UNIT 1

INTRODUCTION TO PROTEINS &

PEPTIDES
• Proteins are the most abundant organic molecules
of the living system.
• They occur in the every part of the cell and
constitute about 50% of the cellular dry weight.
• Proteins form the fundamental basis of structure and
function of life.
• In 1839 Dutch chemist G.J.Mulder while investing the
substances such as those found in milk, egg, found
that they could be coagulated on heating and were
nitrogenous compounds.
• The term protein is derived from a Greek word
proteios, meaning first place.

• Berzelius ( Swedish chemist ) suggested the name

proteins to the group of organic compounds that are
utmost important to life.

• The proteins are nitrogenous macromolecules

composed of many amino acids.
 Biomedical importance of proteins:
• Proteins are the main structural components of the
cytoskeleton. They are the sole source to replace
nitrogen of the body.

• Bio chemical catalysts known as enzymes are

proteins.

• Proteins known as immunoglobulins serve as the

first line of defence against bacterial and viral
infections.
• Several hormones are protein in nature.

• Structural proteins like actin and myosin are

contractile proteins and help in the movement of
muscle fibre.
Some proteins present in cell membrane, cytoplasm
and nucleus of the cell act as receptors.

• The transport proteins carry out the function of

transporting specific substances either across the
membrane or in the body fluids.
• Storage proteins bind with specific substances
and store them, e.g. iron is stored as ferritin.
• Few proteins are constituents of respiratory
pigments and occur in electron transport chain,
e.g. Cytochromes, hemoglobin, myoglobin
• Under certain conditions proteins can be
catabolized to supply energy.
• Proteins by means of exerting osmotic pressure
help in maintenance of electrolyte and water
balance in the body.
 Amino acids

• Amino acids are a group of organic compounds

containing two functional groups – amino and
carboxyl.

• The amino group ( -NH2) is basic while the carboxyl

group ( -COOH ) is acidic in nature

• There are about 300 amino acids occur in nature.

Only 20 of them occur in proteins.
 Structure of amino acids:
• Each amino acid has 4 different groups attached to
α- carbon ( which is C-atom next to COOH). These 4
groups are : amino group, COOH gp,Hydrogen atom
and side Chain (R)

NH3+ C COO-

H
 Optical isomers of amino acids

• If a carbon atom is attached to four different groups,

it is asymmetric and therefore exhibits isomerism.

• The amino acids ( except glycine ) possess four

distinct groups ( R, H, COO-, NH3+) held by a α-
carbon.

• Thus all the amino acids have optical isomers.

• The proteins are composed of L- α-amino acids.

 Classification of amino acids

• Amino acid classification based on the structure

• The 20 standard amino acids found in protein

structure are divided into seven distinct groups.

• 1.Aliphatic amino acids:

• 2.Hydroxyl group containing amino acids:

• 3.Sulfur containing amino acids:

•
• 4.Acidic amino acids and their amides:

• 5.Basic amino acids:

• 6.Aromatic amino acids:

• 7.Imino acids:
 Aliphatic amino acids
• These are monoamino monocarboxylic acids. This
group consists of most simple amino acids.

• A) Glycine - Gly - G
B) Alanine - Ala - A
C) Valine - Val - V
D) Leucine - Leu - L
E) Isoleusine - Ile - I
 Glycine:
• Small, simple amino acid. R – group is hydrogen

• It is a non essential amino acid.

• Glycine is allosteric inhibitor of glutathione

synthetase.

NH3+ C COO-

H
GLYCINE
H
 Alanine:
• It is a non essential amino acid. Alanine is allosteric
inhibitor of glutathione synthetase.

• D-Alanine: is a component of bacterial cell wall.

• Β-Alanine is found in pantothenic acid.

NH3+ C COO-

CH3
ALANINE
 Valine:
• It is essential amino acid.

• It has an aliphatic hydrophobic isopropyl ( 3Carbon )

side chain.
H

NH3+ C COO-

CH

CH3 CH3
Branched chain
 Leucine:
• It is an essential amino acid.

• It has an aliphatic hydrophobic isopropyl ( 4Carbon )

side chain.
H

NH3+ C COO-

CH2
Branched chain
CH

CH3 CH3
 Isoleucine:
• It is an essential amino acid.

• It has an aliphatic hydrophobic isopropyl (4Carbon)

side chain
H

NH3+ C COO-

CH CH3
Branched chain
CH2

CH3
 Glycine

Lysine Isoleucine
Hydroxyl gr. containing amino acids (-OH)

• Serine - Ser - S

• Threonine - Thr - T

• Tyrosine - Tyr - Y
 Serine:

• It is a non essential amino acid.

• It has a alcohol group, is a site for phosphorylation

of many proteins.

+ C COO-
NH3
Hydroxyl
CH2 OH
 Threonine:
• It is a essential amino acid.

• Threonine alcohol side group is a target for

phosphorylation of proteins.
H

NH3+ C COO-

CH OH Hydroxyl
CH3
 3.Sulfur containing amino
acids:
• Cysteine - Cys - C

• Methionine - Met - M
 Cysteine:
• It is a non essential amino acid.

• Cystine, another important sulfur containing amino

acid, is formed by the condensation of two
molecules of cysteine.

NH3+ C COO-
Sulfhydryl
CH2 SH
 Methionine:
• It is an essential amino acid.

• In genetic code methionine is coded by codon AUG.

This codon is called start codon. Methionine is first
amino acid used to build a protein chain.

NH3+ C COO-

CH2
Thioether
CH2

S CH3
 4.Acidic amino acids and their amides:

• Aspartic acid - Asp - D

• Aspargine - Asn - N

• Glutamic acid - Glu - E

• Glutamine - Gln - Q
 Aspartic acid :
• It is a non essential amino acid.

• Aspartic is capable of forming ionic bonds and

involved in chemical reactions.

α
NH3 +
C COO-
β Carboxyl
β CH2 COO-
 Glutamic acid :
• It is a non essential amino acid.

• Vitamin K2 carboxylates glutamate residues in certain

proteins to give carboxy glutamate. This modification
allows protein to bind calcium an essential event in blood
clotting cascade.
H
α
NH3+ C COO-

β CH2 γ Carboxyl

γ CH2 COO-
 Aspargine:
• It is non essential amino acid.

NH3+ C COO-

CH2

C=O Amide
NH2
 Glutamine :
• It is a non essential amino acid.

• It is very important compound in transamination

reactions. H

NH3+ C COO-

CH2

CH2
Amide
C=O

NH2
 5.Basic amino acids:
• Lysine - Lys - K

• Arginine - Arg - R

• Histidine - His - H
 Lysine :
• It is an essential amino acid. These are strongly
polar.

NH3+ C COO-

CH2

CH2

CH2
Amide
CH2 NH3+
 Arginine :
• It is a semi essential amino acid.

• It contain guanidino group and is monocarboxylic acid.

• It is an intermediate in urea cycle and is precursor for nitric

oxide. H
NH3+ C COO-

CH2

CH2

CH2

NH Guanidino
C=NH2+
NH2
 Histidine:
• It is an semi essential amino acid.

• It contains imidazole ring.

H
NH3+ C COO-

CH2

Imidazole
HN N
 6.Aromatic amino acids:

• Phenyl alanine - Phe - F

• Tyrosine - Tyr - Y

• Tryptophan - Trp - W
 Phenyl alanine :

• It is an essential amino acid.

• It contains benzene ring.
H

NH3+ C COO-

CH2

Benzene Ring
 Tyrosine :
• It is a non essential amino acid.

• The hydroxyl group of tyrosine imparts slight

polarity to the side chain and is a site for
phosphorylation in proteins.
H

NH3+ C COO-
CH2

Phenol

OH
 Tryptophan :
• It is a essential amino acid.

• It contains indole ring.

H
NH3+ C COO-

CH2

Indole
N

H
 7.Imino acids :
• Proline containing pyrrolidine ring is a unique amino
acid.

• It has an imino group ( =NH ).Threrefore, proline is an

α-imino acid.
CH2 CH2

H
CH2 C Pyrroline
N COO-

H
 Classification of amino acids based on
polarity:
• Amino acids are classified into 4 groups based on
their polarity.

• 1.Non – polar amino acids:

• These amino acids are also referred to as

hydrophobic.

• They have no change on R-group.

• E.g., alanine, leucine, isoleucine, valine, methionine,

phenyl alanine, tryptophan and proline.
2. Polar amino acids with no charge on R-group:

• These amino acids, as such, carry no charge on the

R-group.

• They however possess groups such as hydroxyl,

sulfhydryl and amide and participate in hydrogen
bonding of protein structure.

• E.g., glycine, serine, threonine, cysteine, glutamine,

aspargine and tyrosine.
 3.Polar amino acids with positive R-
group:

• The three amino acids

• lysine,

• arginine

• histidine
 4. Polar amino acids with negative R- group:

• The dicarboxylic mono amino acids – aspartic acid

and glutamic acid are considered in this group.
 3. Nutritional classification of
amino acids:
• A). Essential or indispensable amino acids:
• The amino acids which cannot be synthesized by the
body and need to be supplied through the diet are
called essential amino acids.

• They are required for proper growth and maintenance

of the individual
• The 10 essential amino acids are
• 1. Arginine -Semi essential

• 2. Valine

• 3. Histidine -Semi essential

• 4. Isoleucine
AVHILLMPTT
• 5. Leucine

• 6. Lysine

• 7. Methionine

• 8. Phenyl alanine

• 9. Threonine

• 10. Tryptophan
B). Non – essential amino acids:
• The body can synthesize about 10 amino
acids to meet the biological needs, hence
they need not be consumed in the diet.

• These are glycine, alanine, serine, cysteine,

aspartate, aspargine, glutamate, glutamine,
tyrosine and proline.
 Based on their metabolic
fate:
• The carbon skeleton of amino acids can
serve as a precursor for the synthesis of
glucose ( glucogenic) or fat ( ketogenic ) or
both.

• From metabolic veiw point, amino acids are

divided into three groups
 1. Glycogenic amino acids:

• These amino acids can serve as

precursor for the formation of glucose
or glycogen. E.g.,alanine, aspartate,
glycine, methionine etc.
 2. Ketogenic amino acids:

• Fat can be synthesized from these

amino acids. Two amino acids leucine
and lysine are ketogenic.
3.Glycogenic and ketogenic Amino acids

• The four amino acids

• Phenyl alanine

• Isoleucine

• Tryptophan

• Tyrosine

• Are precursors for the synthesis of

glucose as well as fat
 New amino acids:

• In addition to 20 L – amino acids that take

part in protein synthesis, recently two more
new amino acids are described. They are

• 1. Selenocysteine – 21st amino acid

• 2. Pyrrolysine – 22 nd amino acid

 1. Selenocysteine
• occurs at the active site of several

enzymes.

• E.g., Thioredixin reductase

• Glutathione peroxidase

• De – iodinase

• Glycine reductase
• Selenoprotein P, a glycoprotein containing 10
selenocysteine residues, found in mammalian blood.
It has an antioxidant function and its concentration
falls in selenium deficiency.

• The stop codon UGA can code for Selenocysteine

• Selenocysteine is enzymatically generated from

serine directly on the t RNA and then incorporated
into proteins.
• 2. Pyrrolysine :
• The STOP codon UAG can code for
pyrrolysine.
 Properties of amino acids:
• A.Physical properties:

• Solubility:Most of the amino acids are

soluble in water and insoluble in organic
solvents.

• Melting points :Amino acids generally melt

at higher temparatures,often above 200ºC.
• Taste : Amino acids may be sweet( Gly, Ala,
Val ), tasteless ( Leu ) or bitter ( Arg, Ile ).

• Monosodium glutamate is a salt of glutamic

acid.

• It is employed as a flavoring agent in food

industry to increase taste and flavor.
• 4. Optical properties : All amino acids except
glycine possess optical isomers due to the presence
of asymmetric carbon atom.

• 5. Amino acids as ampholytes : Amino acids

contain both acidic ( -COOH) and basic ( NH2 )
groups.

• They can donate a proton or accept a proton, hence

amino acids are regarded as ampholytes.
 Zwitter ion or dipolar ion :

• Zwitter ion is a hybrid molecule containing positive

and negative ionic groups.

• The amino acids rarely exist in a neutral form with

free carboxylic and free amino groups.

• In strongly acidic pH, the amino acid is positively

charged ( cation )

• In strongly alkaline pH, the amino acid is negatively

charged ( anion )
• Each amino acid has a characteristic pH at
which it carries both positive and negative
charges and exists as zwitter ion.

• Isoelectric pH is defined as the pH at which

a molecule exists as a zwitter ion or dipolar
ion and carries no net charge.
 Chemical properties:
• The general reactions of amino acids are mostly due
to the presence of two functional groups namely
carboxylic (-COOH) and amino (-NH2) group.

• Reactions due to –COOH group:

• 1. Amino acids form salts (-COONa) with bases and

esters (-COOR´) with alcohols.
• 2. Decarboxylation: Amino acids undergo
decarboxylation to produce corresponding amines.

• E.g., histamine, tyramine, ᵞ- amino butyric acid

(GABA) from the amino acids histidine, tyrosine and
glutamate respectively.

• 3. Reaction with ammonia:

• The carboxyl group of dicarboxylic amino acids

react with NH3 to form amide.

• Aspartic acid + NH3 Aspargine

• Glutamic acid + NH3 Glutamine

 Reactions due to –NH2 group:
• The amino groups behave as bases and combine with
acids to form salts.

• 5.Reaction with ninhydrin:

• The α-amino acids react with ninhydrin to form a purple,
blue or pink colour complex( Ruhemanns`s purple ).

• Amino acid + Ninhydrin Keto acid + NH3 + CO2

+ Hydrindantin
Hydrindantin + NH3 + Ninhydrin Ruhemanns`s
purple
 Colour reactions of Amino acids
Amino acids can be identified by specific Colour
reactions

Reaction Specific group or amino acid

1.Biuret reaction Two peptide linkages
2.Ninhydrin reaction α-amino acids
3.Xanthoproteic reaction Benzene ring of aromatic a.as.
4.Millons reaction Phenolic group (Tyr)
5.Hopkins – Cole reaction Indole ring ( Trp)
6.Sakaguchi reaction Guanidino group (Arg)
7.Nitroprusside reaction Sulfhydryl groups ( Cys)
8.Sulfur test Sulfhydryll groups ( Cys)
9.Pauly´s test Imidazole ring ( His)
.
10.Folin-Coicalteau´ s test Phenoic group ( Tyr)
 7.Transamination:
• Transfer of an amino group from an amino
acid to a keto acid to form a new amino acid
is known as transamination.

• 8.Oxidative deamination:
• The amino acids undergo oxidative
deamination to liberate free ammonia.
 Amino acid derivatives in proteins:
• The 20 standard amino acids can be incorporated
into proteins due to the presence of universal
genetic code.

• Some of these amino acids undergo specific

modifications after the protein synthesis occurs.
These derivatives of amino acids are very important
for protein structure and functions.

• Collagen-the most abundant protein in mammals-

contains 4-hydroxyproline and 5-hydroxy lysine.
• Histones-the proteins found in association
with DNA –contain many methylated,
phosphorulated or acetylated amino acids.

• Carboxyglutamic acid is found in certain

plasma proteins involved in blood clotting.

• Cystine is formed by combination of two

cysteines. It is also a derived amino acid.
 Non-Protein amino acids

α-Amino acids Functions

1.Ornithine Intermediate in the biosynthesis of urea
2.Citrulline Intermediate in the biosynthesis of urea
3.Arginosuccinic acid Intermediate in the biosynthesis of urea
4.Thyroxine Thyroid hormone derived from tyrosine.
5.Triiodothyronine Thyroid hormone derived from tyrosine
6.SAM Methyl donor in biological system.
7.Homocysteine Intermediate in methionine metabolism.
A risk factor for coronary heart
diseases.
8. 3,4-Dihydroxy phenyl alanine ( DOPA) A neurotransmitter, precursor for
melanin
9.Creatinine Derived from muscle and exreted in
urine.
 Non – α-amino acids:

Amino acids Functions

1.β-Alanine Component of pantothenic acid and

coenzyme A.

2.β-Aminoisobutyric acid End product of pyrimidine metabolism.

3. γ-Aminobutyric acid A neurotransmitter produced from

glutamic acid

4.δ-Amino levulinic acid Intermediate in heme synthesis

5.Taurine Found in association with bile acids

 D-Amino acids:

• Certain D-amino acids are also found in the

antibiotics (Actinomycin-D, valinomycin, gramicidin-
S).

• D-Glutamic acid and D-Alanine are present in

bacterial cell wall.

• D-Serine and D-Aspartate are found in brain tissue.

 Peptides and Proteins

• 20 amino acids are commonly found in protein.

• These 20 amino acids are linked together through

“peptide bond forming peptides and proteins.

• The chains containing less than 50 amino acids

are called “peptides”, while those containing
greater than 50 amino acids are called proteins”.
 Peptide bond formation:

• α-carboxyl group of one amino acid (with side chain

R1) forms a covalent peptide bond with α-amino
group of another amino acid (with the side chain R2)
by removal of a molecule of water.

• The result is : Dipeptide (i.e. Two amino acids linked

by one peptide bond).
• The dipeptide can then forms a second
peptide bond with a third amino acid (with
side chain R3) to give Tripeptide.

• Repetition of this process generates a

polypeptide or protein of specific amino acid
sequence.
• Each polypeptide chain starts on the left
side by free amino group of the first amino
acid enter in chain formation.
• It is termed (N- terminus).
• Each polypeptide chain ends on the right
side by free COOH group of the last amino
acid and termed (C-terminus).
 Peptide bond
• The amino acids are held together in a
protein by covalent peptide bonds or linkage
cementing material between amino acids
• When amino group of an amino acid
combines with the carboxyl group of another
amino acid, a peptide bond is formed
• Peptides containing more than 10 amino
acids are referred as polypeptide
 Formation of peptide bond

H H

NH3+ C COO- + NH3+ C COO-

R1 R2

Amino acid 1 Amino acid 2

H2 O

H H

NH3+ -C-CO-NH-C-COO-H

R1 R2
 Functions of peptide bond
• It usually found in trans configuration.
• The peptide bond is a partial double bond.
• N- partially positive, O- partially negative.
• Shorthand to read peptides:
• The amino acids in a peptide or protein are
represented by the 3-letter or one letter
abbreviation.
• This is the chemical shorthand to write peptide.
 Naming of peptides

• The amino acid suffixes –ine (glycine), -an (tryptophan),

-ate (glutamate) are changed to –yl with the exception
of C-terminal amino acid
• E.g, Glutamyl-cysteinyl-glycine
+H3N –glutamate - cysteine - glycine - COO- Amino acids
in a peptide
E - C - G One letter symbols
Glu - Cys - Gly Three letter symbols
Glutamyl - cysteinyl - glycine Peptide name
 Examples of Peptides

• Dipeptide: Two amino acids joined by one

peptide bond.
E.g. Aspartame which acts as sweetening agent
being used in replacement of cane sugar.
• It is composed of aspartic acid and phenyl
alanine.
• Tripeptides: 3 amino acids linked by two
peptide bonds.
• E.g. GSH which is formed from 3 amino
acids: glutamic acid, cysteine and glycine.
• It helps in absorption of amino acids,
protects against hemolysis of RBC by
breaking H2O2 which causes cell damage.
• Polypeptides: 10- 50 amino acids.
• E.g. Insulin, a polypeptide hormone.
 Bonds responsible for protein structure

• Two types of bonds

Covalent and non-covalent bonds

• Covalent bonds:

• The peptide and disulfide bonds are the strong

bonds in protein structure.
• Disulfide bond:

• A disulfide bond (-S-S-) is formed by the

sulfhydryl groups (-SH) of two cystine residues to
produce cystine.

• The disulfide bonds may be formed in a single

polypeptide chain or between different
polypeptides.

• These bonds contribute to the structural

conformation and stability of protein.
 Non-covalent bonds

• Hydrogen bonds (H-bonds):

• The H-bonds are formed by sharing of H-
atoms between the Nitrogen and carbonyl
oxygen of different peptide bonds.
• Each H-bond is weak but collectively they are
strong.
• A large number of H-bonds significantly
contribute to the protein structure.
 Hydrophobic bonds

• The non-polar side chains of neutral amino

acids tend to be closely associated with each
other in proteins.
• These not true bonds.
• The occurrence of hydrophobic forces is
observed in aqueous environment wherein the
molecules are forced to stay together.
• Electrostatic bonds:
• These bonds are formed by interactions
between negatively charged groups of acidic
amino acids with positively charged groups of
basic amino acids.
• Van der Waals forces:
• These are the non-covalent associations
between electrically neutral molecules.
• They are formed by the electrostatic interactions
due to permanent or induced dipoles
UNIT 2
 Structure of proteins

• Proteins have different level of organization;

• Primary structure: The linear sequence of amino

acids forming the backbone of proteins.

• Secondary structure: The spatial arrangement of

protein by twisting of the polypeptide chain.

• Tertiary structure: The three dimensional

structure of a functional protein.
• Quaternary structure: Some of the proteins
are composed of two or more polypeptide
chains referred to as subunits. The spatial
arrangement of these subunits is known as
quaternary structure.
 Structure of proteins

• Primary structure: The linear sequence of

amino acids held together by peptide bonds
in its peptide chain.

• The peptide bonds form the back bone.

• The free –NH2 group of the terminal amino

acid is called as N-terminal end and the free
–COOH end is called C-terminal end.
• It is a tradition to number the amino acids
from N-terminal end as No.1 towards the C-
terminal end.
• Presence of specific amino acids at a
specific number is very significant for a
particular function of a protein.
 Determination of primary structure

• The primary structure comprises the

identification of amino acids with regard to
their quality, quantity and sequence in a
protein structure.
• Determination of primary structure involves
3 stages:

• Determination of amino acid composition.

• Degradation of protein or polypeptide into

smaller fragments.

• Determination of the amino acid sequence

Determination of amino acid composition

• The protein or polypeptide is completely

hydrolyzed to liberate the amino acids
• The hydrolysis may be carried out either by
acid or alkali treatment or by enzyme
hydrolysis.
• Pronase is a mixture of non-specific
proteolytic enzymes that causes complete
hydrolysis of proteins.
 Separation and estimation of amino acids

• The mixture of amino acids liberated by

protein hydrolysis can be determined by
chromatographic techniques.
 Degradation of protein or polypeptide into
smaller fragments.

• Liberation of polypeptides

• Treatment with urea or guanidine hydrochloride

disrupts the non-covalent bonds and
dissociates the protein into polypeptide units.

• Cleaving the disulfide linkages between the

polypeptide units, treatment with performic acid
is necessory.
• Number of polypeptides: The number of
polypeptide chains can be identified by
treatment of protein with dansyl chloride.
• It specifically binds with N-terminal amino
acids to form dansyl polypeptides which on
hydrolysis yield N-terminal dansyl amino
acid.
• The number of dansyl amino acids produced
is equal to the number of polypeptide chains
in a protein.
 Breakdown of polypeptides into fragments

• Polypeptides are degraded into smaller

peptides by enzymatic or chemical methods
• Enzymatic cleavage: The proteolytic
enzymes such as trypsin, chymotrypsin,
pepsin and elastage exhibit specificity in
cleaving the peptide bonds.
• Among these enzymes, trypsin is commonly
used.
• Trypsin hydrolyses the peptide bonds
containing lysine or arginine on the carbonyl
(-C=O ) side of peptide linkage.
• Chemical cleavage: Cyanogen bromide (CNBr )
is commonly used to split polypeptides into
smaller fragments.
• CNBr specifically splits peptide bonds, the
carbonyl side of which is contributed by the
amino acid methionine.
Determination of the amino acid sequence

• Sangers Reagent: Sanger used 1-fluoro-2, 4-

dinitrobenzene (FDNB) to determine insulin
structure.
• FDNB specifically binds with N-terminal amino
acids to form a dinitrophenyl (DNP) derivative of
peptide.
• This, on hydrolysis yields DNP – amino acids ( N-
terminal) and free amino acids from the rest of
the peptide chain.
• DNP amino acids can be identified by
chromatography.
• Edmans reagent: Phenyl isothiocyanate is
Edmans reagent.
• It reacts with the N-terminal amino acid of
peptide to form a phenyl thiocarbamyl
derivativve.
• On treatment with mild acid, phenyl
thiohydrantoin (PTH) –amino acid, a cyclic
compound is liberated.
• PTH amino acid can be identified by
chromatography
• Edmans reagent has an advantage, a peptide
can be sequentially degraded liberating N-
terminal amino acids one after another which
can be identified.
• This is due to the fact that the peptide as a
whole is not hydrolysed but only releases
PTH- amino acids.
 Squenator
• This is an automatic machine to determine
the amino acid sequence in a polypeptide
• It is based on the principle of Edmans
degradation .
• Amino acids are determined sequentially
from N-terminal end
• The PTH-amino acid liberated is identified
by HPLC.
• Sequenator takes about 2 hours to
determine each amino acid.
 Overlapping peptides

• In the determination of primary structure of

protein, several methods are simultaneously
employed.

• This results in the formation of overlapping

peptides

• Overlapping peptides are very useful in

determining the amino acid sequence.
 Reverse sequencing technique
• It is the genetic material (DNA) which ultimately
determines the sequence of amino acids in a
polypeptide chain
• By analyzing the nucleotide sequence of DNA that
codes for protein, it is possible to translate the
nucleotide sequence into amino acid sequence.
• This technique, however, fails to identify the
disulfide bonds and changes that occur in the amino
acids after the protein is synthesized.
 Secondary structure

• The spatial arrangement of protein by

twisting of the polypeptide chain

• The amino acids are located close to each

other in their sequence.

• Two types of secondary structures, α-Helix

and β-sheet
 α-Helix

• α-Helix is the most common spiral structure of

protein
• α-Helical structure was proposed by Pauling
and Corey in 1965.
• The salient feature:
• The α-Helix is a tightly packed coiled structure
with amino acid side chains extending outward
from the central axis.
• The α-Helix is stabilized by extensive hydrogen
bonding

• Formed between H atom attached to peptide N,

and O atom attached to peptide C.

• The H-bonds are individually weak but collectively,

strong enough to stabilize the helix.

• All the peptide bonds, except the first and last in

polypeptide chain, participate in hydrogen
bonding.
• Each turn of helix contains 3.6 amino acids and
travels a distance of 0.54 nm.
• The spacing of each amino acid is 0.15 nm.
• α-Helix is a stable conformation formed
spontaneously with the lowest energy.
• The right handed α-Helix is more stable than the
left handed helix
• Certain amino acids (particularly proline)
disrupt the α-Helix.
• Large number of acidic and basic amino acids are
also interfere with α-Helix structure.
• β-pleated sheet
• An another form of secondary structure in which
two or more polypeptides (or segments of the
same peptide chain) are linked together by
hydrogen bond between H- of NH- of one chain
and carbonyl oxygen of adjacent chain (or
segment).
• Parallel and anti-parallel β-sheets
• The polypeptide chains in the β-sheets may
be arranged either in parallel or anti-
parallel.
• β-Pleated sheet may be formed either by
separate polypeptide chains (H-bonds are
inter chain) or a single polypeptide chain
folding back on to itself (H-bonds are
intrachain)
 Triple helix

• Collagen is rich in proline and hydroxy

proline and cannot form a α-helix or β-
Pleated sheet.
• Collagen forms a triple helix.
• The triple helix is stabilized by both non
covalent as well as covalent bonds.
 Reverse Turns or β-bends

• Since the polypeptide chain of a globular

protein changes direction two or more times
when it folds, the conformation known as
Reverse turns or β-bends.

• Reverse turns usually occur on the surfaces

of globular proteins.
• Super secondary structures

• Varies combinations of secondary structure,

called super secondary structure, are
commonly found in globular proteins.

• These are

• β- α- β unit

• Greek key

• β-meander
• β- α- β unit :

• The β- α- β unit consists of two parallel β-

pleated sheets connected by an intervening
strand of α –helix.

• Greek key :

• A conformation that takes its name from a

design often found on classical greek
pottery.
• β-meander :

• The β-meander consists of five β-Pleated

sheets connected by reverse turns.

• 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.
 Tertiary structure

• The three dimensional structure of a protein.

• It is a compact structure with hydrophobic

side chains held interior while the hydrophilic
groups are on the surface of the protein.

• This type of arrangement ensures stability of

the molecule.
 Bonds of Tertiary structure

• The hydrogen bonds,

• Disulfide bonds(-S-S )
• Ionic interactions (electrostatic bonds) and
• Hydrophobic interactions also contribute to
the tertiary structure of proteins.
• Domains: Used to represent the basic unit of
protein structure (tertiary) and function.
• A polypeptide with 200 amino acids normally
consists of two or more domains.
 Quaternary structure

• Majority of the proteins are composed of

single polypeptide chains.

• Some of the proteins, consists of two or

more polypeptides which may be identical or
unrelated.

• Such proteins are termed as oligomers and

possess quaternary structure
• The individual polypeptide chains are known
as monomers, protomers or subunits.

• A dimer consists of two polypeptides while a

tetramer has four.

• Importance of oligomeric proteins:

• These proteins play a significant role in the

regulation of metabolism and cellular
function.

• E.g. hemoglobin, LDH.

 Bonds responsible for protein structure

• Two types of bonds

Covalent and non-covalent bonds

• Covalent bonds:

• The peptide and disulfide bonds are the strong

bonds in protein structure.
• Disulfide bond:

• A disulfide bond (-S-S-) is formed by the

sulfhydryl groups (-SH) of two cystine residues to
produce cystine.

• The disulfide bonds may be formed in a single

polypeptide chain or between different
polypeptides.

• These bonds contribute to the structural

conformation and stability of protein.
 Non-covalent bonds

• Hydrogen bonds (H-bonds):

• The H-bonds are formed by sharing of H-
atoms between the Nitrogen and carbonyl
oxygen of different peptide bonds.
• Each H-bond is weak but collectively they are
strong.
• A large number of H-bonds significantly
contribute to the protein structure.
 Hydrophobic bonds

• The non-polar side chains of neutral amino

acids tend to be closely associated with each
other in proteins.
• These not true bonds.
• The occurrence of hydrophobic forces is
observed in aqueous environment wherein the
molecules are forced to stay together.
• Electrostatic bonds:
• These bonds are formed by interactions
between negatively charged groups of acidic
amino acids with positively charged groups of
basic amino acids.
• Van der Waals forces:
• These are the non-covalent associations
between electrically neutral molecules.
• They are formed by the electrostatic interactions
due to permanent or induced dipoles
 Methods to determine protein structure

• Determination of secondary and tertiary

protein structures, X-ray crystallography is
most commonly used.
• Nuclear magnetic resonance (NMR) spectra
of proteins provides structural and
functional information on the atoms and
groups present in the proteins
 Methods for the isolation and purification of proteins

• Proteins are fractionated by using different

concentrations of ammonium sulfate or
sodium sulfate.
• Protein fractionation may also be carried out
by ultracentrifugation
• Protein separation is achieved by utilizing
electrophoresis, isoelectric focussing,
immunoelectrophoresis, ion-exchange
chromatography, gel filtration, HPLC.
 Properties of proteins
• Solubility:
• Proteins form colloidal solutions instead of true
solutions in water.
• This is due to huge size of protein molecule.
• Molecular weight:
• The proteins vary in their molecular weights,
dependent on the number of amino acid
residues.
• Majority of proteins/polypeptides may be
composed of 40 to 4,000 amino acids with a
molecular weight ranging from 4,000 to
440,000.
• Shape:
• There is a wide variation in the protein shape.
• It may be globular (insulin), Oval (albumin),
fibrous or elongated (fibrinogen)
• Isoelectric pH:
• At isoelectric pH, the proteins exist as
zwitterion or dipolar ions.
• They are electrically neutral with minimum
solubility, maximum precipitability and least
buffering capacity.
• Precipitation of proteins:
• Proteins exist in colloidal solution due to
hydration of polar groups (-COO-, -NH3+, -OH).
• Proteins can be precipitated by dehydration or
neutralization of polar groups
• Iso-electric precipitation:
• Proteins are least soluble at their iso-electric pH.
• Some of the proteins are precipitated immediately
when adjusted to their iso-electric pH
• E.g. Casein which forms a flocculent
precipitate at pH 4.6 and redissolves in
highly acidic or alkaline solutions.
• When the milk is curdled, the casein forms
the white curd, because lactic acid
produced by the fermentation process
lowers the pH to the iso-electric point of
casein.
 Precipitation by salting out

• When a neutral salt such as ammonium sulphate

or sodium sulphate is added to the protein
solution , the shell of hydration is removed and
the protein is removed and the protein is
precipitated
• This is called as salting out
• Higher the molecular weight of a protein, the
salt required for precipitation is lesser
• Globulins are precipitated by half saturation of
ammonium sulphate; but albumin will need full
saturation
• Precipitation by Organic solvents:
• Organic solvents such as alcohol are good protein
precipitating agents.
• They dehydrate the protein molecule by removing
the water envelope and cause precipitation
• Precipitation by salts of heavy metals:
• Heavy metal ions like Pb2+, Hg2+, Fe2+ , Zn2+ , Cd2+
cause precipitation of proteins.
• These metals being positively charged, when added
to protein solution (negatively charged ) in alkaline
medium results in precipitate formation.
 Precipitation by anionic or alkaloid reagents

• Proteins can be precipitated by trichloroacetic

acid, sulphosalicylic acid, phosphotungstic acid,
picric acid, tannic acid, phosphomolybdic acid etc.
By the addition of these acids, the proteins
existing as cations are precipitated by the anionic
form of acids to produce protein-sulphosalicylate,
protein-tungstate, protein-picrate etc.
 Denaturation
• Defined as an disorganization of native protein
structure
• Denaturation results in the loss of secondary, tertiary
and quarternary structure of proteins due to
cleavage of noncovalent bonds
• This involves a change in physical, chemical and
biological properties of protein molecules
• Note: Primary structure of protein molecule, i.e.,
peptide bond is not affected.
 Denaturatng agents

• Physical agents
• Heat, violent shaking, X-rays, UV radiation
• Chemical agents: Acids, alkalies, organic
solvents (ether, alcohol), salts of heavy
metals (Pb, Hg), urea, salicylate etc.
 Characteristics of denaturation

• The native helical structure of protein is lost

• The primary structure of a protein with peptide
linkages remain intact. i.e.,peptide bonds are not
hydrolyzed
• The protein loses its biological activity
• Denatured protein becomes insoluble in solvent in
which it was originally soluble
• The viscosity of denatured protein (solution)
increases while its surface tension decreases
• Denaturation is associated with increase in
ionizable and sulfhydryl groups of protein.
• This is due to the loss of hydrogen and disulfide
bonds
• Denatured protein is more easily digested.
• This is due to increased exposure of peptide bonds
to enzymes
• Denaturation is irreversible.
• E.g., omelet can be prepared from an egg but the
reversal is not possible
• Careful denaturation is sometimes reversible (known
as renaturation).
Hemoglobin undergoes denaturation in the presence
salicylate.
• By the removal of salicylate, hemoglobin is renatured
• Denatured protein cannot be crystallized
• Coagulation
• Coagulation refers to a semi-solid viscous precipitate
of protein.
• Irreversible denaturation results in coagulation
• Coagulation is optimum and requires lowest
temparature at isoelectric pH
• E.g., Albunins and globulins
• Heat coagulation test is commonly used to detect the
presence of albumin in urine
• Flocculation:
• It is the process of protein precipitation at isoelectric
pH
• The precipitate is referred to as flocculum
• Flocculation is reversible.
• On the application of heat, flocculam can be
converted into an irreversible mass, coagulum.
 Classification of proteins

• Three major types of classifying proteins

based on their function, chemical nature and
solubility and nutritional importance

• Functional classification of proteins

• Chemical nature and solubility

• Nutritional importance
 Functional classification of proteins

• Structural proteins: Keratin of hair and nails,

collagen of bone
• Enzymes or catalytic proteins: Hexokinase,
pepsin
• Transport proteins: Hemoglobin, serum
albumin
• Hormonal proteins: Insulin, growth hormone
• Contractile proteins: Actin, myosin

• Storage proteins: Ovalbumin, glutelin

• Genetic proteins: Nucleoproteins

• Defense proteins: Immunoglobulins

• Receptor proteins: For hormones and

viruses
 Proteins

Simple Conjugated Derived

Globular Scleroproteins Nucleoproteins Primary Secondary

Glycoproteoins
Proteoses
Albumins Collagen Coagulated

Globulins Lipoproteins Peptones

Proteans
Elastins
Glutilins Phosphoproteins Metaproteins Poly
Keratins peptides
Prolamines Chromoproteins
Histones Peptides
Metalloproteins
Globines

Protamines
 Simple proteins

• A) Globular proteins:
• These are spherical or oval in shape, soluble in
water or other solvents and digestible
• Albumins:
• They are soluble in water and coagulated by heat.
E.g serum albumin, ovalbumin (egg), lactalbumin
(milk)
• Globulins:
• These are insoluble in pure water, but soluble in
dilute salt solutions and coagulated by heat.
• They are precipitated by half saturation with
ammonium sulphate or by full saturation with sodium
chloride. E.g., serum globulins, egg globulins
• Glutelins:
• These are plant proteins, insoluble in water or neutral
salt solutions, soluble in dilute acids or alkalies
• They are rich in glutamic acid.
• They are large molecules and can be coagulated by
heat. e.g., glutelin (wheat), oryzenin (rice).
• Prolamines:
• They are soluble in 70-80% alcohol, but insoluble in
pure water.
• They are rich in proline but lack in lysine. E.g.,
gliadin (wheat), zein (maize)
• Histones:
• These are basic proteins, rich in arginine and
histidine, with alkaline isoelectric pH.
• They are soluble in water, dilute acids and salt
solutions but insoluble in ammonia
• They form conjugated proteins with nucleic acids
(DNA) and porphyrins.
E.g., nucleohistones, chromosomal nucleoproteins
• Globins:
• These are generally considered along with histones.
Globins are not basic proteins and are not
precipitated by NH4OH
• Protamines:
• These are soluble in water, dilute acids and alkalies.
They are not coagulated by heating..
• They contain large number of arginine and lysine
residues, and are strongly basic. These are also
found in association with nucleic acids
 Scleroproteins or Albuminoids
• These are fibrous proteins with great stability and
very low solubility and form supporting structures
of animals
• Collagens, are connective tissue proteins lacking
tryptophan
• Elastins, these proteins are found in elastic tissues
such as tendons and arteries
• Keratins, these are present in exoskeletal structures
e.g. hair, nails, horns.
• Human hair has a higher content of cysteine.
 Conjugated proteins
• They are combinations of proteins with a non-protein
part, called prothetic group
• These are
• Glycoproteins:
• Glycoproteins are the proteins with carbohydrate
moiety as the prosthetic group.
• The term mucoprotein is used if the carbohdrate
content is more than 4%.
• Blood group antigens and many serum proteins are
glycoproteins
• Lipoproteins:

• These are the proteins loosely combined with lipid

components.

• They occur in blood and on cell membranes

• Serum lipoproteins, membrane lipoproteins

• Nucleoproteins:

• These are the proteins attached to nucleic acids,

e.g.Histones.

• The DNA carries negative charges, which combines

with positively charged proteins
 Chromoproteins

• These are the proteins with colored prosthetic

groups
• Hemoproteins: All hemoproteins are
Chromoproteins which carry heme as the prosthetic
group
• Hemoglobin: Respiratory protein found in RBCs
• Cytochromes: These are the mitochondrial enzymes
of the respiratory chain
• Catalase:
• This enzyme decomposes H2O2 to water and O2
• Others
• Flavoproteins:
• Is a cellular oxidation-reduction protein which has
riboflavin a constituent of B-complex vitamin as its
prosthetic group.
• This is yellow in color
• Visual purple:
• Is a protein of retina in which the prosthetic group is
a corotenoid pigment which is purple in colour
• Phosphoproteins:
• These contain phosphorous. E.g. casein and vitellin
of egg yolk.
• The phosphoric acid is esterified to the hydroxyl
groups of serine and threonine residues of proteins
• Metalloproteins:
• They contain metal ion as their prothetic group.
• Several enzymes contain metallic elements such as
Fe, Co, Mn, Zn, Cu, Mg, etc
E.g., Ferritin (Fe), Carbonic anhydrase (Zn),
Ceruloplasmin (Cu)
 Derived proteins

• The derived proteins are of two types

• Primary derived proteins:
• These are the denatured or coagulated or first
hydrolysed products of proteins
• Secondary derived proteins:
• These are the degraded (due to breakdown of
peptide bonds) products of proteins
 Primary derived proteins

• Proteans:
• These are earliest products of protein hydrolysis by
enzymes, dilute acids, alkalies etc.
• They are insoluble in water
• E.g., Myosan (from myosin), Edestan (from elastin)
Meta proteins:
• These are the second stage products of protein
hydrolysis by treatment with slightly stronger acids
and alkalies. E.g., acid and alkali metaproteins.
• Coagulated proteins:
• These are the denatured proteins produced by agents
such as heat, acids, alkalies etc, E.g., cooked proteins,
coagulated albumin (egg white )
• Secondary derived proteins:
• These are the degraded (due to breakdown of peptide
bonds) products of proteins
• Proteoses or albumoses:
• These are hydrolytic products of proteins which are
soluble in water and coagulated by heat and precipitated
by saturation with ammonium sulphate
• Peptones:
• These are hydrolytic products of proteoses.
• They are soluble in water, not coagulated by heat
and not precipitated by saturation with ammonium
sulphate.
• They can be precipitated by phosphotungstic acid
• Peptides:
• Peptides are composed of very small number of
amino acids joined as peptide bonds.
• They are named according to the number of amino
acids present in them
• Dipeptide: made up of two amino acids
• Tripeptides: made up of three amino acids
• Peptides are water soluble and not coagulated
by heat
• Hydrolysis: The complete hydrolytic decomposition
of a protein generally follows these stages
• Protein Protean Metaprotein
Peptides Peptone Proteose
Amino acids
 Nutritional classification of proteins
• Complete proteins or Nutritionally rich proteins:
These proteins have all the essential amino acids in
the required proportion.
• Also called as first class proteins. E.g., egg albumin,
casein of milk
• Partially incomplete proteins:
• These proteins are partially lacking one or more
essential amino acids and can promote moderate
growth. E.g., wheat and rice proteins.
• Limiting amino acids Lysine and Threonine
• Incomplete or poor proteins:
• They lack in many essential amino acids
Hence they do not promote growth at all
• e.g., gelatin (lacks Trp), zein ( lacks Trp, Lys)
 Biologically Important Peptides

• When 10 or less number of amino acids are

joined together, it is called as an
oligopeptide
• Some of them are biologically active
• Glutathione:
• It is tripeptide composed of 3 amino acids.
• Chemically, glutathione is γ-glutamyl-
cystinyl-glycine.
• It exists in reduced or oxidized states.
• Functions:
• Glutathione serves as a coenzyme for certain
enzymes e.g., prostaglandin PGE2 synthetase,
glyoxylase
• It prevents the oxidation of sulfhydryl (-SH ) groups of
several proteins to disulfide (-S-S-) groups.
• This is essential for protein function
• Glutathione in association with glutathione reductase
participate in the formation of correct disulfide bonds
in several proteins
• Glutathione maintains RBC membrane structure and
intigrity
• Glutathione protects hemoglobin from getting
oxidized by agents such as H2O2
• Glutathione is involved in the transport of amino
acids in the intestine and kidney tubules via γ-
glutamyl cycle or Meister cycle
• Glutathione involved in the detoxification process
• Toxic amounts of peroxides and free radicals
produced in the cells are scavanged by glutathione
peroxidase (a selenium containing enzyme)
• Thyroprophin releasing hormone(TRH):
• It is tripeptide secreted by hypothalamus
• TRH is stimulated by pituitary gland to release
thyrotrophic hormone
• Oxytocin:
• It is a hormone secreted by posterior pituitary gland
and contains 9 amino acids (nonapeptide).
• Oxitocin causes contraction of uterus
• Vasopressin (antidiuretic hormone,ADH):
• ADH is also a nonapeptide secreted by posterior
pituitary gland
• It stimulates kidney to retain water and thus
increases blood pressure.
• Angiotensins:
• Angiotensin is a decapeptide (10 amino acids) which
is converted to angiotensin II (8 amino acids)
• Angiotensin II stimulates the release of aldosterone
from adrenal gland
• Methionine enkephalin:
• It is a pentapeptide found in brain and has opiate like
function
• It inhibits the sense of pain
• Bradykinin:

• They are nona and decapeptides.

• Act as powerful vasodilators

• Produced from plasma proteins by snake venom

enzymes

• Peptide antibiotics:

• Antibiotics such as gramicidin, bacitracin, tyrocidin

and actinomycin are peptide in nature

• GIT hormones:

• Gastrin, secretin etc. are serves as hormones

Thank you