydroxy amino acids: The hydroxyl amino acids have hydroxyl group in
their R group and include serine, tyrosine and threonine.

 Sulphur containing amino acids: These contain sulphur in their

R-group. This class includes methionine and cysteine.

 Acidic amino acids: These are amino acids containing an additional

-COOH group in the side chain e.g., aspartic acid, glutamic acid. Thus
these are dicarboxylic acids.

 Basic amino acids: The basic amino acids contain additional basic -NH2
groups or a heterocyclic ring. Arginine, lysine and histidine fall into this
group.

 Aromatic amino acids: In this class aromatic groups are present as the
side chain group. The examples are phenylalanine, tyrosine and
86 tryptophan.
Unit 4 Amino Acids

 Imino amino acid: This amino acid is different from others in that there is
an imino group in place of amino group. The only example in this category
is proline.

(B) Classification of amino acids based on the polarity of R groups

You may again have a look at Table 4.1. You would observe the amino acids
placed into different classes on the basis of polarity. These are:

 Nonpolar R group: The nonpolar group has generally a nonpolar

aliphatic or a non-polar aromatic side chain. Examples are Glycine,
Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan,
Methionine, Proline

 Polar uncharged R group: The uncharged polar groups have side

chains containing hydroxyl, amide or sulphydryl groups. Examples are
Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glutamine

 Polar (Negatively charged) group: The charged polar group can be

acidic when the side chain contains a carboxyl group. Examples are
Aspartic acid and Glutamic acid.

 Polar (positively charged) R group: The charged polar group can be

basic when the side chain contains a basic group. Examples are Lysine,
Arginine, Histidine

Though tyrosine and tryptophan have nonpolar side chains, they can also be
put in the uncharged polar group. Their feeble polarity arises from the
presence of –OH group in tyrosine side chain and –NH group in the tryptophan
side chain. Polar side chains, whether charged or uncharged, have a strong
tendency to interact with polar molecules like water, which is the normal
medium in which most of the proteins function. Nonpolar side chains, on the
other hand, avoid or repel water molecules and tend to seek a nonpolar
environment. This difference in interaction of polar and nonpolar side chains
with water is of great significance for the folding of a linear protein chain into
diverse structures, which are characteristic of different functions in the
organism.

(C) Essential and Nonessential amino acids

There is yet other way amino acids are classified and that is on the basis of
their source in the living system. Our body can synthesise only ten of the
amino acids listed in Table 4.1 as is indicated by an asterisk. The rest are not
synthesized in humans but are synthesized in either plants or bacteria and
must be supplied by our diet. Such amino acids which cannot be synthesised
by humans are known as essential amino acids. The essential amino acids
are leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan,
threonine, valine and histidine. Proteins that contain all the ten essential amino
acids are called adequate proteins. Milk and animal proteins are an excellent
source of all amino acids, whereas plant proteins lack one or more of the
essential amino acids. However, the protein in soybeans is an exception, and
is an adequate protein. 87
Block 2 Amino Acids, Peptides and Proteins

You should remember that essential or nonessential classes of amino acids do

not relate to their significance. In fact all the twenty amino acids are necessary
for normal functioning of the body. The amino acids, arginine and histidine
belong to the list of semi-essential amino acids because the body does not
always require dietary sources for it.

The variation in the nature of side chain as explained above is the cause of
variation in the physical properties of the amino acids. We will now study their
physical properties in the following section. Before proceeding, try to answer
the following SAQ.

SAQ 3
Tick [√ ] mark the appropriate answer.

(a) Amino acids owe their variation to the

i) carboxyl group [ ]

ii) chiral carbon [ ]

iii) α-amino group [ ]

iv) nature of their side chains [ ]

(b) Sulphur containing amino acids are:

i) methionine, serine [ ]

ii) threonine, serine [ ]

iii) methionine, cysteine [ ]

iv) threonine, cysteine [ ]

4.3 PHYSICAL PROPERTIES OF AMINO ACIDS

The amino acid structure, which we have shown up to this stage, is how it
would appear in an organic, nonpolar solvent. You would certainly wonder as
to how this molecule would appear in an aqueous solution i.e. in a polar
medium, since the molecule contains an acidic as well as a basic group. So let
us study the structure of an amino acid in a polar medium.

4.3.1 Zwitterionic Structure

An amino acid is usually represented in its unionised form to show the
presence of both amino and the carboxyl groups as you have seen in the
previous section. However, these are represented in the ionised form which
predominates under physiological conditions of pH around 7. The
understanding of the ionised structure of amino acid will help us in
understanding some of the properties of proteins.

Thus, an amino acid like glycine can be represented in its unionised and
88 ionised forms as given below.
Unit 4 Amino Acids
+
NH 2 NH3

H C COOH H C COO–

H R

Unionised amino acid Dipolar amino acid

Structure of glycine in ionised and unionised form

We have already mentioned that amino acids contain at least one carboxyl
group and one amino group. Both the groups are capable of ionisation in an
aqueous solution. Thus, in water, amino acids can act as both acids and
bases. Molecules which exhibit this property are known as amphoteric
molecules. In other words, this means an amphoteric molecule can accept as
well as donate a hydrogen ion (H+). At or around neutral pH, an amino acid
forms a dipolar ion or a zwitterion (Z-ion), as the acidic carboxyl group
donates a hydrogen ion and the basic amino group accepts one.

H H H
+ +
+ H + H
H3 N C COOH H3N C COO – H2 N C COO –

R R R
cation zwitterion anion
(at low pH) (at high pH)
Thus, a zwitterion has a positive as well as a negative charge within the same
molecule at neutral pH. As the normal biological environment in which amino
acids are generally present is aqueous medium at near neutral pH, they are
found as zwitterions only. However, generally we show these molecules in the
unionised form for the sake of convenience. Some amino acids in their
zwitterion form are given below.

O O O
OH
–
O– O– O
+ + + O
NH3 NH3 NH3

Phenylalanine Alanine Aspartic Acid

The existence of amino acid molecules in the zwitterionic form leads to an

important property in these molecules. Let us study it in the following
subsection.

4.3.2 Isoelectric Point

You would have observed from the above that zwitterions have one positive
and one negative charge and are, thus, electrically neutral. Therefore, these
dipolar ions do not migrate in an electric field. The pH at which amino acids
(and also proteins) carry no net charge is called its isoelectric point (pI). For
example, the isoelectric point for glycine is pH 6.0, which means that at this
pH, glycine will be in its zwitterionic form.
89
Block 2 Amino Acids, Peptides and Proteins

Let us now describe briefly how the amino acid molecules (and consequently
the proteins) behave in acidic or basic medium. These amphoteric compounds
can react with an acid or a base as shown in the following reaction.
H O H O H O
+ –
H –
OH –
R C C OH R C C O R C C O
NH3+ NH3+ NH2
Acidic pH pH around neutrality Basic pH
(zwitterion)

Net charge= +1 Net charge= 0 Net charge= -1

Isoelectric point
-
We can observe from these reactions that addition of H+ or OH to a zwitterion
results in a net positive or a negative charge on the molecule. Thus
depending on the pH of the aqueous solution, amino acids (as also the
proteins) can exist in different ionic forms.

This property of amino acids (and hence the proteins) to behave as weak
acids (due to –COOH group) or as weak bases (due to –NH2 group) is
employed effectively in nature. These molecules act as buffers in body fluids.
Thus, one of the important functions served by proteins in the blood is to
maintain the pH of blood in the narrow range of 7.35 to 7.45.

We may mention here that only those amino acids with ionisable side chains
(such as glutamic acid or lysine) or amino acids at the extreme ends in the
protein with free α-amino and α-carboxyl group, contribute to the acid-base
properties of a protein molecule. This is because the amino acids forming the
polypeptide chain of proteins (discussed in Unit 6 of this block) do not have
free α-amino and α-carboxyl groups, except at the two terminals of the
polypeptide chain. Some of the side chains (R groups) which are capable
of ionisation will increase the number of ionised forms possible for that
particular amino acid. This is highly important in the overall structure and
function of proteins. The tendency of a group to ionise is expressed in terms of
pKa value, which is analogous to that of pH. The lower the pH of a solution, the
more acidic it is and larger is its hydrogen ion concentration. In a similar way,
lower pKa value for an ionisable group implies stronger acidic nature of that
group and thus more readily it will ionise.

Let us look into the titration curves in amino acids that help in indicating the
charged state of the molecules on the basis of the pH of the solution. Before
going to the next subsection try to answer the following SAQ.

SAQ 4
Tick [√ ] mark the appropriate answer.
Amino acids are called amphoteric because they have
i) a basic group [ ]
ii) an acidic group [ ]
iii) both acidic and basic groups [ ]
iv) charged group as a side chain [ ]
90
Unit 4 Amino Acids

4.3.3 Titration Curves in Amino Acids

When the pH of given volume of a sample solution is changed by successive
addition of acid or alkali, a titration curve is obtained. The curves are usually
plots of pH against the volume of titrant added or more correctly against the
number of equivalents added per mole of the sample. This curve empirically
defines several characteristics. The precise number of each characteristic
depends on the nature of the acid being titrated. The characteristics are:

1) the number of ionising groups

2) the pKa of the ionising group(s)

3) the isoelectric pH value

The ionic form of the amino acid present in an aqueous solution depends on
the pH of the solution. Titration curves of amino acids are very useful for their
identification. Titration curve of an amino acid is obtained when a solution of
an amino acid in a strong acid, such as HCl is titrated against a strong base of
the same strength as NaOH. The pH is recorded after each addition, and a
graph is plotted between change in pH at y-axis and the amount of base at x-
axis as shown in Fig. 4.2. Due to the presence of different ionisable groups,
different amino acids have different types of curves. Let us understand the
titration curve of amino acids with an example of simplest structured amino
acid viz. glycine.

You now know that glycine has one -COOH and one –NH3 group in fully
protonated form with a net positive charge. In Fig 4.2 you can see two stages
corresponding to the dissociation of these two groups. Initially glycine is fully
protonated (species I) with pH approximately 1 and has two ionisable groups
capable of giving protons. As the titration proceeds and pH rises glycine
undergoes dissociation and becomes a Z-ion (species II). On plotting the
titration curve you can see that by addition of NaOH the pH gradually rises and
reaches a flat zone at pH 2.3 (pK1=2.34). This is the stage when –COOH has
- +
half dissociated into COO and H . This flat zone indicates a buffering zone.
As the titration proceeds a sharp rise in pH can be seen from pH 4.8. This is
because at this pH, glycine existing as a Z-ion is unable to provide proton to
neutralise the base. With further addition of NaOH and rise of pH, a second
flat zone over a pH range of 9-10 can be noted (pK2=9.6). At this range the
NH3 group deprotonates and generate an anionic form of the amino acid
(species III). This is the second buffering zone of glycine. The midpoint
between two pK values is pI which is 5.97.

+ +
NH3 NH3 NH2
pK 1 pK 2
CH2 CH2 CH2

COOH COO– COO –

Species I Species II Species III

91
Block 2 Amino Acids, Peptides and Proteins

Fig 4.2: Titration curve of glycine (0.1M) at 25C

The ionic species predominating at key points in the titration are shown above
the graph. The shaded boxes, centered at about pK1 =2.34 and pK2=9.60
indicate the regions of greatest buffering power. In general, the isoelectric
points of the amino acids can be computed by the following formula,

pl =1/2 (pKi + pKj)

where, pKi and pKj refer to the pK1 and pK2 values i.e., the pKa values for the
main chain –COOH and the α- NH3 group respectively for the neutral amino
acids. However, for acidic amino acids these refer to pK1 and pKR while for the
basic amino acids these represent pKR and pK2 values respectively, pKR being
the pKa value of the side chain functional group.

Now let us understand the technique of separation of amino acids in the next
section. Before proceeding further attempt the following SAQ.

SAQ 5
A zwitterion consists of:
i) a positive charge. [ ]
ii) a negative charge. [ ]
iii) both positive and negative charges. [ ]
iv) nil charge. [ ]

4.4 SEPARATION OF AMINO ACIDS

The amino acids earlier separated from the mixture of amino acids are
obtained on hydrolysis of plant and animal proteins. The extracted protein is
then purified by dialysis and should not contain any ionic impurities. It is then
dried and subjected to acidic or alkaline hydrolysis or to enzymatic
degradation with proteolytic enzymes. The separation of the amino acids from
92 protein hydrolysate is then carried out with the help of separation procedures
Unit 4 Amino Acids

like electrophoresis, ion-exchange chromatography or paper chromatography

etc. We will discuss two of the simplest methods of separation of amino acids
that is paper chromatography and paper electrophoresis in the following
subsections.

4.4.1 Paper Chromatography

In paper chromatography, amino acids are separated as the consequence of
differences in their partition coefficients between water and an organic solvent.
In this technique the amino acid solution is placed on one side of the sheet of
a moist filter paper with the help of a capillary tube, which is then placed in a Ninhydrin reacts with
jar as shown in Fig. 4.3 (a). The organic solvent contained in the jar migrates amino group of amino
upward by capillary action across the paper, carrying the amino acids with it acids to form a deep
blue coloured solution.
along one edge. When the solvent reaches the marked top of the paper, the
paper is removed and dried in the air. The spots remain invisible at this stage.
These can be made visible by spraying the paper with ninhydrin solution when
different amino acids appear at different places depending on their interaction
with the spraying agent as shown in Fig. 4.3 (b). Structure of ninhydrin

Fig.4.3: (a) Chromatography jar (b) Amino acids with spraying

agent

4.4.2 Paper Electrophoresis

Paper electrophoresis involves placing a mixture of proteins or amino acid in
an electric field at constant pH. Molecules with a net charge will move towards
the opposite electrodes, whereas the dipolar ions, with a net zero charge will
not migrate. Thus, it is the separation of amino acids based on the migration of
amino acids in an electric field. In the experimental procedure the amino acid
mixture is spotted onto a sheet of filter paper, the paper is wet with a buffered
salt solution and placed between two electrodes (cathode and anode) and
high voltage applied. At the neutral pH, the acidic amino acids will have a net
negative charge and will migrate toward the anode i.e., positive pole while the
basic amino acids will migrate towards the cathode i.e., the negative
pole. Electrically neutral amino acids will not migrate much, unless the pH is
made acidic or basic.

The front and top view of the paper electrophoresis apparatus is given in
Fig. 4.4.
93
Block 2 Amino Acids, Peptides and Proteins

Fig. 4.4: Paper electrophoresis showing migration of amino acid

SAQ 6
Which of the following reagents is used in the identification of amino acids by
paper chromatography?

i) Nessler’s reagent ii) Benedict’s reagent iii) Fehling’s solution iv) Ninhydrin

4.5 SUMMARY
 Amino acids are the monomeric units of proteins that are large complex
molecules responsible for a multitude of functions essential to life. The
amino acids are called -amino acids because of the presence of amino
group at the - carbon atom i.e. the carbon atom next to the carboxyl
group. The amino acids are commonly represented by a three letter
symbol or one letter symbol.

 All the amino acids, with the exception of proline, have a common
structural feature. They have the amino and carboxyl groups attached to
the same carbon atom. The carbon also carries a hydrogen atom and a
fourth group represented as the R-group or the side chain. The side chain
is characteristic of each amino acid.

 With the exception of glycine, all other amino acids show optical activity
and it is the L isomer that is found in the living beings. These are classified
based on chemical composition into aliphatic, hydroxy, sulphur containing
acidic, basic and aromatic types. On the basis of polarity of side chain,
these are classified into nonpolar, polar (uncharged), polar negatively
charged) and polar (positively charged) types.

 The carboxyl group and amino group of amino acid are capable of
ionisation in an aqueous solution. Therefore, in water, these act as acids
and bases. Molecules which exhibit this property are known as
amphoteric molecules. As an amphoteric molecule can accept as well as
donate a hydrogen ion (H+) at or around neutral pH, an amino acid forms
a dipolar ion or a zwitterion (z-ion). The acidic carboxyl group donates a
hydrogen ion and the basic amino group accepts one.

 The ionisable side chains of amino acid residues in proteins contribute to

94 their acid-base properties and also to their buffering capacity. These
Unit 4 Amino Acids

properties are depicted by the titration curves of amino acids that are very
useful for their identification.

 Two techniques used for separation of amino acids are paper

chromatography and paper electrophoresis. The chromatographic
technique makes use of ninhydrin as the spraying agent while
electrophoresis uses an electric field to separate amino acids.

4.6 TERMINAL QUESTIONS

1. List the ten essential amino acids.

2. Describe the structure of a typical amino acid.

3. Classify amino acids based on the chemical composition.

4. What is Z-ion and how does it affect titration of amino acids.

5. Name and describe the technique used to separate amino acids.

6. Give an example of the amino acid with the following in the side chain.

i) basic group ii) acidic group iii) aliphatic group iv) aromatic group

7. Write the three letter symbol representation the following amino acids:

i) Valine ii) Leucine iii) Isoleucine iv) Tyrosine

v) Alanine vi) Phenylalanine

4.7 ANSWERS
Self Assessment Questions

1. a) amino acids b) -amino acid c ) glutamine

2. a) it does not have a chiral carbon atom

b) left hand side c) L d) glyceraldehyde

3. a) (iv) b) (iii)

4. iii)

5 iii)

6 iv)

Terminal Questions
1. leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan,
threonine, valine, histidine and arginine.

2. The amino acids have a common structural motif. They have an amino
group at one end and a carboxyl group at the other end, both of which are
attached to the α-carbon atom i.e. the carbon atom next to the carboxyl
group. The α-carbon also carries a hydrogen atom. The fourth group on
this carbon is known as the side chain and is denoted as R. 95
Block 2 Amino Acids, Peptides and Proteins

3. Amino acids can be classified based on their chemical composition as

follows:

 Aliphatic amino acids: The aliphatic amino acids have aliphatic chain
as R group

 Hydoxy amino acids: The hydroxyl amino acids have hydroxyl group in
their R group and include serine, tyrosine and threonine.

 Sulphur containing amino acids: contain sulphur in their R-group. This

group has methionine and cysteine.

 Acidic amino acids: These are dicarboxylic acids containing an

additional-COOH group in the side chain

 Basic Amino acids: The basic amino acids contain additional basic-NH2
groups or heterocyclic ring.

 Aromatic amino acids: Aromatic groups are present as R group.

4. The carboxyl group and amino group in an amino acid can ionise in an
aqueous solution. These can accept as well as donate a hydrogen ion and
can act as acids and bases. Thus these act as amphoteric molecules. At
or around neutral pH, an amino acid forms a dipolar ion or a zwitterion (z-
ion). Thus a zwitterion has a positive as well as a negative charge within
the same molecule at neutral pH. The slope in titration curve is observed
when at a particular pH, amino acid exists in a Z- ion form and therefore is
unable to provide proton to neutralise the base.

5. Paper electrophoresis is the technique used for the separation of amino

acids. It is based on the migration of amino acids in an electric field. An
amino acid mixture is spotted onto a sheet of filter paper, the paper is wet
with a buffered salt solution and placed between two electrodes and high
voltage applied. At neutral pH, the acidic amino acids will have a net
negative charge and will migrate towards the anode while the basic amino
acids will migrate towards the cathode. Electrically neutral amino acids will
not migrate much unless the pH is made acidic or basic.

6. i) arginine, lysine and histidine

ii) aspartic acid, glutamic acid

iii) glycine, alanine, valine, leucine

iv) phenylalanine, tyrosine and tryptophan

7. i) Val iv) Tyr

ii) Leu v) Ala

iii) Ile vi) Phe

96