Proteins

• Proteins are the most abundant biological macromolecules, occurring in all cells and all
parts of cells.

• It is a polymer of amino acids and constitutes the largest fraction of cells.

• Proteins also occur in great variety; thousands of different kinds, ranging in size from
relatively small peptides to huge polymers with molecular weights in the millions, may be
found in a single cell.

• Moreover, proteins exhibit enormous diversity of biological function and are the most
important final products of the information pathways.

• Proteins are the molecular instruments through which genetic information is expressed.

Amino acids (AA): Building blocks of proteins

• AA is compound containing carbon, hydrogen, oxygen and nitrogen.

side chain (R-group) all bonded to the same carbon atom (the 𝛂 carbon). All 20
• They have a carboxyl group, an amino group a hydrogen atom and a distinctive

of the common amino acids are 𝛂-amino acids.

• They differ from each other in their side chains, or R groups, which vary in structure,
size, and electric charge, and which influence the solubility of the amino acids in
water.

• With four different groups connected to the tetrahedral -carbon atom, 𝛂 -amino acids
are chiral: they may exist in one or the other of two mirror-image forms, called the L
isomer and the D isomer

• The common amino acids of proteins have been assigned three-letter abbreviations
and one-letter symbols which are used as shorthand to indicate the composition and
sequence of amino acids polymerized in proteins.
 •

• More than 300 amino acids are present in the cells, however only 22 amino acids
participate in protein synthesis.

• Some amino acids are more abundant in proteins than other amino acids.

• Abundant AA-Leucine, serine, lysine and glutamic

• Rare AA- Tryptophan and methionine

Amino acids: Classification

On The Basis Of Structure:

1. Amino acids with aliphatic side chains: These amino acids contain
aliphatic groups e.g. glycine, leucine, valine etc.
2. Amino acids containing hydroxyl groups: These amino acids
contain hydroxyl groups e.g. serine, threonine and tyrosine.
3. Sulfur containing amino acids: These are Sulphur containing amino
acids e.g. cysteine, methionine.
4. Acidic amino acids and their amide: Aspartic acids and glutamic
acids are dicarboxylic monoacids while asparagine and glutamine are
their respective amides derivatives.
5. Basic amino acids: Lysine, arginine, and histidine are dibasic
monocarboxylic acids.
6. Aromatic amino acids: Phenylalanine, tyrosine, and tryptophan are
aromatic amino acids.
7. Imino acids: Proline is imino acids.

On The Basis Of Polarity:

1. Non-polar amino acids: Example- methionine, proline, alanine,

leucine, valine, etc.
 2. Polar amino acids with no charge on R group: Example-
threonine, glutamine, glycine, serine, threonine, glutamine.
3. Polar amino acids with positive charge on R group: Example-
histidine, and, lysine, arginine.
4. Polar amino acids with negative charge on R group: Example-
aspartic and glutamic acid.

On The Basis Of Nutritional Classifications:

1. Essential amino acids: Amino acids which are not synthesized by

body and needs to be supplied through diet. E.g. arginine, valine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan
histidine, isoleucine.
2. Non-essential amino acids: Amino acids that can be synthesized by
the diet. E.g. glutamine, tyrosine, and proline, glycine, alanine, serine,
cysteine, aspartate, asparagine, glutamate.

On The Basis Of Metabolic Fate:

1. Glycogenic amino acids: Type of amino acid that can serve as

precursor for the synthesis of glucose and glycogen. E.g. alanine,
aspartate, methionine.
2. Ketogenic amino acids: Amino acids that are involved in the
synthesis of fat. E.g. leucine and lysine.
3. Glycogenic and ketogenic amino acids: There are four amino acids
that are involved in the synthesis of both fat and glucose. E.g.
isoleucine, phenylalanine, alanine, tryptophan.

Amino acids: Structure

The additional carbons in an R group are commonly designated 𝛃, 𝛄, 𝛅, 𝛆 and so

forth, proceeding out from the 𝛂 carbon.
•

• For all the common amino acids except glycine, the carbon is bonded to four different
groups: a carboxyl group, an amino group, an R group, and a hydrogen atom (In
glycine, the R group is another hydrogen atom).
 • The 𝛂-carbon atom is thus a chiral centre. Because four different groups can occupy
two unique spatial arrangements, and thus amino acids have two possible
stereoisomers.

• Since they are non-superimposable mirror images of each other, the two forms
represent a class of stereoisomers called enantiomers

• All molecules with a chiral centre are also optically active (they rotate plane-
polarized light).

PROTEIN STRUCTURE

The structure of proteins can be divided into four levels of organization:

1. Primary Structure
 The primary structure of a protein consists of the amino acid sequence along the
polypeptide chain.
 Amino acids are joined by peptide bonds.
 Because there are no dissociable protons in peptide bonds, the charges on a polypeptide
chain are due only to the N-terminal amino group, the C-terminal carboxyl group, and the
side chains on amino acid residues.
 The primary structure determines the further levels of organization of protein molecules.

2. Secondary Structure
 The secondary structure includes various types of local conformations in which the atoms
of the side chains are not involved.
 Secondary structures are formed by a regularly repeating pattern of hydrogen bond
formation between backbone atoms.
 The secondary structure involves α-helices, β-sheets, and other types of folding patterns
that occur due to a regularly repeating pattern of hydrogen bond formation.
 The secondary structure of protein could be :
 Alpha-helix
 The α-helix is a right-handed coiled strand.
 The side-chain substituents of the amino acid groups in an α-helix extend to the outside.
 Hydrogen bonds form between the oxygen of the C=O of each peptide bond in the strand
and the hydrogen of the N-H group of the peptide bond four amino acids below it in the
helix.
 The side-chain substituents of the amino acids fit in beside the N-H groups.
 Beta-helix

 The hydrogen bonding in a ß-sheet is between strands (inter-strand) rather than within
strands (intra-strand).
 The sheet conformation consists of pairs of strands lying side-by-side.
 The carbonyl oxygens in one strand hydrogen bond with the amino hydrogens of the
adjacent strand.
 The two strands can be either parallel or anti-parallel depending on whether the strand
directions (N-terminus to C-terminus) are the same or opposite.
 The anti-parallel ß-sheet is more stable due to the more well-aligned hydrogen bonds.

3. Tertiary Structure
 The tertiary structure of a protein refers to its overall three-dimensional conformation.
 The types of interactions between amino acid residues that produce the three-dimensional
shape of a protein include hydrophobic interactions, electrostatic interactions, and hydrogen
bonds, all of which are non-covalent.
 Covalent disulfide bonds also occur.
 It is produced by interactions between amino acid residues that may be located at a
considerable distance from each other in the primary sequence of the polypeptide chain.
 Hydrophobic amino acid residues tend to collect in the interior of globular proteins, where
they exclude water, whereas hydrophilic residues are usually found on the surface, where
they interact with water.

4. Quaternary Structure
 Quaternary structure refers to the interaction of one or more subunits to form a functional
protein, using the same forces that stabilize the tertiary structure.
 It is the spatial arrangement of subunits in a protein that consists of more than one
polypeptide chain.

PROTEIN FUNCTION
PROTEIN: SOURCE
 A LIST OF PROTEIN DEFICIENCY DISEASES

Proteins are substances that are part of cells, tissues and organs throughout the body,
according to the Centers for Disease Control. Protein deficiency is common among people
who live in developing countries, those who live in impoverished communities in developed
countries and in the elderly who lack access to nutritious food. Protein deficiency also affects
people who are born with a genetic disorder to produce certain proteins, and people with
diseases that cause them to lose appetite and experience muscle breakdown.

A LIST OF PROTEINDEFICIENCY DISEASES

Proteins are substances that are part of cells, tissues and organs throughout the body,
according to the Centers for Disease Control. Protein deficiency is common among people
who live in developing countries, those who live in impoverished communities in developed
countries and in the elderly who lack access to nutritious food. Protein deficiency also affects
people who are born with a genetic disorder to produce certain proteins, and people with
diseases that cause them to lose appetite and experience muscle breakdown.

DEFICIENCIES OF PROTEIN C AND PROTEIN S

Deficiencies of protein C and protein S are inherited conditions that cause abnormal blood
clotting. Deficiency of protein C occurs in about 1 out of 300 people. Deficiency of protein S
affects 1 in 20,000 people. Symptoms for these deficiencies include redness, pain, tenderness
or swelling in the affected area. People with these protein deficiencies need to be careful
about activities that increase risk of blood clots, such as prolonged sitting, bed rest, and long-
time travel in cars and airplanes. Protein S deficiency causes ischemic stroke.

CACHEXIA

Cachexia is a condition that involves protein deficiency, depletion of skeletal muscle and an
increased rate of protein degradation. Cachexia causes weight loss and mortality and is
associated with cancer, AIDS, chronic kidney failure, heart disease, chronic obstructive
pulmonary disease and rheumatoid arthritis

PHYSICAL PROPERTIES OF PROTEIN

1. Colour and Taste: Proteins are colourless and usually tasteless. These are homogeneous
and crystalline.

2. Shape and Size: The proteins range in shape from simple crystalloid spherical structures
to long fibrillar structures. Two distinct patterns of shape have been recognized :

A. Globular proteins—These are spherical in shape and occur mainly in plants,

esp., in seeds and in leaf cells. These are bundles formed by folding and crumpling
of protein chains. e.g., pepsin, insulin, ribonuclease etc.

B. Fibrillar proteins—These are thread-like or ellipsoidal in shape and occur

generally in animal muscles. e.g., fibrinogen, myosin etc.

3. Molecular Weight: The proteins generally have large molecular weights ranging between
5 × 103 and 1 × 106

[Link] Nature: Because of their giant size, the proteins exhibit many colloidal
properties, such as :

I. Their diffusion rates are extremely slow.

II. They may produce considerable light-scattering in solution, thus resulting in

visible turbidity (Tyndall effect).
[Link]: Denaturation refers to the changes in the properties of a protein. In
other words, it is the loss of biologic activity. In many instances the process of
denaturation is followed by coagulation— a process where denatured protein molecules
tend to form large aggregates and to precipitate from solution.

Denaturation may be brought about by a variety of agents, both physical and chemical. The
physical agents include mechanical action (like shaking), heat treatment cooling and
freezing operations, rubbing, high hydrostatic pressures, (5,000 to 10,000 atm.), ultraviolet
rays, etc. The chemical agents, that cause denaturation, are many ionizing radiations
(like X-rays, radioactive and ultrasonic radiations), organic solvents (acetone, alcohol),
aromatic anions (salicylates), some anionic detergents (like sodium dodecyl sulphate), etc.

6. Amphoteric Nature: Like amino acids, the proteins are amphoteric, i.e., they act as
acids and alkalies both. These migrate in an electric field and the direction of
migration depends upon the net charge possessed by the molecule. The net charge is
influenced by the pH value. Each protein has a fixed value of isoelectric point (pl) at which it
will move in an electric field. Isoelectric point (or isoionic point) is the pH value at which the
number of cations is equal to that of anions. Thus, at isoelectric point, the net electric charge
of a protein is always zero. But the total charge on the protein molecule (sum of
positive and negative charges) at this point is always maximum. Thus, the proteins are
dipolar ions or internal salts or zwitterions (German for ‘ion of both kinds’ ; amphoteric ions)
at pl.

7. Ion Binding Capacity. Being amphoteric in nature, the proteins can form salts with both
cations and anions based on their net charge. In fact, a mixture of different proteins
at a given pH (except at pl) will include cations and anions both and the salts of protein-
protein combinations will be formed. This occurs in tissues since both acidic and
basic proteins are present. Many ions form insoluble salts with proteins and serve as
excellent precipitating agents for proteins.

8. Solubility. The solubility of proteins is markedly influenced by pH. Solubility is lowest at

isoelectric point and increases with increasing acidity or alkalinity. This is because when the
protein molecules exist as either cations or anions, repulsive forces between ions are
high, sinceall the molecules possess excess charges of the same sign. Thus, they will be
more soluble than in the isoelectric state.

A.‘Salting-in’ effect. Globulins are sparingly soluble in water but their solubility is
greatly increased by the addition of neutral salts like NaCl. This phenomenon is
commonly described as salting-in’ effect.

B.‘Salting-out’ effect. Proteins are precipitated from aqueous solution by high

concentrations of neutral salts. This is the ‘salting-out’ process. Divalent and
trivalent ions are more effective than univalent ions. The salts commonly used for
this purpose are Na2.SO4, (NH4)2.SO4, magnesium salts and phosphates.
9. Optical Activity. All protein solutions rotate the plane of polarized light to the left,
i.e.,these are levoratotory. For example, the specific rotation [α]D for ovalbumin is near —
30° over the pH range between 3.5 and 11. However, at lower or higher pH values
the rotation becomes more negative, e.g., at pH 13, the [α]D is about —60°. The
rotation is further increased by subjecting proteins to high temperatures.

CHEMICAL PROPERTIES

A. HYDROLYSIS

Proteins are hydrolyzed by a variety of hydrolytic agents.

1. By acidic agents. Proteins, upon hydrolysis with conc. HCl (6–12N) at 100–110°C
for6 to 20 hrs, yield amino acids in the form of their hydrochlorides.

2. By alkaline agents. Proteins may also be hydrolyzed with 2N NaOH. Alkaline

hydrolysisis, however, less used as it is highly disadvantageous:

(a) It leads to the destruction of certain amino acids like arginine, cysteine,
cystine, serine,threonine etc.

(b) It also causes loss of optical activity (or racemization) of the amino acids.

3. By proteolytic enzymes: Under relatively mild conditions of temperature and

acidity, certain proteolytic enzymes like pepsin and trypsin hydrolyze the proteins. Enzyme
hydrolysis is used for the isolation of certain amino acids like tryptophan. Two important
drawbacks with this type of hydrolysis are:

(a) It requires prolonged incubation.

(b) Hydrolysis is incomplete.
B. REACTIONS INVOLVING COOH GROUP
1. Reaction with alkalies (Salt formation). The carboxylic group of amino acids can
release a H+ ion with the formation of carboxylate (COO—) ions. These may be
neutralised by cations like Na+ and Ca2+ to form salts. Thus, amino acids react with
alkalies to form salts. Sodium salt of glutamic acid (monosodium glutamate) is used
commercially as a flavouring agent. It imparts a meat-like flavour to soups, for
[Link] glutamate (MSG)or ‘ajinomoto’
2. Reaction with alcohols (Esterification). With alcohols, corresponding esters are
produced. The esters, so obtained, are volatile in contrast to the free amino acids.
The reaction was, for the first time, used by Emil Fischer for the isolation of amino acids in
pure form from protein hydrolysates by the fractional distillation in vacuum of their ethyl
esters.
3. Reaction with amines. Amino acids react with amines to form amides.
C. REACTIONS INVOLVING NH2 GROUP
1. Reaction with mineral acids (Salt formation). When either free amino acids or proteins
are treated with mineral acids like HCl, the acid salts are formed. The basic amino
acids, arginine and lysine react with CO2 in the presence of air to form carbonate
salts. Because of this property, these are usually stored and also sold in the form of their
monochlorides.
2. Reaction with formaldehyde. With formaldehyde, the hydroxy-methyl derivatives are
formed. These derivatives are insoluble in water and resistant to attack by
microorganisms. Because of this action, formaldehyde is the principal reagent in
embalming fluids and is used to harden and preserve certain fibres (Aralac, Vicara)
obtained from globular proteins. This reaction is the basis of the Sorensen titration
method for determining the purity of the individual amino acids.
3. Reaction with benzaldehyde. Schiff's bases are formed.
4. Reaction with nitrous acid (Van Slyke reaction). The amino acids react with HNO2 to
liberate N2 gas and to produce the corresponding α-hydroxy acids. This reaction is
characteristic of aliphatic primary amines and has been utilized by Van Slyke (1912) as the
basis for his `nitrous acid' method for the estimation of amino acids by measuring the
volume of N2 gas liberated. The amino acids proline and hydroxyproline, however,
do not respond to this reaction.
5. Reaction with acylating agents (Acylation). Acylation is brought about by many
acid chlorides ([Link], [Link]) and acid anhydrides ([Link]—O—OC.CH3,
phthalic anhydride), when amino acids in alkaline medium react with them.

FUNCTIONL PROPERTIES OF PROTEIN