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CHAPTER-1 PROTEIN

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CHAPTER-1
PROTEIN
Sunanda Biswas

Introduction
Protein is common, important and most abundant biological
macromolecules present in all cells. It is composed of amino acids
which are commonly called building blocks of protein. Proteins,
from the Greek proteios, meaning ‘of the first rank’ named by
Jons J. Berzelius in 1938. Protein plays important roles on body
building function which includes all the major structural and
functional aspects of the body. Protein contains Carbon,
Hydrogen, Oxygen and Nitrogen as the major components while
Phosphorus and Sulfur are minor components. All proteins are
polymers of amino acids. About 3000 molecular species of
proteins have been identifies which contributes average 17% of
wet cell mass.
Amino acids
Chemistry
Amino acids are the fundamental units of protein. To form larger
protein molecule amino acids bond together. Structure of amino
acids has been represented in Fig. 1.

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 Fig.1: Structure of Amino acids
Each α- amino acid contains a central carbon atom, called the α-
carbon, connected to a side chain (R group), a hydrogen, a
nitrogen containing amino group, a carboxylic acid group-hence
the name “amino acid”. The ionization state of an amino acids
varies with pH and forms different ionic forms-acidic (Fig 2a),
Zwitterion (Fig 2b) or basic (Fig 2b) due to its amphoteric nature.
Each form varies in structure, size and electric charge as well as
solubility in water.

At low pH At neutral pH At high pH

acidic zwitterion basic
a b c

Fig 2 Amphoteric Nature of Amino acids

Optical isomerism is another important character of amino acids.
When side chain R is other than H, the α-carbon atom (C next to

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the carboxyl group) becomes asymmetric with four different
groups, this type of compounds show optical isomerism- the
ability to rotate the plane of polarization of plane polarized light.
Depending on the configuration of atoms around the asymmetric
carbon, the amino acids rotate in dextrorotatory (D) or
levorotatory (L) configuration. Only L-amino acids are
constituents of proteins which are generally obtained from
mammalian sources. D-amino acids have been identified in some
peptide antibiotics of microbial origin.
Classification
There are twenty different amino acids which are commonly
found in proteins. These amino acids have been classified based
on their polarity of the side chain or R groups. (Table 1)
They have also been classified based on nutritional aspects as
Essential (His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val) and Non-
essential (Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, Pro, Tyr, Ser).
Among these amino acids some amino acids are conditionally
essential (Arg, Cys, Gln Gly Pro, Tyr). Monoaminodicarboxylic
acids and diaminomonocarboxylic acids are acidic (Asp, Glu) and
basic (Arg, His, Lys) amino acids respectively.

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 Table 1: Classification of amino acids with their code
Group Subgroups Name of the 3- 1-
amino acids lette lette
r r
code code
Amino Amino acids with Alanine Ala A
acids nonpolar aliphatic Glycine Gly G
with sidechains Valine Val V
nonpolar Leucine Leu L
sidechain Isoleucine Ile I
s Methionine Met M
Amino acids with Phenylalanin Phe F
nonpolar aromatic e
sidechains Tryptophan Trp W
Imino acids Proline Pro P
Amino Amino Hydroxy Serine Ser S
acids acids amino
Threonine Thr T
with with acids
Tyrosine Tyr Y
polar uncharge
Sulfur- Cysteine Cys C
sidechain d polar
containin
s sidechain
g amino
s
acid
Amino Asparagine Asn N
acid Glutamine Gln Q
amides
Amino Amino Histidine His H
acids acid with Arginine Arg R
with positivel Lysine Lys K
charged y charged
polar sidechain
sidechain s
s Amino Glutamic Glu E
acids acid
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 with Aspartic Asp D
negativel acid
y charged
polar
sidechain
s
Peptides:
The amide bond which covalently link two amino acids to form a
dipeptide is termed as peptide bond or peptide link. This link
forms between the carbonyl group of an amino acid and the
nitrogen of the next amino acid by removing one water (H2O)
molecule as shown in Fig. 3.

R1 and R2 represent the side chains of two different amino acids

Fig. 3 Formation of Peptide Bond

Amino acids that have been incorporated into peptides are called
amino acid residues. Depending on the number of peptide bonds,
the2, 3, 4, 6-10 numbers of amino acid residues linked with

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peptide bond are called as di, tri, tetra, oligo and polypeptides.
Large numbers of amino acid residues linked with peptide chains
are called proteins. The amino acid residue with a free amino
group at one end of the chain is called the N-terminal residue and
the other end with a free carboxylic acid group is known as C-
terminal residue. Names of peptides are derived from names of
amino acid residues. Generally, names of peptides are usually
numbered from their N-terminal end. Properties of shorter chain
length peptides and proteins are different from each other.

Protein structure
The levels of protein structural organization have been divided
into four orders, one leading to the other.
The sequence of amino acids in its peptide chain is known as
primary structure of a protein. It can be well understood from the
primary structure of protein that whether all the amino acids are
present or not, if so in what concentration as well as the sequence
arrangement of amino acids. Primary structure mostly formed by
covalent peptide bonds linking the α- carboxyl carbon of each
amino acids with α-nitrogen of the next amino acids (Fig. 4).

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 Fig 4 Primary Structure of Protein
The secondary structure consists of the three-dimensional
conformation of the protein. Depending on hydrogen bond, amino
acid residues close-up, hold and stabilize this three-dimensional
form of the protein. Detailed studies in chemistry confirmed three
different types of secondary structure of protein viz. α- helix, β-
pleated and triple helix.

The α-helix is right-handed, compact and rigid coiling like

structure as shown in Fig. 5a. Each turn of the helix has a pitch of
540 pm and contains about 3, 6 amino acid residues. The alpha
helix is stabilized by a hydrogen bond between the H-atom
attached to electronegative nitrogen atom of a peptide linkage and
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the electronegative carbonyl oxygen atom of the fourth amino
acid on the amino-terminal side of that peptide bond. The amount
of helix content differs in various proteins.

The β-pleated sheet is a less common, nonhelical, fully extended,

slightly zig-zag, sheet like structure shown in Fig 5b. It is
stabilized by regular hydrogen bonds between α-amide N of a
peptide bond of one peptide strand and the α- carbonyl O of
another peptide linkage of an adjacent peptide strand.
The triple helix structure is formed by three congruent
geometrical helices of polypeptide chains with same axis winding
around one another to form a kind of stiff cable like structure (Fig
5c). It is also known as super helical structure. Examples of triple
helices include collagen like proteins, triplex DNA, triplex RNA.
This type of structure is stronger and relatively rigid compared to
others.

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 Fig 5a: α- helix Fig. 5b: β-pleated Fig 5c: Triple
helix
Fig 5 Secondary Structures of Protein
A protein needs to adopt a final and stable 3-dimentional shape in
order to function properly. The tertiary structure refers to this
configuration of a protein subunit in three-dimensional space
(Fig. 6). The forces that stabilize the tertiary structure are
intermolecular disulfide bonds between cystein residues,
electrostatic interactions between ionic groups of opposite charge
and also hydrogen bonding.

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Fig 6 Schematic representation of the Tertiary Structure of a
protein
Tertiary structure of a protein plays a role in catalytic activity of
proteins like enzymes and in how the hormone regulates receptor
activation. Physiologic alterations of mature tertiary protein
structure occur much more rapidly than does primary sequence.
Many proteins consist of more than one polypeptide chain which
is known as subunit. Quaternary structure refers spatial
arrangement of these subunits of a naïve protein (Fig 7). Due to
presence of two or more subunits, it is called as oligomers. The
subunits are then held together mainly by hydrogen bonds and
ionic bonds between polar amino acid sidechains of surface
residues, and some hydrophobic interactions between nonpolar

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amino acid sidechains of the contact regions. Hemoglobin is the
simple example of quaternary structure. It contains two α and two
β chains. The four chains are held together to give a globular
shape. Quaternary structure allows a protein to have multiple
functions and also to undergo complicated conformational
changes.

Fig 7 Quaternary Structure of a protein

The protein structure and functions of protein also changed by
prosthetic groups attached to them. These types of proteins are
called conjugated protein. According to nature of the prosthetic
group, generic names of those proteins as presented in Table 2.

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 Table 2: Different types of Conjugated proteins
Type of conjugated protein Prosthetic group
Metalloproteins Metal ions ( Zn2+, Fe2+, Cu2+)
Haemoproteins Haeme group
Glycoproteins Carbohydrates
Lipoproteins Lipid
Nucleoproteins Nucleic acid (DNA, RNA)

Properties of proteins
Proteins are macromolecules having high molecular weight. They
are amphoteric in nature and its isoelectric pH depends on relative
numbers of ionic sidechains in the amino acid residues as well as
their ionization exponents (pK).
The relationship of protein with water is complex. Protein
solubility is variable and is affected by the number of polar and
non polar groups and their arrangement along the molecule.
Generally proteins are soluble only in strongly polar solvents such
as water, glycerol or formic acid. In less polar solvents such as
ethanol, proteins are rarely soluble. The solubility depends on
various factors such as salt concentration, pH, molecular size and
hydration of proteins.

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Proteins can be denatured by agents such as heat, X-rays, UV
rays, urea that cause unfolding of polypeptide chains without
causing hydrolysis of peptide bonds.

Classification of proteins
Proteins are classified in many ways. According to on their
overall shape it may be classified as globular proteins and fibrous
proteins.
Globular proteins are compact and spherical and usually are
soluble in water. As globular proteins have less rigid structure,
they show more functional properties compared to fibrous.
Enzymes, hormones, antibodies, transport proteins and respiratory
proteins are included in this group.
Fibrous proteins are long and are less soluble in water. Structural
proteins like collagen, elastin, keratin and fibrin are examples of
this type of proteins.
Based on biologic functions, proteins are classified as enzymes
(dehydrogenases, kinases), storage proteins (ferritin, myoglobin),
structural proteins (collagen, proteogkycans), regulatory proteins
(DNA-binding proteins, peptide hormones), protective proteins
(blood clotting factors, immunoglobulins), transport proteins
(hemoglobin, plasma lipoproteins) and contractile or motile
proteins (actin, tubulin).

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Depending on composition, proteins are also classified as simple
protein i.e composed of amino acids only, conjugated proteins i.e.
composed of simple protein with nonproteins and derived proteins
i.e. produced from natural proteins by various physical and
chemical factors.

Functions of proteins
As mentioned before that protein contributes average 17% of wet
cell mass and they are the agents of biological functions. Proteins
provide energy like carbohydrate and fat, but its major role as
structural component of muscle mass and other tissues. Enormous
number of proteins exhibit diversified functions as described here.
The most varied and most highly specialized proteins with
catalytic activity are known as enzymes. Each enzyme is very
specific in its function and acts only in a particular metabolic
reaction. About all the chemical reactions in a living system and
every step in metabolism and is catalyzed by enzymes.
A protein that serves the function of moving metal ions, organic
molecules or gases like oxygen by binding them referred as
transport protein. On the cell membrane different transport
proteins are located as they enable transport of nutrients like
glucose, amino acids etc across it. Membrane transport proteins
take up metabolite molecules on one side of a membrane,

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transport them across the membrane, and release them on the
other side. Each transport protein is designed to transport a
specific substance as needed. Examples include actin and myosins
are essential for locomotion of the whole organism as they are
involved in muscle contraction.
The structural proteins serves as structural component of the body
are keratins, collagens and elastins, which form structural
framework of various organs and connective tissues and provide
strength and mechanical support. Examples include collagen
present in tendons and cartilage provides tensile strength and
elastin, present in the ligament provides elastic property to organs
like uterus, arteries, and lung.
The immunoglobulins or antibodies produced by the lymphocytes
of vertebrates acts as protective protein. They specifically
recognize and neutralize the invading microorganisms as well as
any other intruding antigen.
Specific proteins bind and reservoir specific substances in cells or
in extracellular fluid are known as storage proteins. Ferritin acts
as a storage protein as it stores iron in spleen and liver cells.
A number of hormones as either peptide or protein in nature
regulate metabolic functions of a cell. Proteins also maintain
ionic and fluid distribution on two sides of membrane.

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Protein synthesis
Protein synthesis is one of the most fundamental biological
processes by which individual cells construct their specific
proteins. This process of protein synthesis takes place in multiple
ribosomes simultaneous and all throughout the cell cytoplasm.
This process is very complex and is determined by the genetic
information present in the DNA. Through this process biological
cells generate new proteins, as well as save loss of cellular
proteins via degradation or export. Briefly protein synthesis
process is carried out in two steps – Transcription and
Translation.
Transcription is the first step in which the pattern of one gene is
copied from DNA sequence to RNA sequence. In this process the
information in the DNA is transferred to the cytoplasm by way of
synthesis of a template called messenger RNA (mRNA) by the
enzyme RNA polymerase. Mainly three steps take place to
complete this process. In the initiation step RNA polymerase
enzyme binds to a region of a gene called promoter and makes a
strand of mRNA with a complementary sequence of base. In the
next elongation step addition of nucleotides occurs to the mRNA
strand, RNA polymerase reads the unwound DNA strand and
builds the mRNA molecule. In the termination step sequence in
the gene stops and mRNA detaches from DNA.

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After leaving nucleus, mRNA undergoes several modifications as
removal of introns, adding of an adenine based poly- A tail to
protect the ends of the mRNA molecule before translation.
In translation, occurs in cytoplasm, mRNA along with transfer
RNA (tRNA) and ribosomes work together to produce proteins.
In the initiation step mRNA anchored by ribosomes, which
contain ribosomal nucleic acid (rRNA), the translation begins
with the reading of the first triplet. Small tRNA molecules bring
in the individual amino acids and attach them to the mRNA. In
elongation step this pattern continues and forms a chain of amino
acids. In termination step, when a stop signal is reached, the entire
complex disassociates. Ribosomes, the mRNA, the tRNA and the
enzymes are then splits back for another translational event.
The peptide now dissociates may undergo post translational
modifications like cleavage of signal peptide, methylation,
hydroxylation, glycosilation etc. in the golgi apparatus and finally
the protein is either released into the extracellular fluid or stored
in granules inside the cell for future release.
Protein quality
The quality of protein is determined by the quantity of essential
amino acids present in it. Protein quality evaluation aims to
determine the capacity of food protein sources and diets to meet
the protein and essential amino-nitrogen requirements, i.e. to

41
satisfy the metabolic needs for amino acids and nitrogen. Animal
proteins except gelatin posses all essential amino acids in
adequate amounts but plant proteins show limiting in one or more
amino acids. So, animal based foods tend to be good sources of
complete protein compared to plant based foods. Inadequacy of
even a single essential amino acid will grossly interfere with body
protein synthesis. Quality of protein is influenced by the amino
acid content, amino acid imbalance, interference of non available
carbohydrates and trypsin inhibitors and influence of heating and
processing.
Evaluation of Protein Quality
Following methods are used to assay the nutritional quality of
proteins a) chemical score, b) biological value, c) net protein
utilization and d) protein efficiency ratio.

a) Chemical score: The chemical scoring is one of the

easiest methods if all appropriate instruments for
measuring amino acid content of a protein are available. It
is measured by determining the ratio of an mg of the
limiting amino acid for that protein compared it with same
amount of standard protein like hen’s egg protein
multiplied by 100. Information on the most limiting
amino acid is generally satisfied at the chemical score.

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 Chemical score
mg of limiting amino acid⁄g test protein
= X 100
mg of same amino acid⁄g egg protein
b) Biological value (BV): Biological value of a protein is
measured as the percentage of absorbed nitrogen that is
retained for use in growth of maintenance.
Retained nitrogen
Biological value = X 100
Absorbed nitrogen
After feeding one group a non-protein diet while another group
test diet containing 10 percent for 10 days, urinary, faecal
nitrogen are analyzed. The biological value can be arrived at by
the following equation.

I − (F − 𝐹𝑚 ) − (U − 𝑈𝑒 )
BV = X 100
I − (F − 𝐹𝑚 )

Where, I, F, U are dietary, faecal and urinary nitrogen on the test

diet and Fm and Ue are faecal and urinary nitrogen on a protein
free diet.
c) Net protein utilization (NPU): Formula of net protein
utilization is –

Retained nitrogen
Net protein utilisation = X 100
Absorbed nitrogen
Losses of nitrogen in digestion is not accounted in biological
value, net protein utilization includes that. It is equal to BV x

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availability. In human it is calculated from nitrogen balance
studies.
d) Protein efficiency ratio (PER): Protein efficiency ratio
is defined as the weight gain per gram of protein intake.
This parameter was measured in groups of young albino
rats are fed for a period of 4 weeks on different protein
diets at 10 percent protein. PER is calculated in following
formula by taking records of the gain in body weight and
protein intake of rats.

Gain in weight (g)

PER =
Protein intake (g)

The WHO and the US FDA adopted Protein Digestibility

Corrected Amino Acid Score (PDCAAS) as the official assay for
evaluating protein quality. It represents the amino acid score after
correcting digestibility.

References:
 Ferrier D.R. Biochemistry. 6th Edition Lipincott, Williams
& Wilkins 2013
 Das D. Biochemistry. 14th Edition. Academic Publishers
2010.

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  Lehniniger Principles of Biochemistry. Eds. David L.
Nelson & Michael M. Cox 6th Edition Publishers W.H.
Freeman, Lipincott, Williams & Wilkins 2012
 Satyanarayana U. Biochemistry. Books & Allied (P)Ltd.
2008.
 Stryer L.(Ed). Biochemistry, 2nd Edition CBS publishers and
Distributors (India), 1981.
 Textbook of Human Nutrition. Eds Bamji MS,
Krishnaswamy K and Brahmam GNV 4th Edition Publishers
Oxford & IBH Publishing Co. Pvt.Ltd.

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