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Amino acids:
 Amino acids are the building blocks for proteins.
 Amino acids are the basic structural units of proteins consisting of an amino
group, (-NH2) a carboxyl (-COOH) group, a hydrogen (H) atom and a distinctive
side chain (R). All of the substituents in amino acid are attached to a central α
carbon atom.

 Amino acids, or amino-carboxylic acids, are organic

carboxylic acids in which at least one hydrogen atom of the
hydrocarbon chain is replaced by an amino group.

 Although there are around 300 naturally occurring amino

acids, About 60 different amino acids and their derivatives
are found in the humans; still, not all of them serve as
constituents for proteins.

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 Nearly all proteins are made from 20 amino acids

 Based on this, amino acids are divided into two groups: proteogenic, or proteinogenic
amino acids, which are normally components of proteins & non-proteogenic amino
acids, usually not incorporated into proteins.
 Some proteins contain additional amino acids that arise by the post-translational
modification of an amino acid already present in a peptide. Examples include the
post-translational conversion of proline and lysine to hydroxyproline and
hydroxylysine; the conversion of glutamate to γ-carboxyglutamate. These amino
acid are some times referred to as rare amino acids.
 Other amino acids do not participate in the protein synthesis; they occur in the cells
either in a free state (as metabolites), or are part of non protein compounds.
 ɤ-Aminobutyric acid occurs in a free state and functions as an inhibitory
neurotransmitter; β-alanine is a component of a vitamin, pantothenic acid.
 All amino acids found in living systems, plant and animal proteins are L-α-amino
acids. Glycine is the only amino acid, which is optically inactive and cannot be
resolved into D- or L-form because of symmetry on the α-carbon.

 L-Amino acids are classified into α, β, ϒ-types, depending on the position of the carbon
bearing an –NH2 group with respect to –COOH group.
Basic structure of amino acid:
All amino acids (except proline) include five basic parts:
 A central carbon atom
 A basic amino group (-NH2)
 An acidic carboxyl group (-COOH)
 A hydrogen atom (-H)
 A distinctive side chain (-R)
The side chain can be a hydrogen atom, aliphatic, aromatic or
heterocyclic group.
 At physiological pH (pH = around 7), both the α- amino and
carboxyl group are ionized resulting the charged form of amino
acids called zwitterions.

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Classification of amino acids:

Several classifications of amino acids are currently adopted:
1. Electrochemical classification: based on the acid-basic properties of amino acids.
2. Structural classification: based on the structural features of side-chain of amino acids.
3. Nutritional classification: based on the nutritional requirement of amino acids for the
organism.
4. Classification based upon Fate of amino acids
5. Classification based upon participation in protein synthesis
Electrochemical classification:
According to their electro-chemical (or acid-basic) properties, amino acids are divided
into three groups:
a) Acidic
b) Basic and
c) Neutral

a) Acidic amino acids:

Acidic amino acids are those having additional carboxylic groups in the side-chain,
which provide for enhanced acidic properties in this group of amino acids.
Examples:
Aspartic acid
Glutamic Acid
b) Basic amino acids:
Amino acids carrying an additional amino group (Two –NH2 groups) which contribute
to their enhanced basic properties.
Examples:
Arginine, Lysine, Hydroxylysine,

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c) Neutral amino acids:

Their side-chain radicals exhibit neither acidic, nor basic properties.
Examples:
Glycine, Alanine, valine, leucine, Phenyl alanine, Tyrosine etc.

On the basis of the composition of the side chain or R group (Structural classification):
Based on the composition of the side chain, amino acids, may be grouped into
following categories:
(i) Simple (aliphatic) amino acids.
These have no additional functional group in the side chain, e.g., glycine, alanine,
valine, leucine and isoleucine.

(ii) Hydroxy amino acids:

These contain a hydroxyl group in their side chain, e.g., serine and threonine.

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(iii) Sulfur-containing amino acids.

These possess a sulfur atom in the side chain, e.g., cysteine and methionine.

(iv) Acidic amino acids.

These have a carboxyl group in the side chain, e.g., aspartic acid and glutamic acid.

(v) Amino acid amides.

These are derivatives of acidic amino acids in which one of the carboxyl group has
been transformed into an amide group (-CO.NH2), e.g., asparagine and glutamine.

(vi) Basic amino acids.

These possess an additional amino group in the side chain, e.g., lysine and arginine.

(vii) Heterocyclic amino acids.

These amino acids have, in their side chain, a ring which possesses at least one atom
other than the carbon, e.g., tryptophan, histidine and proline.

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(viii) Aromatic amino acids

These have a benzene type aromatic ring in the side chain, e.g., phenylalanine and
tyrosine.

Nutritional Classification
This classification is based on the nutritional importance of amino acids for the
organism.
1. Essential Amino Acids:
 Amino acids which are not synthesized in the body and must be provided in the diet
to meet an animal’s metabolic needs are called essential amino acids.
 Ten amino acids are grouped under this category.
 Deficiency in anyone of these amino acids leads to growth failure and may even be
life threatening.
Essential Amino Acids for humans are:
Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine,
Threonine, Tryptophan, Valine

2. Semi-essential amino acids

Two amino acids are grouped under semi-essential amino acids since they can be
synthesized within the organism but their synthesis is not in sufficient amounts. In that
case they should also be provided in the diet. Thus, 8 amino acids are absolutely
essential while 2 are semi-essential.
Semi essential amino acids include:
Arginine and Histidine
3. Non- Essential Amino Acids
These amino acids are biosynthesized in adequate amounts within the organism and not
required to be provided through diet.
These amino acids are derived from carbon skeletons of lipids and carbohydrates during
their metabolism or from the transformation of essential amino acids.
Non-Essential Amino Acids include:
Alanine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine,
Proline, Serine, Tyrosine

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Classification Based on the Fate of Amino acid:

I. Glucogenic Amino Acids
 Those amino acids in which their carbon skeleton gets degraded to pyrurate, α
ketoglutarate, succinyl CoA, fumrate and oxaloacetate and then converted to Glucose
are called Glucogenic amino acids. OR/
 Amino acids whose catabolism yields pyruvate or one of the intermediates of citric
acid cycle i.e. substrates of gluconeogenesis & therefore can give rise to GLUCOSE are
called Glucogenic amino acids.
These include:-
Alanine, cysteine, glycine, Arginine, glutamine, Isoleucine, tyrosine.
II. Ketogenic Amino Acids
 Amino acids whose catabolism yields acetyl CoA or acetoactyl CoA and finally give
rise to ketone bodies are called ketogenic amino acids.
 Leucine & lysine are the only exclusively ketogenic amino acids & therefore cannot
give rise to glucose.

III. Ketogenic and glucogenic Amino Acids

 amino acids that can be converted in to pyruvate (intermediate of
glucose synthesis) and can also potentially form acetoacetate (ketone
body intermediate) are called mixed ketogenic and glucogenic amino
acids.
 Amino acids that give rise to both glucose and ketone body precursors
are called as being glucogenic and Ketogenic amino acids.
 Isoleucine, Tyrosine, Phenylalanine and Tryptophan
Classification Based on Participation in Protein Synthesis:
1. Standard amino acids:
Amino acids that take part in protein biosynthesis inside the body are
called primary or standard amino acids. Out of the 300 or so naturally
occurring amino acids, 20 are recognized to be standard amino acids.

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The standard amino acids are:

Glycine Alanine Proline Valine
Methionine Phenylalanine Leucine Isoleucine
Tyrosine Tryptophan Serine Threonine
Asparagine Glutamine Lysine Cysteine
Glutamate Arginine Histidine Aspartate

2. Non-Standard Amino Acids:

A nonstandard amino acid is an amino acid that occurs naturally in cells but do not
participate in peptide synthesis. Some nonstandard amino acids are constituents of
peptides, but they are generated by modification of standard amino acids in the peptide
molecule (post-translational modification).
 Among the non-standard amino acids, 4-hydroxyproline a derivative of proline, 5-
hydroxylysine derivative of lysine, both are found in collagen, a fibrous protein of
connective tissues.
 GABA acts as neurotransmitter.
 β-alanine is a part of vitamin called pantothenic acid.
 N-methyllysine occurs in myosin

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Properties of Amino Acids:

Physical Properties
Physical state:
 Amino acids are white crystalline substances. They have high melting points varying
from 200-300 ºC or even more.
Solubility:
 Most of them are soluble in water and insoluble in non-polar organic solvents (e.g.,
chloroform and ether).
 Aliphatic and aromatic amino acids, particularly those having several carbon atoms,
have limited solubility in water but readily soluble in polar organic solvents.
Taste:
 They are tasteless (Leucine), sweet (Glycine, Alanine, Valine) or bitter (Arginine,
Isoleucine). Some are having good flavor.
Isomerism:
 The α-carbon of an amino acid is attached to four different chemical groups and is,
therefore, a chiral carbon atom (Glycine is an exception).

 Amino acids with an asymmetric center at the α-carbon can exist in two forms,
designated D and L, that are mirror images of each other. The two forms in each pair
are termed stereoisomers, optical isomers, or enantiomers.
 All amino acids found in proteins are of the L configuration. However, D-amino acids
are found in some antibiotics and in bacterial cell walls.
 In D-amino acids – NH2 group is on the right hand while in L-amino acids it is oriented
to the left.

Amphoteric Nature and Isoelectric pH:

Each amino acid has at least two ionizable groups
• The –NH2 group
• The –COOH group (In addition, charged groups can also be present in the side chain
of some amino acid).

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• At specific pH, the amino acids carry both the charges in equal number and thus exist as
dipolar ion or Zwitterions. At this point, the net charge on the amino acid is zero. The pH
at which the amino acid exists as Zwitterion (with no net charge) is called isoelectric pH or
isoelectric point.
• This property in which amino acids act both as an acid and as base is known as
amphoteric nature of amino acids.
• On the acidic side of its pI, amino acids exist as a cation by accepting a proton and
becomes positively charged (cationic form).
• On the basic side of its pI, the –COOH group of the amino acid acts as proton donor
and the amino acid becomes negatively charged by loosing a proton (anionic form).

Chemical Properties:
The general reactions of amino acids are mostly due to the presence of two functional
groups namely carboxyl (-COOH) group and amino (-NH2) group.
A) Due to Carboxylic (—COOH) Group
1. Formation of esters:
Amino acids are readily esterified by acid-catalyzed reactions with alcohols, to
produce the corresponding esters. The esters, so obtained, are volatile in contrast to
the free amino acids.

2. Formation of amides:
Amino acids react with amines to form amides. For example

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The -COOH group of dicarboxylic amino acids (other than α carboxyl) can combine
with ammonia to form the corresponding amide. For example

3. Reaction with Bases:

Amino acids react with bases to form the corresponding salts.

4. Reduction to amino alcohol:

This is achieved in presence of lithium aluminium hydride.

5. Decarboxylation:
Action of specific amino acid decarboxylases, dry distillation or heating with
Ba(OH)2 changes the amino acid into its corresponding amine. Thus some
important amines are produced from amino acids. For example:

II. Properties Due to Amino (–NH2) Group

1. Salt formation with acids:
The basic amino group reacts with mineral acids such as HCl to form salts like
hydrochlorides.

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2. Reaction with acylating agents:

The amino group of amino acids can be acylated with acid anhydrides or acid halides to
form N-acyl amino acids.

3. Reaction with Nitrous acid (Van Slyke reaction):

The amino acids react with HNO2 to liberate N2 gas and to produce the corresponding
α-hydroxy acids.

This reaction is characteristic of aliphatic primary amines and has been utilized by Van
Slyke (1912) as the basis for his `nitrous acid' method for the estimation of amino acids
by measuring the volume of N2 gas liberated. The imino acids, proline and
hydroxyproline, however, do not respond to this reaction.

4. Reaction with CO2:

The amino acid present in an alkaline solution may react with CO2 through NH2
group to form a carbamino acid.

5. Reaction with formaldehyde:

Formaldehyde reacts with the -NH2 group to form a methylene compound.

This reaction is the basis of the Sorensen titration method for determining the
purity of the individual amino acids.

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6. Reaction with benzaldehyde.

On reaction with benzaldehyde, amino acid form Schiff 's bases.

III. Properties of Amino acids Due to Both NH2 and COOH

1. Formation of co-ordination complexes
The amino acids form co-ordination complexes with certain heavy metals and other
ions. These include Co++, Cu++, Mn++ and Ca++. An example of co-ordination
complex of glycine and Cu++ is given below.

2. Formation of peptide bond:

αcarboxyl group of one amino acid reacts with α amino group of another amino
acid to form a peptide bond or CO-NH bridge. Proteins are made by
polymerization of amino acids through peptide bonds

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Peptide bond:
A peptide bond is a chemical bond formed between two amino acids when the carboxyl
group of one amino acid reacts with the amino group of the other amino acid,
releasing a molecule of water (H2O).
Depending on the number of amino acid molecules composing a chain, the peptides
may be termed as:
 Dipeptides (containing 2 amino acid units)
 Tripeptides (containing 3 amino acid units)
 Oligopeptides (containing not more than 10 amino acids
 Polypeptides (made up of more than 10 amino acids)
N- and C-terminals
In a polypeptide chain, the terminal amino acid with the free amino group is called
as the N-terminal amino acid (N-terminal) & the one with the free carboxyl group
at the other end is termed as C-terminal amino acid.
Coincidentally, the protein biosynthesis also starts from the N-terminal amino acid.

PROTEINS (proteiosG = pre-eminent or first)

Proteins are polypeptides with >50 amino acid residues1 with a molecular weight
greater than 5000. OR/
Proteins are compounds of high molecular weight made up of α-amino acids linked to
one another by peptide linkages.
Classification of proteins:
Proteins are classified;
A. On the basis of shape and size
B. On the basis of functional properties
C. On the basis of composition
D. On the basis of solubility

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A.On the basis of shape and size

1. Fibrous proteins:
When the ratio of length to breadth of a protein molecule is more than 10, it is called a
fibrous protein.
They are long and fiber like and their molecule consists of several coiled cross-linked
polypeptide chains. They are insoluble in water and highly resistant to enzyme digestion.
Examples: α-keratin from hair, elastin, collagen.
2. Globular protein:
When the ratio of length to breath of a protein molecule is less than 10, it is called as
globular protein (axial ratio usually 3 to 4 or less).
These are globular or ovoid in shape, and soluble in water. As a class, globular proteins are
more complex in conformation than fibrous proteins, have a far greater variety of
biological functions and are dynamic rather than static in their activities. Tertiary and
quaternary structures are usually associated with this class of proteins. Protein
hormones, blood transport proteins, antibodies, storage proteins and nearly all enzymes
are globular proteins.
Examples: Myoglobin, hemoglobin, ribonuclease, albumin, lysozyme, chymotrypsin.

B. On the basis of functional properties:

i. Defence proteins:
These proteins are involved in defense mechanisms of organisms. For example,
immunoglobulins and snake venoms;
ii. Contractile proteins:
Contractile proteins are involved in muscle contraction and relaxation. For example,
actin and myosin
iii. Transport proteins:
These are involved in the transport of small molecules inside the organism, like
hemoglobin, myoglobin, albumin, ferritin, lipoproteins.
iv. Structural proteins:
These serve as structural materials. For example, myosin of muscles, keratin of skin
and hair and collagen of connective tissues.
v. Catalytic Proteins:
These proteins have catalytic functions. For example; different enzymes like
lysozymes, chymotrypsin, kinases, transaminases

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vi. Regulatory Proteins:

These proteins regulate cellular or physiological activity.
For example: Peptide hormones like insulin and growth hormone.
vii. Secretary Proteins:
Fibroin is a protein secreted by spiders and silkworms to form webs and cocoons.
viii. Storage Proteins:
These proteins have the function of storing amino acids as nutrients and as building
blocks for the growing tissues. Storage proteins are source of essential amino acids,
which cannot be synthesized by human beings.
For example, Glutelin, Casein in milk and ovalbumin in egg.
ix. Genetic proteins:
For example, histones
x. Receptor proteins:
They function as receptors for hormones, viruses

III. On the basis of composition (chemical nature):

A. Simple proteins:
These are proteins which on complete hydrolysis yield only amino acids (since they are
made up of amino acids only).
e.g. Albumins, Globulins, Trypsin, Chymotrypsin, Pepsin, Insulin
B. Conjugated proteins:
These are proteins which in addition to amino acids contain a non-protein group called
prosthetic group in their structure.
Conjugated Protein = Protein part + Prosthetic group.
Conjugated proteins can further be divided into:
 Nucleoprotein (a protein containing nucleic acids as prosthetic group) e.g. Histones
 Lipoprotein (a protein containing lipids as prosthetic group) e.g. Lipovitellin of egg, HDL,
LDL
 Phosphoprotein (a protein containing phosphate groups) e.g. Casein of milk
 Metalloprotein (a protein containing metal ions like Fe2+) e.g. Ferritin
 Glycoprotein (a protein containing carbohydrates) e.g. Immunoglobulins
 Flavoproteins (a protein containing FAD/FMN as prosthetic group) e.g. Succinate
dehydrogenase

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C. Derived proteins:
They are formed from simple and conjugated proteins by physical and chemical means
(by the action of heat, acids, alkalis/ enzymes). It is not a well-defined class of proteins.
They include two types of derivatives:
 Primary-derived Proteins
They are formed from native proteins by the action of heat or alcohol etc. They are
synonymous with denatured proteins. There is little or no hydrolytic cleavage of
peptide bonds.
For example, Coagulated proteins like cooked meat and cooked albumin, Fibrin from
fibrinogen, Proteans, Metaproteins
 Secondary-derived Proteins
These proteins are formed by the progressive hydrolytic cleavage of the peptide bonds
of protein molecule. They are roughly grouped into proteoses, peptones and peptides
according to average molecular weight.

A. On the basis of solubility:

Proteins classification according to their solubility is as follows;
1. Albumins: Soluble in water and salt solutions and coagulated by heat.
Examples: Albumin of plasma, egg albumin and lactalbumin of milk.
2. Globulins: Sparingly soluble in water but soluble in salt solutions.
Examples: Globulins of plasma, ovoglobulins of egg, lactoglobulin of milk.
3. Glutelins: Soluble in dilute acids & alkalies but insoluble in neutral aqueous solutions.
Examples: Glutenin of wheat, oryzenin of rice, zein of maize.
4. Protamins: Soluble in ammonia and water (NH4OH).
Examples: Salmine from salmon fish, sturine of sturgeon.
5. Histones: Soluble in water and dilute acids.
Example: Histones present in chromatin.
6. Prolamines: It is water insoluble but soluble in ethanol (70%).
Examples: gliadin (from wheat)
7. Sclero proteins: Insoluble in water and dilute acids and alkalies, salt solutions and
organic solvents and soluble only in hot strong acids.
Examples: Collagen, elastin and keratin.

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C. Nutritional classification of proteins

The nutritive value of proteins is determined by the composition of essential amino
acids (described already). From the nutritional point of view, proteins are classified
into 3 categories.
1. Complete proteins:
They are also called as nutritionally rich proteins or first class proteins. These
proteins have all the ten essential amino acids in the required proportion by the
human body to promote good growth. e.g. egg albumin, milk casein.
2. Partially incomplete proteins:
These proteins partially lack one or more essential amino acids, and can promote
moderate growth. e.g.
wheat and rice proteins (limiting Lys, Thr). Proteins from pulses are deficient in
methionine.
3. Incomplete proteins:
These proteins completely lack one or more essential amino acids. Hence they do
not promote growth at all e.g. gelatin (lacks Trp), zein (lacks Trp, Lys).

STRUCTURE/STRUCTURAL ORGANISATION OF PROTEINS

Proteins are the polymers of L-α-amino acids. Every protein has a
unique three-dimensional structure, which is referred to as its
native conformation. This protein conformation (structure) is
complex and can be divided into 4 levels of organization
 Primary,
 Secondary,
 Tertiary and
 Quaternary structures.

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A. Primary Structure:
Primary structure refers to the number and linear sequence of amino acids held together by
peptide bonds in the peptide chain.
 The peptide bonds form the backbone and side chains of amino acid residues project
outside the peptide backbone.
 In a polypeptide chain, the terminal amino acid with the free amino group is called as
the N-terminal & the one with the free carboxyl group at the other end is termed as C-
terminal. It is a tradition to number the amino acids from N-terminal end as No. 1
towards the C-terminal end.

Primary Structure of Insulin

It consist of two polypeptide chains A and B with 21 and 30 amino acids, respectively.
The two chains are covalently linked by inter chain and intra chain disulfide bonds. The
A chain has N-terminal glycine and C-terminal asparagine. The B chain has
phenylalanine and alanine as N-and C-terminal residues, respectively.

 Presence of specific amino acids at a specific number is very significant for a

particular function of a protein. Any change in the sequence is abnormal and may
affect the function and properties of protein as well as higher orders of structural
organization of that protein. For example:
 In sickle cell anemia, Hb S has valine instead of glutamic acid at position 6.

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B. Secondary Structure:
 The secondary structure refers to the way the peptide is folded into an ordered structure
owing to hydrogen bonding between peptide groups of the same chain or adjacent
polypeptide chains. OR/

 The three dimensional structure of the polypeptide chain by way of folding or coiling
resulting in a helically-coiled, zigzag, linear or mixed form is called secondary structures
of proteins.

 The folding of protein chain can be ordered or disordered. The ordered secondary
structures are α-helix and β-pleated sheet. The disordered secondary structures are
random coil and reverse turn or β-turn.

The linkages or bonds involved in the secondary structure formation are;

o Hydrogen bonds and
o Disulfide bonds.

 Hydrogen bond:
These are weak noncovalent bonds and involves sharing a single hydrogen by two
electronegative atoms such as O and N. Hydrogen bonds are formed in secondary
structure by sharing H-atoms between carbonyl oxygen and nitrogen of amide group
of different peptide bonds.
The hydrogen bonds in secondary structure may form either an α-helix or β-pleated
sheet structure.
 Disulphide bonds:
These are formed between two cysteine residues. They are strong, high energy
covalent bonds.
The following are the important types of secondary structures of proteins;

1. α-Helix:
α-Helical structure was proposed by Pauling and Corey (1951).
In α-helix, a single protein chain is twisted in such a way to resemble a coiled helical
spring.

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 In alpha helix the polypeptide bonds form the

back-bone and the side chains of amino acids
extend outward.
 The helical coils are stabilized by the regular
formation of hydrogen bonds parallel to the axis
of the helix between NH and C=O groups of the
main chain (carbonyl O of 1st amino and amide N
of 4th amino acid residues). Thus in α-helix intra
chain hydrogen bonding is present.
 3.6 amino acid residues are present in one
complete turn.
 The distance between two equivalent points on
turn is 0.54 nm which is called the pitch and
width of the helix is 5.0 Å.
Types:
The α-helices can be either right handed or left handed.

 Left handed α-helix is less stable than right handed α-helix because of the steric
interference between the C=O and the side chains.
 Only the right handed α-helix has been found in protein structure.

 Small or uncharged amino acid residues such as alanine,

leucine and phenylalanine are often found in α-helix.
 More polar residues such as arginine, glutamate and
serine may repel (too many + or – charged groups near
each other in space are unfavorable -electrostatic
repulsion) and destabilize helix.
 The cyclic amino acid proline has no free hydrogen to
contribute to helix stability. α-helixes in long protein
chains often end at a place where proline residues occur
in the primary structure. Proline is therefore,
sometimes called an α-helix disrupter or “helix
breaker” for this reason.

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 The proteins of hair, nail, skin contain a group of proteins called keratins which are
rich in α-helical structure.
 Myoglobin and hemoglobin are predominantly in α-helical structure.
2. β-Pleated Sheet Structure:
 This is the second type of structure proposed by
Pauling and Corey.
 The beta-pleated sheet consists of peptide chains
arranged side by side to form a structure that
resembles a piece of paper folded into many pleats.
 β-pleated sheet structure is formed when hydrogen
bonds are formed between the carbonyl oxygens
and amide hydrogens of two or more adjacent
extended polypeptide chains.
 The adjacent chains in β-pleated sheet structure are
either parallel or antiparallel.

Parallel Beta sheets:

 If the polypeptide strands run in the same direction, N-terminus to C-terminus. The N-
terminus of one beta strand will be opposite the N-terminus of the other beta strand.
Antiparallel Beta sheets:
 If the polypeptide chains run in opposite direction,
the β-pleated sheet are said to be antiparallel.
 The parallel arrangement is less stable because the
geometry of the individual amino acid molecules
forces the hydrogen bonds to occur at an angle,
making them longer and thus weaker.

 In both parallel and antiparallel β-

pleated sheet structures, the side
chains of adjacent amino acids are
on opposite sides of the sheet.

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 An anti-parallel beta-pleated sheet also

forms when a polypeptide chain sharply
reverses direction and loops back on itself.
This can occur in the presence of two
consecutive proline residues, which create
an angled kink in the polypeptide chain
and bend it back upon itself.
 Generally glycine, serine and alanine are
most common to form β-pleated sheet.
Proline occurs in β-pleated sheet although
it tends to disrupt the sheets by
producing kinks.
 Albumin and hemoglobin of blood
contains β-pleated sheet. Silk fibroin, a
protein of silkworm is rich in β-pleated
sheets.

3. Triple Helix:
 Triple helix is formed from three peptide chains, each a helix, which are wound
around each other as a rope.
 This form is best exemplified by Collagen, which is rich in proline and hydroxyproline
and cannot form α-helix or β-pleated sheet.
 Collagen contains long chains of the three-residue repeating unit Gly-X-Y, in which X
is often Pro, and Y is often 4-hydroxyPro (Hyp).

 Each polypeptide strand forms a left-

handed helix with about three
residues per turn. Three such parallel
chains then wrap around each other
in a gentle right-handed coil (pitch of
10.0 Å).

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The triple helix is stabilized by both noncovalent as well as covalent

bonds.
Inter-chain hydrogen bonds between different peptide chains are formed
which are almost perpendicular to the long axis of helix. Strength and
insolubility of collagen fibrils is also due to intra- and inter-chain
covalent cross links between (Lys and His) residues.
4. Bends, turns , Loops and super-secondary Motifs:
In addition to the α-helix and β-sheets, which accounts for about 50%
of the secondary structure of a globular protein, the other half is
constructed by bends, turns, loops and other conformational features, of
which β-bend is the commonest. These are discussed below:

a) Turns and bends (β-Bends/β-Turns/Reverse turns/Hairpin turn/hairpin loop):

Turns and bends refer to short segments of amino acids that join two units
of secondary structure, such as two adjacent strands of an antiparallel β
sheet.
 A β turn involves four amino acid residues, in which the first residue is
hydrogen-bonded to the fourth, resulting in a tight 180-degree turn.
They are usually found on the surface of protein molecules and often
include charged residues such as Ser, Asp as well as proline and Gly.

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b) Loops:
The loops also connect adjacent regions of secondary structure, but have a larger
number of amino acid residues than bends.
 Bends and loops possess important biological properties e.g. contributing to
enzyme activities (participate in catalysis) and provide the epitopes that are sites
of recognition and binding for antibodies.
c) Super-secondary structures (motifs):
When different Secondary structures (α-helix, β-pleated sheet, β-turns etc.) group
together in specific geometric arrangements they form super-secondary structures
or motifs. They are very important for biological function. Various super secondary
structures found in globular proteins are;
i) β-α-β Units:
The β-α-β unit consists of two parallel β-pleated sheets
connected by an intervening strand of α- helix.

2. Greek Key:
The Greek key motif consists of four adjacent antiparallel strands and their
linking loops. Its three antiparallel strands are connected by hairpins, while the
fourth is adjacent to the first and linked to the third by a longer loop. It was
named after a pattern common to Greek ornamental artwork and pottery.
3. β-meander:
The β-meander consists of five β-pleated sheets linked together by hairpin loops.
The β-meander contains nearly as many hydrogen bonds as an α-helix, and its
common occurrence probably reflects the stability conferred by this extensive
hydrogen bonding.

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C. Tertiary structure:
 The tertiary structure of a protein refers to the
bending and folding of the protein into a
specific three-dimensional shape.
 The polypeptide chain with secondary structure
may be further folded, super-folded twisted
about itself forming a specific three-dimensional
shape, which is called tertiary structure.
 It is that conformation which is biologically
active and protein in this conformation is called
as native protein.
 The tertiary structure is due to interaction of
amino acids located far apart (away from each
other) but brought closer to each other by
folding.

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The bonds responsible for interaction between groups of amino acids are as follows:
1. Hydrophobic interactions:
The non-polar side chains of neutral amino acids tend to associate in proteins. These
are called as hydrophobic interactions. Normally occur between nonpolar side chains
of alanine, leucine, methionine, isoleucine and phenylalanine. They constitute the
major stabilizing forces for tertiary structure forming a compact three-dimensional
structure.
2. Hydrogen bonds:
Normally formed by the polar side chains of the amino acids. For example: hydroxyl
group of serine, threonine, the amino groups and carbonyl oxygen of glutamine and
aspargine, the ring nitrogen of histidine participates in internal hydrogen bond
formation.
3. Ionic or electrostatic interactions (salt bridges):
These bonds are formed between oppositely charged groups of amino acid side chains.
The έ-amino groups of lysine is positively charged and second (non-α) carboxyl group
of aspartic acid is negatively charged at physiological or body pH. These interact
electrostatically to stabilize tertiary structure of protein.

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4. Van der Waal Forces:

These are the weak interactions between uncharged groups of protein molecule. They
also contribute to the stability of proteins.
5. Disulfide bonds:
These are the S–S bonds between -SH groups of distant cysteine residues.
D. Quaternary Structure:
 When a protein consists of two or more polypeptide chains, each of which has its own
primary, secondary, and tertiary structure and held together by non-covalent interactions
or by covalent cross-links, it is referred to as the quaternary structure of the protein.
 The assembly is often called as oligomer and each constituent peptide chain is called as
monomer or subunit.
 The monomers of oligomeric protein can be identical or quite different in primary,
secondary or tertiary structure.
 The protein will lose its function when the subunits are dissociated.
 The forces that keep the quaternary structure are the same hydrogen bonds, electrostatic
bonds, hydrophobic bonds, van der Waals forces and occasionally, disulphide bonds.

 Examples: The enzyme creatine phosphokinase (CPK) is an example of protein with

two monomers (dimer).
 Hemoglobin molecule, which consists of four separate polypeptide chains, exhibits
quaternary structure. Haemoglobin and lactate dehydrogenase (LDH) are tertramers
consisting of four monomers.
 Apoferritin, an iron binding and storage protein contains 24 identical subunits.
 Myoglobin has no quaternary structure since, it is composed of a single polypeptide
chain.

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Color Reactions of Proteins

Proteins give specific color reactions with certain reagents which have significant importance
in the qualitative detection and quantitative estimation of proteins & their constituent
amino acids.
1. Biuret Reaction
A compound, which is having more than one
peptide bond when treated with Biuret
reagent, produces a violet color. This is due
to the formation of coordination complex
between four nitrogen atoms of two
polypeptide chains and one copper atom.
 Only Histidine can give a positive reaction.
Significance:
 Group test for detection of proteins
The name of the reaction is derived from the organic
compound, biuret, obtained by heating urea at high
temperature which gives this test positive.

2. Ninhydrin reaction:
 All amino acids (except proline and hydroxyproline), proteins or
protein derivatives containing free amino group and a free
carboxyl group react with ninhydrin to give a blue-violet/purple
colored compound called Rheumann’s purple.
 Proline and hydroxyproline, give a yellow color with ninhydrin.
Significance:
This test is given by α-amino acids, proteins and dipeptides.
It can be used to detect α-L-amino acids.

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3. Xanthoproteic Reaction
 The aromatic amino acids such as tyrosine and tryptophan present in the protein give
yellow precipitate when heated with conc. HNO3 due to the formation of Nitro-
derivative. On addition of alkali, the precipitate turns orange since the salts of these
derivatives are orange in color.
 Gelatin which contains no tryptophan and very little tyrosine, gives only a faint color.
Significance:
Specific test for aromatic amino acids either free or
within protein molecules.

4. Millon’s reaction:
 Millon’s reagent contains mercuric sulfate in H2SO4 and sodium nitrite solution. Sodium
nitrite reacts with Sulfuric acid to form nitrous acid (reacting acid). If protein are
present, they get precipitated by the mercuric sulfate (formation of white ppt). The
reacting groups (phenol group of tyrosine) which get exposed on boiling, reacts with
nitrous acid to form Nitroso derivatives which then forms red color precipitate with
mercury.
Significance:
This is a specific test for tyrosine (the only phenol containing amino acid). This test is
also given by those proteins which yield tyrosine on hydrolysis.

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5. Hopkins-Cole Reaction
In Hopkins-Cole Reaction, the indole group of tryptophan reacts with glyoxylic acid in
the presence of concentrated H2SO4 to give a purple/violet ring at the junction of two
layers.
Significance:
 The reaction is characteristic of tryptophan.
 Protein containing tryptophan also gives this test positive.
 Gelatin and collagen do not contain tryptophan and hence do not give this test
positive.

6. Nitroprusside reaction:
Proteins with free -SH group of cysteine give reddish color with sodium
nitroprusside in ammoniacal solution. Many proteins give this test positive after heat
coagulation or denaturation indicating the liberation of free -SH groups.
Significance:
Specific test for free cysteine, and cysteine or proteins containing these.

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7. Sakaguchi test:
This is a specific test for detection of amino acid containing guanidium group (e.g free
arginine or arginine bound in protein).
Sakaguchi reagent consists of alcoholic α-naphthol and a drop of sodium hypobromite
/hypochlorite.
Guanidine group of arginine (-HN=C-NH2) is responsible for the formation of red
colour.
Significance:
For the determination of free or bound arginine.

8. Lead acetate test:

When cysteine or cysteine containing proteins are boiled with strong alkali, organic
Sulphur splits and forms sodium sulphide, which on addition of lead acetate produces
lead sulphide as a black precipitate.
Significance:
For the determination of cysteine and cystine.

Na2S + Pb (CH3COO)2 →(black precipitate) PbS

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9. Pauly’s test:
Red color develops due to coupling of diazotized sulfanilic acid in alkaline solution
with phenol and imidazole groups.

Significance:
The reaction is specific for tyrosine and histidine.

10. Sullivan reaction:

Red color is produced when cysteine containing protein is heated with sodium 1, 2
naphthoquinone -4-sulfonate in the alkaline medium in presence of Na2S2O4.
Significance:
This is a specific test for the detection of cysteine and cystine in proteins.

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Biological and pharmaceutical importance of proteins

1. Takes important part in the formation of protoplasm which is essential for all forms
of life.
2. Biochemical catalysts known as enzymes are proteins.
3. Proteins known as immunoglobulins serve as the first line of defence against bacterial
and viral infections.
4. Several hormones are protein in nature.
5. Some proteins have role in contraction of muscles.
Example: actin and myosin are contractile proteins and help in the movement of
muscle fiber.
6. Some proteins present in cell membrane, cytoplasm and nucleus of the cell act as
receptors.
7. The transport proteins carry out the function of transporting specific substances
either across the membrane or in the body fluids.
8. Storage proteins bind with specific substances and store them, e.g. iron is stored as
ferritin.

9. Few proteins are constituents of respiratory pigments and occur in electron transport
chain or respiratory chain, e.g. cytochromes, hemoglobin, myoglobin.
10. Under certain conditions proteins can be catabolized to supply energy.
11.Protein-based drugs are fastest growing class of drugs. Insulin was the first
pharmaceutical protein produced using genetic engineering. It is widely used by
diabetics.
12. Pharmaceutical applications of proteins also include the use of biopharmaceuticals
like
 Antibodies
 Vaccines
 Hormones
 Enzymes
 Human serum albumins
 mAb

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Importance of Amino Acids

Apart from being constituents of proteins and peptides, amino acids serve variety of
functions.
(a) Some amino acids are converted to carbohydrates & are called as glucogenic amino
acids.
(b) Specific amino acids give rise to specialized products, e.g.
 Tyrosine forms hormones such as thyroid hormones, (T3, T4), epinephrine and
norepinephrine and a pigment called melanin.
 Tryptophan can synthesize a vitamin called niacin.
 Glutamate, cysteine and glycine synthesize glutathione.
 Histidine changes to histamine on decarboxylation.
 Serotonin is formed from tryptophan.
(c) Some amino acids such as glycine and cysteine are used as detoxicants of specific
substances. E.g. paracetamol poisoning
(d) Methionine is utilized in the body to transfer methyl group to various substances by
transmethylation.

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