1Proteins:

Proteins are essential constituents of protoplasm. They

differ from carbohydrates and lipids by always containing
nitrogen and sometimes phosphorus and sulphur.
Proteins contain: carbon-54%; hydrogen-7%; nitrogen-
16%; oxygen-22%. Some may contain, sulphur-1%; while
others, phosphorus-0.6%.

Proteins are among the most abundant organic

molecules; in most living systems they make up 50
percent or more of the dry weight. Only plants, with their
high cellulose content, are less than half protein. The
protein molecule is built up by the union of a large
number of amino acids. The amino acids should be
considered as the units with which the protein molecule
is composed.
It is to be specially noted that the term protein is applied
only to the complex protein molecule responding to the
characteristic tests of protein, e.g., copper-protein
(biuret) test, etc., due to the presence of two or more
peptide linkages whereas amino acids do not respond to
the characteristic tests of protein; hence they are called
one of the non-protein nitrogenous (NPN) constituents.

2 Functional Importance of
Proteins:

Protein has got multiple functions of which certain

physiological importance’s are described below:

(a) Protein acts as a growth material for the organism,

(b) Structures of living materials are composed of
different types of protein molecules,

(c) It also acts as a part of fuel of the organism,

(d) All the pituitary hormones, hypothalamic-releasing

factors (R.F.), certain placental hormones, pancreatic
hormones, etc., are proteins in nature,

(e) Similarly all enzymes are proteins in nature.

3 Structure of Proteins:

Fibrous Proteins:
In general, fibrous proteins have a regular, repeated
sequence of amino acids and so a regular, repetitious
structure. An example is collagen, which makes up about
one-third of all the protein in vertebrates. The basic
collagen molecule is composed of three very long
polymers of amino acids-about 1,000 amino acids per
chain.

These three polymers, which are made up of repeating

groups of amino acids, are held together by hydrogen
bonds linking amino acids of different chains in a tight
coil. The molecules can coil so tightly because every third
amino acid is glycine , the smallest of the amino acids.

Collagen performs many functions in the body. Consider

a cow. Tendons, which link muscle to bone, are made up
of collagen fibers in parallel bundles; thus arranged, they
are very strong but do not stretch. The cow’s hide, by
contrast, is made up of collagen fibrils arranged in an
interlacing network laid down in sheets.

Even its corneas-the transparent coverings of the

eyeballs-are composed of collagen. Boiling in water
disperses the polymers of collagen into shorter chains,
which we know as gelatin. Other fibrous proteins include
elastin , present in the elastic tissue of ligaments, silk,
and keratin.

Structural Uses of Globular Proteins:

Some structural proteins are globular. For example,

microtubules, which function in a variety of ways inside
the cell, are made up of globular proteins. They are long
hollow tubes-so long that their entire length can seldom
be traced in a single microscopic section.

They apparently act as internal skeletons, stiffening parts

of the cell body. They also may serve as tracks along
which substances can move inside the cell.

The formation of a new cellulose cell wall in a plant can

be predicted by the appearance at the site of large
numbers of microtubules; when cellulose fibrils are being
laid down outside a plant cell membrane, as a cell wall
forms or grows, it is possible to detect microtubules
inside the cell aligned in the same direction as the fibrils
outside.

Chemical analysis shows that each microtubule consists

of a very large number of subunits, each of which is a
globular protein made up of two polypeptide chains. The
two polypeptides fit together because of their
complementary configurations, forming approximately
spherical subunits. The subunits assemble themselves
into tubules, adding on length as required. When their
job is over, they separate. Assembling requires an input
of energy but just how it is triggered is not known.

4. Properties of Proteins:

Proteins are colloidal in nature but many of them can be

crystallised. They are generally soluble in water, weak
salt solutions, dilute acids and alkalies. Each protein has
got a particular isoelectric point at which it is
precipitated. These precipitates again dissolve when the
reaction of the medium is shifted from isoelectric point
or pH.

During precipitation proteins do not undergo any

intramolecular change. They simply separate out because
the medium is not favourable for solution. Most of the
proteins undergo coagulation by heat or acid.
Coagulation involves intramolecular change. There is
another kind of change which the proteins undergo,
called denaturation.

This may be done by many kinds of chemical or physical

treatment, such as shaking, change of temperature,
change of reaction, addition of neutral salts, etc. The
exact nature of change undergone during denaturation is
not known. It is probable that the molecular
arrangement alters. All proteins show the characteristic
properties of colloids.
Proteins not only differ from one another in the number
and variety of amino acids they contain but also in
chemical structure, physical and physiological properties.
There are infinite varieties of proteins, each one differing
from the rest in certain characteristics. The proteins of a
particular tissue of an animal are quite different from
those of the same tissue of another species.

Moreover, in the same species, each type of tissue

contains proteins which are distinct from those found in
the other tissues. No two proteins are found to be
exactly the same in their physiological properties. This is
another characteristic of proteins as a class, which are
not found with the carbohydrates or fats. Vegetable
proteins differ from animal proteins in the fact that the
former is generally poorer in essential amino acids.
5. Identification of Proteins:

Proteins can be identified by three groups of tests:

1. Colour reaction,

2. Coagulation reaction, and

3. Precipitation reaction.

1. Colour Reaction:

It depends upon the characteristic radicles in the

molecule.
i. Biuret Tests (Piotrowski’s Rose’s):

To 2 to 3 ml of the protein solution in a test tube, when

an equal volume of concentrated caustic soda solution
and one drop or two drops of 1% copper sulphate
solution are added, a distinct violet colour develops. This
reaction is due to the presence of peptide linkage in the
protein molecule.

ii. Xanthoproteic Reaction:

To 2 to 3 ml of the protein solution when few drops of

concentrated nitric acid are added, a white precipitate
forms. It is boiled for a minute the precipitate turns
yellow and partly dissolves to furnish a yellow solution. It
is then cooled and concentrated caustic soda is added till
the reaction is alkaline. The yellow colour change to an
orange one. This reaction is due to the presence of the
phenyl group in the protein molecule.
iii. Millon’s Reaction:

To 5 ml of the protein solution when few drops of

Millon’s reagent are added-a white precipitate forms
which on heating changes to a brick-red coagulum. This is
due to the presence of the tyrosine in the protein
molecule.

iv. Molisch’s Reaction:

To 3 or 4 ml of protein solution, three or four drops of an

alcoholic solution of thymol are added. The solution is
well mixed and to it few drops of concentrated sulphuric
acid are added when a distinct purple or violet ring
develops at the junction of the two liquids. The reaction
is due to the presence of carbohydrate radicles in the
protein molecule.

v. Adamkiewicz’s Reaction:
To 3 or 4 ml of protein solution excess of glacial acetic
acid is added and heated. It is cooled and few ml of
concentrated sulphuric acid are allowed to flow down
the side of the inclined test tube, a purple colour
develops at the junction of two liquids within a short
time. The reaction is due to presence of tryptophan in
protein molecule.

vi. Nitroprusside Reaction:

To a solution of protein, add sodium nitroprusside in

ammoniacal solution. A reddish colour is produced. This
is due to the presence of sulphydryl group in the protein
molecule. (Disulphide groups are to be reduced first to
sulphydryls by suitable reducing agent to give this
reaction.)

2. Coagulation Reaction:
Albumin and globulin solution when heated, fine flocculi
appear. They are denatured.

3. Precipitation Reaction:

In a protein solution the proteins may be precipitated by;

(a) Acids, e.g. tannic acid, picric acid, trichloro-acetic acid

and phosphotungstic acid,

(b) Salts of heavy metals, and

(c) Alcohol.

Formation of the Protein Molecule:

The synthesis of protein molecule takes place by the
union of the -NH2 group of one amino acid with the -
COOH group of another.

For instance:

In this way infinite number of amino acids may join up in

straight chains. This junction, CO-NH, through which the
amino acids become joined together, is called the
peptide linkage.

It is obvious that in such a simple chain there will be one

free -NH2 group at one end and another free -COOH
group at the other. It sometimes so happens that such a
straight chain bends upon itself and the basic radicle at
one end combines with the acid radicle on the other by
forming the same peptide linkage.

In this way a ring structure is produced. These

compounds are called diketopiperazines. The following
scheme – shows the formation of diketopiperazine by
the condensation of two molecules of glycine.

Amino Acids:

The amino acid is an organic acid in which one or more

hydrogen atoms are replaced by -NH, group. Thus it
contains at least a free amino group (-NH2) and a
carboxyl group (-COOH). The empirical formula is R-
CH.NH2, COOH. Amino acids are amphoteric in reaction
and form salts with both acids and bases.

The amino acids in the body are almost all a-amino acids.
They should be regarded as derivatives of saturated fatty
acids in which the amino group is attached to that carbon
atom which is situated in the a-position, i.e. next to the
COOH group. Amino acids are colurless, crystalline
substances, soluble in water, easily diffusible and except
glycine all are optically active.
[10:53 PM, 8/9/2024] Aditya: Isoelectric pH:

Each of the amino acids has at least one carboxyl group (-

COOH) and one amino group (-NH2). For this reason they
are called ampholytes. This characteristic of amino acid is
also present in intact protein molecule due to the
presence of two terminal free amino acids. However
there may be more than one acidic or basic group
depending on whether they are dicarboxylic or diamino
acids. Due to amphoteric nature, in acid solution protein
molecule is positively charged and in alkaline one
negatively.

At certain pH the number of positive charge is equal to

the number of negative charge and protein remains as
zwitterion form. The isoelectric pH or point of a protein is
the pH at which the protein does not migrate in an
electric field.
At this particular pH the protein molecule does not move
either to the positive or negative pole, if placed in an
electric field. All proteins, at its isoelectric pH, have got
least osmotic pressure, swelling capacity, viscosity,
solubility and mobility or migrating power. The proteins
are thus precipitated at its isoelectric pH.

6. Protein Molecule:

Some evidence has accumulated to suggest that the

amino acids in the protein molecule are arranged in a
definite pattern. This is more indicated by the fact that
many proteins form well-defined crystals. From X-ray and
other observations the existence of two types of proteins
is revealed; (he fibrous form with elongated molecules
and another rounded form with globular molecule.
Keratin is an example of the fibrous type. Keratin may
exist in two forms α and β.
By stretching α-keratin, β-keratin is obtained. When the
stretch is removed, α-kertatin becomes reconverted into
β-keratin. If however the stretched hair is subjected to
steam, it loses its power of recovery and becomes set
permanently as β-keratin. In this way permanent waving
of hair is done. X-ray photographs suggest that the
keratin molecule consist of thin bundles of polypeptide
chains joined together in a zigzag manner.
[10:55 PM, 8/9/2024] Aditya: Wrinch suggests that the
globular type of proteins are also formed in similar lines
but vary in detail. She holds that in a folded polypeptide
chain a linkage occurs between the NH of one peptide
link with the CO of the neighbouring one. Thus a poly-
peptide chain of this folded type may be closed into
hexagonal loops. Six amino acids in a closed polypeptide
chain would give a pattern in which the centre is a
hexagon.

Such a molecule is called cyclol. A series of cyclols with

18, 30, 42, 54 etc., amino acids could be formed resulting
in a sheet like molecule with repeating pattern. Extension
of the sheet may occur in any plain and in this way
globular types of protein molecules are formed.

7. Post-Translational Processing of
Proteins:

Post-translational modifications are the chemical

modifications that most of the proteins which undergo
before becoming functional in different body cells. It
plays a crucial role in generating the heterogeneity in
proteins and also helps in utilizing identical proteins for
different cellular functions in different cell types. The
modifications occurring at the peptide terminus of the
amino acid chain play an important role in translocating
them across biological membranes. Translocated
proteins carry an N-terminal extension of about twenty
amino acids, termed a signal peptide; it binds to a
receptor in the membrane as soon as it is synthesized
and emerges from the ribosome.
The signal peptide is recognized by a multi-protein
complex termed the signal recognition particle (SRP).
This signal peptide is removed following passage through
the endoplasmic reticulum membrane. These include
secretory proteins in prokaryotes and eukaryotes and
also proteins that are intended to be incorporated in
various cellular and organelle membranes such as
lysosomes, chloroplast, mitochondria and plasma
membranes.

Sometimes in eukaryotes different types of functional

proteins are produced by proteolytic cleavage at multiple
points in the protein chain, in which one gene codes for
multiple products. The best studied example is the
complex of polypeptide hormones produced by the
pituitary gland.

The major post translational modifications are:

I. Proteolytic Cleavage:

Most proteins undergo proteolytic cleavage following

translation. The simplest form of this is the removal of
the initiation methionine. Many proteins are synthesized
as inactive precursors that are activated under proper
physiological conditions by limited proteolysis. Inactive
precursor proteins that are activated by removal of
polypeptides are termed proproteins. Certain proteins
particularly of the enzyme class are synthesized as
inactive precursors called zymogens. Zymogens are
activated by proteolytic cleavage such as is the situation
for several proteins of the blood clotting cascade.

The preproprotein insulin secreted from the pancreas

has a prepeptide. After cleavage of the 24 amino acid
signal peptide the protein folds into proinsulin, which is
further cleaved yielding active insulin, composed of two
peptide chains linked together through disulfide bonds.
II. Chemical Modification:

The chemical modification mainly includes methylation,

sulfation, phosphorylation, lipid addition, and
glycosylation.

(a) Glycosylation:

Many proteins, particularly in eukaryotic cells, are

modified by the addition of carbohydrates, a process
called glycosylation. Glycosylation in proteins results in
addition of a glycosyl group to asparagine, hydroxylysine,
serine, or threonine.

(b) Acylation:
Acylation involves of the addition of an acyl group,
usually at the N-terminus of the protein. In most cases
the initiator methionine is hydrolyzed and an acetyl
group is added to the new N-terminal amino acid. Acetyl-
CoA is the acetyl donor for these reactions.

(c) Methylation:

The most common methylations are on the s-amine of

lysine residues, occurs on nitrogen and oxygen. The
activated methyl donor is S-adenosylmethionine (SAM).
Methylation of the oxygen of the R-group carboxylates of
gutamate and aspartate also takes place and forms
methyl esters. Proteins can also be methylated on the
thiol R- group of cysteine. Methylation of histones in
DNA is an important regulator of chromatin structure
and consequently of transcriptional activity.

(d) Phosphorylation:
Post-translational phosphorylation occurs as a
mechanism to regulate the biological activity of a protein
in animal cells. In animal cells, serine, threonine and tyro-
sine are the amino acids subject to phosphorylation. As
an example, the activity of numerous growth factor
receptors is controlled by tyrosine phosphorylation.
Other relevant examples are the phosphorylations that
occur in glycogen synthase and glycogen phosphorylase
in hepatocytes in response to glucagon release from the
pancreas. Phosphorylation of synthase inhibits its
activity, whereas, the activity of phosphorylase is
increased. These two events lead to increased hepatic
glucose delivery to the blood.

(e) Sulfation:

Sulfate modification of proteins occurs at tyrosine

residues such as in fibrinogen and in some secreted
proteins (e.g.: gastrin). The universal sulfate donor is 3′-
phosphoadenosyl-5′-phosphosulphate (PAPS). Since
sulfate is added permanently it is necessary for the
biological activity and not used as a regulatory
modification like that of tyrosine phosphorylation.

(f) Vitamin C-Dependent Modifications:

Modifications of proteins that depend upon vitamin C as

a cofactor include proline and lysine hydroxylations and
carboxy terminal amidation. The hydroxylating enzymes
are identified as prolyl hydroxylase and lysyl hydroxylase.
The donor of the amide for C-terminal amidation is
glycine. The most important hydroxylated proteins are
the collagens. Several peptide hormones such as oxytocin
and vasopressin have C-terminal amidation.

(g) Vitamin K-Dependent

Modifications:

Vitamin K is a cofactor in the carboxylation of glutamic

acid residues. The result of this type of reaction is the
formation of a γ- carboxyglutamate (gamma-
carboxyglutamate), referred to as a gla residue. The
formation of gla residues within several proteins of the
blood clotting cascade is critical for their normal function.
The presence of gla residues allows the protein to
chelate calcium ions and thereby render an altered
conformation and, biological activity to the protein. The
coumarin-based anticoagulants, warfarin and dicumarol
function by inhibiting the carboxylation reaction.

(h) Selenoproteins:

Selenium is a trace element and is found as a component

of several prokaryotic and eukaryotic enzymes that are
involved in redox reactions. The selenium in these
selenoproteins is incorporated as a unique amino acid,
selenocysteine, during translation. A particularly
important eukaryotic selenoenzyme is glutathione
peroxidase. This enzyme is required during the oxidation
of glutathione by hydrogen peroxide (H2O2) and organic
hydroperoxides.
8. Molecular Weights of Proteins:

Protein molecules are large and have varying molecular

weights. Their molecular weights cannot be determined
by ordinary methods. The most important method for
their determination is based upon the measurement of
sedimentation rates in the ultracentrifuge. They can also
be determined by osmotic pressure measurement, light
scattering, etc.

Molecular weights of some of the typical proteins from

different sources are as follows: salmine (salmon sperm)
– 5,600; cytochrome (heart) -156,000; lactalbumin (milk)
-17,400; gliadin (wheat) – 27,400; pepsin (stomach) –
35,500; insulin (pancreas) – 40,900; haemoglobin
(human) – 63,000; myogen (muscle) -150,000;
thyroglobulin (thyroid) – 628,000; myosin (muscle) -
1,000,000; virus (tomato) – 7,600,000; mosaic virus
(tobacco) – 60,000,000.

It has been shown by Svedberg that by a change in the

protein concentration or in the pH or in the salt
concentration of the medium, proteins in solution, may
easily dissociated into smaller component units. If the
original condition is resorted the fragments may re-
associate and form the original molecule. This shows that
proteins of higher molecular weights are built up by
conglomeration of smaller units.

Virus Proteins:

These are nucleoproteins of special interest. Several

varieties of virus proteins with very high molecular
weights have been isolated in crystalline form, from the
juice of plants suffering from virus diseases. The virus
protein of tobacco mosaic disease has a molecular
weight of about 60,000,000, an isoelectric point or pH
3.49 and contains about 5% nucleic acid. It is a highly
interesting fact that even minute amount of pure
recrystallised virus proteins induces the virus disease
when injected into healthy plants.

One millionth of a milligram of tobacco mosaic protein

invariably infects a healthy plant and from such infected
plants large quantities of the same protein can be
recovered. This astonishing property of the virus
proteins, in directing the metabolism of the plant cells so
that they synthesise more of the same protein, is unique
in a chemical compound and is suggestive of the
reproduction of a living organism rather than the
elaboration of a non-living chemical molecule. The
evidence accumulated so far indicates that these giant
molecules are purely of chemical nature although we
have no absolute proof that they are dead.

Electrophoresis:
The property of migration of protein molecules in an
electric field has been utilised by Tiselius for
electrophoretic method for the separation of protein
molecules. Protein solution in an alkaline buffer has
maximum number of negative charges and so the protein
molecules move towards the positive pole. Different
protein molecules, due to the presence of charged
particles in different amounts, move towards the pole at
different rates which can be optically measured.

This complicated procedure has been simplified by

application on paper. Here a strip of filter paper is moist-
ened with suitable buffer and placed in between two
glass plates The free ends of the paper dip into two small
reservoirs of buffer solution, and either positive or
negative electrode is placed in each of them. On passing
electric current the protein molecules migrate towards
the anode. After definite time interval, the paper is taken
put and washed, and a suitable stain is applied to localise
the presence of difference in protein molecules

Classification of Proteins

Based on the molecular shape, proteins can be classified

into two types.

1. Fibrous Proteins:

When the polypeptide chains run parallel and are held

together by hydrogen and disulfide bonds, then the fiber-
like structure is formed. Such proteins are generally
insoluble in water. These are water-insoluble proteins.
Example – keratin (present in hair, wool, and silk) and
myosin (present in muscles), etc.

2 Globular Proteins:

This structure results when the chains of polypeptides

coil around to give a spherical shape. These are usually
soluble in water.

Example – Insulin and albumins are common

examples of globular proteins.

Levels of Protein Structure

1. Primary Structure of Protein

The Primary structure of proteins is the exact ordering of
amino acids forming their chains.
The exact sequence of the proteins is very important as it
determines the final fold and therefore the function of
the protein.
The number of polypeptide chains together form
proteins. These chains have amino acids arranged in a
particular sequence which is characteristic of the specific
protein. Any change in the sequence changes the entire
protein.
The following picture represents the primary protein
structure (an amino acid chain). As you might expect, the
amino acid sequence within the polypeptide chain is
crucial for the protein’s proper functioning. This
sequence is encrypted in the DNA genetic code. If
mutation is present in the DNA and the amino acid
sequence is changed, the protein function may be
affected.
The protein ‘s primary structure is the amino acid
sequence in its polypeptide chain. If proteins were
popcorn stringers designed to decorate a Christmas tree,
a protein ‘s primary structure is the sequence in which
various shapes and varieties of popped maize are strung
together.

Covalent, peptide bonds which connect the amino acids

together maintain the primary structure of a protein.

All documented genetic disorders, such as cystic fibrosis,

sickle cell anemia, albinism, etc., are caused by mutations
resulting in alterations in the primary protein structures,
which in turn lead to alterations in the secondary ,
tertiary and probably quarterly structure.

Amino acids are small organic molecules consisting of a

chiral carbon with four substituents. Of those only the
fourth the side chain is different among amino acids.

2. Secondary Structure of Protein

Secondary structure of protein refers to local folded
structures that form within a polypeptide due to
interactions between atoms of the backbone.

The proteins do not exist in just simple chains of

polypeptides.
These polypeptide chains usually fold due to the
interaction between the amine and carboxyl group of the
peptide link.
The structure refers to the shape in which a long
polypeptide chain can exist.
They are found to exist in two different types of
structures α – helix and β – pleated sheet structures.
This structure arises due to the regular folding of the
backbone of the polypeptide chain due to hydrogen
bonding between -CO group and -NH groups of the
peptide bond.
However, segments of the protein chain may acquire
their own local fold, which is much simpler and usually
takes the shape of a spiral an extended shape or a loop.
These local folds are termed secondary elements and
form the proteins secondary structure

(a) α – Helix:

α – Helix is one of the most common ways in which a

polypeptide chain forms all possible hydrogen bonds by
twisting into a right-handed screw with the -NH group of
each amino acid residue hydrogen-bonded to the -CO of
the adjacent turn of the helix. The polypeptide chains
twisted into a right-handed screw.

(b) β – pleated sheet:

In this arrangement, the polypeptide chains are stretched

out beside one another and then bonded by
intermolecular H-bonds. In this structure, all peptide
chains are stretched out to nearly maximum extension
and then laid side by side which is held together by
intermolecular hydrogen bonds. The structure resembles
the pleated folds of drapery and therefore is known as β
– pleated sheet

3. Tertiary Structure of Protein:

This structure arises from further folding of the

secondary structure of the protein.
H-bonds, electrostatic forces, disulphide linkages, and
Vander Waals forces stabilize this structure.
The tertiary structure of proteins represents overall
folding of the polypeptide chains, further folding of the
secondary structure.
It gives rise to two major molecular shapes called fibrous
and globular.
The main forces which stabilize the secondary and
tertiary structures of proteins are hydrogen bonds,
disulphide linkages, van der Waals and electrostatic
forces of attraction.
 4. Quaternary Structure of Protein

The spatial arrangement of various tertiary structures

gives rise to the quaternary structure. Some of the
proteins are composed of two or more polypeptide
chains referred to as sub-units. The spatial arrangement
of these subunits with respect to each other is known as
quaternary structure.

The exact amino acid sequence of each protein drives it

to fold into its own unique and biologically active three-
dimensional fold also known as the tertiary structure.
Proteins consist of different combinations of secondary
elements some of which are simple whereas others are
more complex. Parts of the protein chain, which have
their own three-dimensional fold and can be attributed
to some function are called “domains”. These are
considered today as the evolutionary and functional
building blocks of proteins.
Many proteins, most of which are enzymes contain
organic or elemental components needed for their
activity and stability. Thus the study of protein evolution
not only gives structural insight but also connects
proteins of quite different parts of the metabolism