1.

CELL

The cell is the fundamental unit of life. All organisms are built from cells. All animal
tissues including human are also organized from collections of cells.
Types of Cells
In general two types of cells exist in nature. They are:
1. Prokaryotic cells
2. Eukaryotic cells

Prokaryotic cell Eukaryotic cell

1 Smaller in size 1 to 10 um Larger in size 10 to 100µm

2 Mainly unicellular Mainly multicellular.

3 Single membrane, surrounded by rigid Lipid bilayer membrane with protein

cell wall

4 Anaerobic or aerobic Aerobic

5 Not well defined nucleus, only a nuclear Nucleus well defined,4 to 6 µm in diameter,
zone with DNA contains DNA and surrounded by a perinuclear
membrane

6 Histones absent Histones present

7 No nuclei Nucleolus present, rich in RNA

8 Cytoplasm contains no cell organelles Membrane bound cell organelles are present

9 Ribosomes present free in cytoplasm Ribosomes studded on outer surface of

endoplasmic reticulum present

10 Mitochondria absent .Enzymes of Mitochondria present. ”Power house” of the cell.

energy metabolism bound to Enzymes of energy metabolism are located in
membrane mitochondria

1
11 Golgi apparatus absent. Storage Golgi apparatus present...flattened single
granules with polysaccharides membrane vesicle

12 Lysosomes absent Lysosomes present...single membrane vesicle

containing packets of hydrolytic enzymes

13 Cell division usually by fission, Cell division...by mitosis

14 Cytoskeleton....absent Cytoskeleton....present

15 RNA and protein synthesis in same RNA synthesized and processed in nucleus.
compartment Proteins synthesized in cytoplasm

16 Examples are bacteria, cyanobacteria, Examples are Protists, fungi, plants and animal cells
rickettsii

Eukaryotic cell

2
 2. CELL ORGANELLES
The eukaryotic cell is subdivided by membranes. On the outside, it is enclosed by a plasma
membrane. Inside the cell, there is a large space containing numerous components in solution—
the cytoplasm. Additional membranes divide the internal space into compartments (confined
reaction spaces). Well defined compartments of this type are known as organelles.

1. Nucleus:
The nucleus contains more than 95 per cent of the cell’s DNA and is the control
center of the eukaryotic cell.

• Nuclear envelope: A double membrane structure called the nuclear envelope

separates the nucleus from the cytosol.

• Nuclear pore complexes: These are embedded in the nuclear envelope. These
complex structures control the movement of proteins and the nucleic acid,
ribonucleic acids (RNAs), across the nuclear envelope.

• Chromatin: DNA in the nucleus is coiled into a dense mass called chromatin

• Nucleolus: A second dense mass closely associated with the inner nuclear
envelope is called nucleolus.

• Nucleoplasm: Nucleoplasm of nucleus contains various enzymes such as DNA

polymerases, and RNA polymerases, for m-RNA and t-RNA synthesis.

Functions

• DNA replication and RNA transcription of DNA occur in the nucleus.

Transcription is the first step in the expression of genetic information and is the
major metabolic activity of the nucleus.
3
 • The nucleolus is non-membranous and contains RNA polymerase, RNA-ase,
ATPase and other enzymes but no DNA polymerase. Nucleolus is the site of
synthesis of ribosomal RNA (r-RNA).

• Nucleolus is also the major site where ribosome subunits are assembled.

2. Mitochondrion:
Mitochondrion is the power house of cell.

• Number: The number of mitochondria in a cell varies dramatically. A

mammalian liver cell contains from 800 to 2500 mitochondria.

• Size: They vary greatly in size. A typical mammalian mitochondrion has a

diameter of 0.2 to 0.8 μ and a length of 0.5 to 1.0 μm.

• Shape: The shape of mitochondrion is not static.

Structure and Functions

The mitochondrion is bounded by two concentric membranes that have markedly

different properties and biological functions.
4
Mitochondrial Membranes

(a) Outer mitochondrial membrane: The outer mitochondrial membrane consists mostly
of phospholipids and contains a considerable amount of cholesterol. The outer
membrane also contains many copies of the protein called Porin.

Functions of Porin and other Proteins

(i) These proteins form channels that permit substances with molecular weights
of less than < 10,000 to diffuse freely across the outer mitochondrial
membrane.

(ii) Other proteins in the outer membrane carry out various reactions in fatty
acid and phospholipid biosynthesis and are responsible for some oxidation
reactions.

(b) Inner mitochondrial membrane: The inner mitochondrial membrane is very rich in
proteins and the ratio of lipid to proteins is only 0.27:1 by weight. It contains high
proportion of the phospholipid cardiolipin. In contrast to outer membrane, the inner
membrane is virtually impermeable to polar and ionic substances. These substances enter
the mitochondrion only through the mediation of specific transport proteins.

• Cristae: The inner mitochondrial membrane is highly folded. The tightly

packed inward folds are called “cristae”.

(c) Intermembrane space: The space between the outer and inner membranes is known
as the intermembrane space. Since the outer membrane is freely permeable to small
molecules, the intermembrane space has about the same ionic composition as the
cytosol.

(d) Mitochondrial matrix: The region enclosed by the inner membrane is known as the
mitochondrial matrix.

5
  Composition of matrix: The enzymes responsible for citric acid cycle and fatty
acid oxidation are located in the matrix. The matrix also contains several
strands of circular DNA, ribosomes and enzymes required for the
biosynthesis of the proteins coded in the mitochondrial genome. The
mitochondrion is not, however, genetically autonomous, and the genes
encoding most mitochondrial proteins are present in nuclear DNA.

Functions

 The oxidation of pyruvate, amino acids, fatty acids (by β-oxidation), and the
tricarboxylic acid (TCA) cycle occurs in matrix. The synthesis of glucose, urea,
and heme also occur partially in the matrix.

 The matrix also contains mitochondrial RNA and DNA (mtRNA and mtDNA)
and mitochondrial ribosomes.

 The mitochondrion is specialized for the rapid oxidation of NADH (reduced

NAD) and FADH2 (reduced FAD) produced in the reactions of glycolysis, the
citric acid cycle and the oxidation of fatty acids. This is done with the help of
electron transport chain and ATP synthases enzyme. The energy produced is
trapped and stored as ATP, for future use of energy in the body.

6
CLINICAL ASPECT

 A disease known as Luft’s disease involving mitochondrial energy

transduction has been reported.
 Mutations in mtDNA are responsible for several diseases, including some
cases of mitochondrial myopathies and Leber hereditary optic
neuropathy, a disease in which bilateral loss of central vision occurs as a
result of neuroretinal degeneration, including damage to the optic
nerve.
 Age related degenerative disorders such as Parkinson’s disease;
cardiomyopathy may have a component of mitochondrial damage.

3. Endoplasmic reticulum (ER):

Eukaryotic cells are characterized by several membrane complexes that are
interconnected by separate organelles. These organelles are involved in protein synthesis,
transport, modification, storage and secretion.

Varying in shape, size and amount, the endoplasmic reticulum (ER) extends from
the cell membrane, coats the nucleus, surrounds the mitochondria and appears to

7
connect directly to the Golgi apparatus. These membranes and the aqueous channels
they enclose are called cisternae.

Types: There are two kinds of endoplasmic reticulum (ER):

(i) Rough surfaced ER: They are coated with ribosomes. Near the nucleus, this
type of ER merges with the outer membrane of the nuclear envelope.

(ii) Smooth surfaced ER: They do not have attached ribosomes.

Functions

(a) Function of rough ER:

Rough ER synthesizes membrane lipids, and secretory proteins. These

proteins are inserted through the ER membrane into the lumen of the
cisternae where they are modified and transported through the cell.

(b) Function of smooth ER:

Smooth endoplasmic reticulum is involved:

(i) In lipid synthesis and

(ii) Modification and transport of proteins synthesized in the rough ER

A number of important enzymes are associated with the endoplasmic reticulum of

mammalian liver cells. These include the enzymes responsible for the synthesis of
sterol, triacylglycerol (TG), Phospholipids (PL) and the enzymes involved in
detoxification of drugs. Cytochrome P450 which participates in drug hydroxylation
resides in the ER.

8
4. Golgi complexes (or Golgi apparatus):
They are also called Dictyosomes. They are a unique stack of smooth surfaced
compartments or cisternae. The ER is usually closely associated with the Golgi complexes,
They contain flattened, fluid filled golgi sacs. The Golgi complex has a Proximal or Cis
compartment, a medial compartment and a distal or trans compartment. The complex
serves as a unique sorting device, which receives newly synthesized proteins, all
containing signal or transit peptides from the ER. Those proteins with no signal or transit
peptides regions are rejected by the Golgi apparatus without processing it further and
remain as cytoplasmic protein.

Functions

(i) On the proximal or cis side, the Golgi complexes receive the newly
synthesized proteins by ER via transfer vesicles.

(ii) The post-translational modifications take place in the Golgi lumen

(median part) where the carbohydrates and lipid precursors are
added to proteins to form glycoproteins and lipoproteins
respectively.

(iii) On the distal or trans side they release proteins via modified
membranes called secretory vesicles. These secretory vesicles move
to and fuse with the plasma membrane where the contents may be
expelled by a process called exocytosis.

9
 5. Lysosomes:
Lysosomes are cell organelles found in cells which contain packet of enzymes.

• Size: Mean diameter is approximately 0.4 μ. They are surrounded by a lipoprotein

membrane.
• Lysosomes are found in all animal cells, except erythrocytes, in varying numbers
and types.
• pH: pH inside the lysosomes is lower than that of cytosol. The lysosomal enzymes
have an optimal pH around 5. Acid phosphatase is used as a marker enzyme for
this organelle.
• As long as the lysosomal membrane is intact, the encapsulated enzymes can act
only locally. But when the membrane is ruptured, the enzymes are released into
the cytoplasm and can hydrolyse external substrates (biopolymers).

CLINICAL ASPECT

1. Allergic responses and arthritic conditions: Released enzymes from ruptured

lysosomal membrane can hydrolyze external biopolymers (substrates) leading
to tissue damage in many types of allergic responses and arthritic conditions.

2. In Gout:: Urate crystals are deposited around joints. These crystals when
phagocytosed cause physical damage to lysosomes and release of enzymes
producing inflammation and arthritis.

3. Inherited disorders:
10
  Nieman-Pick disease sphingomyelinase deff  sphingomylin
accumulates in brain and spleen.It produces mental retardation and
enlargement of liver and spleen. It is fatal in early life.
 Gaushers disease
4. I-Cell disease:

I-cell disease is a rare condition in which lysosomes lack all of the normal
lysosomal enzymes. The disease is characterised by severe progressive
psychomotor retardation and a variety of physical signs, with death often
occurring in the first decade.

Cultured cells from patients with I-cell disease was found to lack almost all of
the normal lysosomal enzymes. The lysosomes thus accumulate many
different types of undegraded molecules forming inclusion bodies.

6. Peroxisomes:
Peroxisomes are small organelles also called Microbodies, present in eukaryotic cell. The
particles are approximately 0.5 μ in diameter. They are bounded by a single membrane. Like
mitochondria, peroxisomes can replicate by division. However, they are dependent on the import
of proteins to function. They contain no DNA.

Functions
(i) Peroxisomes are involved in oxidative
reactions using molecular oxygen. These
reactions produce the toxic chemical
hydrogen peroxide (H2O2), which is
subsequently used or degraded within the
peroxisome by catalase and other
enzymes.

(ii) Peroxisomes function in the oxidation of very long chain fatty acids
(containing 20 or more carbons) to shorter chain fatty acids, the
conversion of cholesterol to bile acids, and the synthesis of ether lipids
called plasmalogens.
CLINICAL ASPECT

Peroxisomes may be absent in inherited disorder Zellweger’s syndrome.

B. Cytoplasm (Cytosol)
This is the simplest structure of the cell. Organelles free sap is called as cytosol.
Many metabolic reactions take place in cytosol where substrates and cofactors interact
with various enzymes. There is no specific structure for cytosol. It has a high protein
contents. The actual physiochemical state of cytosol is poorly understood. A major role of
cytosol is to support synthesis of proteins on the rough endoplasmic reticulum by
supplying cofactors and energy.

11
 Cytosol also contains free ribosomes. They contain many different types of proteins and
ribosomal RNA or r-RNA. They exist as 2 subunits and act as the site of protein synthesis.

3. CELL MEMBRANE
Membranes are composed of lipids, proteins and carbohydrates.

(a) Lipids: Lipids are the basic structural components of cell membranes. Lipid molecules
have a ‘polar’ or ionic head hence hydrophilic and the other end is a ‘nonpolar’ and
hydrophobic tail. Hence they are amphipathic.

Types of Lipids Present in Bio membranes

1. Fatty acids: They are major components of most membrane lipids. The
nonpolar tails of most membrane lipids are long chain fatty acids attached to
polar head groups, such as glycerol-3-P. Oleic acid is the most abundant
unsaturated fatty acid in animal membrane lipids; others are arachidonic acid,
linoleic and linolenic acids. The degree of unsaturation determines the fluidity of
the membranes.

2. Glycerophospholipids: They are another group of major components of

biomembranes. Phosphatidylethanol amine (cephalin), phosphatidylcholine
(Lecithin) and phosphatidylserine are among the most of common
glycerophospholipids.

3. Sphingolipids: They comprise another group of lipids found in biological

membranes especially in the tissues of nervous system. There are three types of
sphingolipids; sphingomyelin, cerebrosides and gangliosides. About 6 per cent of
the membrane lipids of grey matter cells in the brain are gangliosides.

4. Cholesterol: Cholesterol is another common component of the

biomembranes of animals. It is oriented with its hydrophilic polar heads
exposed to water and its hydrophobic fused ring system and attached
hydrocarbon groups buried in the interior between phospholipids. Cholesterol
helps to regulate fluidity of animal membranes.

12
(b) Proteins: Main types of membrane proteins are:

1. Integral membrane proteins (also called intrinsic membrane proteins): These

proteins are deeply embedded in the membrane. Thus portions of these proteins are in
Van der Waals contact with the hydrophobic region of the membrane.

2. Peripheral membrane proteins (also called extrinsic proteins): These may be weakly
bound to the surface of the membrane by ionic interactions or by hydrogen bonds that
form between the proteins and the ‘polar’ heads of the membrane lipids. They may also
interact with integral membrane proteins. They can be removed without disrupting the
membrane.

3. Transmembrane proteins: Some of the integral proteins span the whole breadth of
the membrane and are called as transmembrane proteins. The hydrophobic side chains
of the amino acids are embedded in the hydrophobic central core of the membrane.
These proteins can serve as receptors for hormones, neurotransmitters, tissue specific
antigens, growth factors, etc.

(b) Carbohydrates: Some of the proteins and lipids on the external surface of the
membrane contain short chains of carbohydrates (oligosaccharides) that extend into
the aqueous medium.
 Carbohydrates therefore constitute 2 to10% of the weight of plasma membranes.
 This hydrophilic carbohydrate layer, called the glycocalyx, protects the cell against
digestion and restricts the uptake of hydrophobic compounds.
 Specific carbohydrate chains on the glycolipids serve as cell recognition molecules
 Glycophorin is a major integral membrane glycoprotein of human erythrocytes.

13
FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE
The fluid mosaic model of membrane structure proposed by Singer and Nicholson in 1972
is now accepted widely.

All biological membranes are constructed according to a standard pattern.

 They consist of a continuous bilayer of amphipathic lipids approximately 5 nm

thick, into which proteins are embedded.

 In addition, some membranes also carry carbohydrates (mono- and

oligosaccharides) on their exterior, which are bound to lipids and proteins.

 Membrane lipids are strongly amphipathic molecules with a polar hydrophilic

“head group” and an apolar hydrophobic “tail.”In membranes, they are primarily
held together by the hydrophobic effect and weak Van der Waals forces, and are
therefore mobile relative to each other. This gives membranes a more or less
fluid quality. The fluidity of membranes primarily depends on their lipid
composition and on temperature. Heat promotes lipid bilayer from gel
(crystalline) state to fluid state. The cholesterol content also influences
membrane fluidity. It increases the fluidity of semi crystalline, closely-packed
membranes and it stabilizes fluid membranes that contain a high proportion of
unsaturated lipids.

 Like lipids, proteins are also mobile within the membrane. If they are not fixed in
place by special mechanisms, they float within the lipid layer as if in a two-
dimensional liquid; biological membranes are therefore also described as being a
“fluid mosaic.”

 Lipids and proteins can shift easily within one layer of a membrane, but switching
between the two layers (“flip/flop”) is not possible for proteins and is only
possible with difficulty for lipids (with the exception of cholesterol).

14
Effects of Fluidity of Membrane

The fluidity of membrane significantly affects its functions:

 As membrane fluidity increases, its permeability to water and other small

hydrophilic molecules also increases.

 As fluidity increases, the lateral mobility of integral proteins also increases.

FUNCTIONS OF CELL MEMBRANE:

It is a physical barrier to separate the inside and outside of the cells. Its function is to
maintain Homeostasis. Its selective permeability allows the cell to maintain a constant internal
environment. Plasma membranes form compartments within cells.

Its functions are to:

1. Regulate cell volume (control water in/ out)

2. Maintain intracellular pH (H+ regulation)
3. Selectively regulate intracellular ions (composition e.g. Na+ , K+ )
4. Concentrate metabolic fuel (nutrients, ATP, etc)
5. Concentrate and move building blocks (amino acids, etc)
6. Remove toxic compounds (detoxification, trap and/or pump out)
7. Generate ionic gradients to maintain excitability of nerve and muscle cells
8. Control the flow of “information” within the cell, between cells and their environment.

FUNCTIONS OF CELL MEMBRANE

15
 4. TRANSPORT OF MOLECULES ACROSS THE PLASMA
MEMBRANE
An essential role of biomembranes is to allow movement of all compounds necessary for
the normal function of a cell across the membrane barrier. These compounds include a vast array
of substances like sugars, amino acids, fatty acids, steroids, cations and anions to mention a few.
These compounds must enter or leave the cells in an orderly manner for normal functioning of
the cell.

ION CHANNELS

Ion channels are transmembrane channels, pore like structures composed of

proteins. Specific channels for Na+, K+, Ca++, and Cl– have been identified. All ion
channels are basically made up of transmembrane subunits that come together to form a
central pore through which ions pass selectively. All channels have gates, and are
controlled by opening and closing.

Types of Gates

Two types of gated channels. They are:

a. Ligand gated channels: In this a specific molecule binds to a receptor

and opens the channel.

b. Voltage gated channels: These channels open or close in response to

changes in membrane potential.

IONOPHORES

Certain microorganisms can synthesize small organic molecules, called

ionophores, which function as shuttles for the movement of ions across the membrane.

16
 WATER CHANNELS (AQUAPORINS)

In certain cells, e.g. in red blood cells, and cells of the collecting ductules of the
kidney, the movement of water by simple diffusion is enhanced by movements of water
through water channels, composed of tetrameric transmembrane proteins called
aquaporins. About five distinct types of aquaporins have been recognised.

GAP JUNCTION

Certain cells develop specialized regions on their membranes for intercellular

communications which are in close proximity.

Function: They mediate and regulate the passage of ions and small molecules up to 1000
to 2000 mol wt, through a narrow hydrophilic core connecting the cytosol of adjacent
cells.

Structure:They are composed of proteins called connexin which contains four membrane
spanning alpha helixes.

TYPES OF TRANSPORT MECHANISMS

The following are three important mechanisms for transport of various compounds across
the bio-membrane:

(a) Passive or simple diffusion

17
 (b) Facilitated diffusion and
(c) Active transport

(a) PASSIVE OR SIMPLE DIFFUSION:

It depends on the concentration gradient of a particular substance across the

membrane. The solute passes from higher concentration to lower concentration till
equilibrium is reached. The process neither requires any carrier protein nor energy. It
operates unidirectionally.

Examples: water, gases, pentose sugars.

Factors affecting net diffusion:

• Concentration gradient: The solutes move from high to low concentration.
• Electrical potential: Solutes move toward the solution that has the opposite
charge. The inside of the cell usually has a negative charge.
• Hydrostatic pressure gradient: Increased pressure will increase the rate and
force of the collision between the molecules.
• Temperature: Increased temperature will increase particle motion and thus
increase the frequency of collisions between external particles and the
membrane.
• Permeability coefficient: Net diffusion also depends on the permeability
coefficient for the membrane.

(B) FACILITATED DIFFUSION:

It is similar to passive diffusion in that solutes move along the concentration

gradient. But it differs from passive diffusion in that it requires a carrier or transport
protein. Hence the rate of diffusion is faster than simple diffusion. The process does not
require any energy and can operate bidirectionally.

Example: D-fructose is absorbed from intestine by facilitated diffusion.

18
(C) ACTIVE TRANSPORT:

Active transport occurs against a concentration gradient and electrical gradient.

Hence it requires energy. About 40 per cent of the total energy requirement in a cell is
utilized for active transport system. It requires the mediation of specific carrier or
transport proteins.

Types of transport system: Transport systems can be classified as follows:

1. Uniport system: This system involves the transport of a single solute molecule
through the membrane.

Example: Glucose transporters in various cells.

2. Co-transport system: D-Glucose, D-Galactose and L-amino acids are transported into
the cells by Na+ - dependant co-transport system. Na+ is not allowed to accumulate
in the cells and it is pumped out by “sodium pump”.

(i) Symport system: It is a co-transport system in which the transporter

carries the two solutes in the same direction across the membrane.

(ii) Antiport system: It is a type of cotransport system in which two solutes or

ions are transported simultaneously in opposite directions.

Example: Chloride and bicarbonate ion exchange in lungs in red blood cells.

Cystic Fibrosis
Inheritence: A recessive genetic disorder, prevalent among whites in N America and certain
parts of Northern Europe.
Clinical Features: The disease is characterised by:
• Chronic bacterial infections of the respiratory tract and sinuses
• Fat maldigestion due to pancreatic exocrine insufficiency
• Infertility in males due to abnormal development of the vas deferens, and
• Elevated levels of chloride in sweat, greater than > 60 mmol/L.
Defect: Cystic fibrosis transmembrane protein (CFTR) is a cyclic AMP dependant regulatory
protein for chloride channel. Genetic mutation produces an abnormal CFTR, which
produces an abnormality of membrane Cl– permeability resulting to increased viscosity of
many bodily secretions.
Prognosis: It is bad, life threatening and serious complication is recurrent lung infections
due to overgrowth of bacteria in viscous secretions. Efforts are in progress to use gene
therapy to restore the activity of CFTR protein.
19
TRANSPORT OF MACROMOLECULES

The mechanism of transport of macromolecules (ligands) such as proteins,

hormones, immunoglobulins, low density lipoproteins (LDL) and even viruses takes place
across the membrane by two independant mechanisms

1. Exocytosis

2. Endocytosis.

1. Exocytosis: Most cells release macromolecules to the exterior by the process called
exocytosis. This process is also involved in membrane remodeling when the components
synthesized in the Golgi apparatus are carried in vesicles to the plasma membrane. The
movement of the vesicle is carried out by cytoplasmic contractile elements in the
microtubular system.

Mechanism: The inner membrane of the vesicle fuses with the outer plasma membrane,
while cytoplasmic side of vesicle fuses with the cytoplasmic side of plasma membrane.
Thus, the contents of vesicles are externalised. The process is also called reverse
pinocytosis. The process induces a local and transient change in Ca++ concentration
which triggers exocytosis.

Types of macromolecules released by exocytosis:

They fall into 3 categories.

I. They can attach to the cell surface and become peripheral proteins, e.g.
antigens.
II. They can become part of extracellular matrix, e.g. collagen and
glycosaminoglycans (GAGs)
III. Hormones like insulin, parathormone (PTH) and catecholamines are all
packaged in granules, processed within cells to be released upon
appropriate stimuli.

3. Endocytosis: All eukaryotic cells are continuously ingesting parts of their plasma
membrane. Endocytic vesicles are formed when segments of plasma membrane
invaginates enclosing a minute volume of extracellular fluid (ECF) and its contents.
The vesicle then pinches off as the fusion of plasma membranes seal the neck of the
vesicle at the original site of invagination. The vesicle fuses with other membrane
structures and thus transports of its contents to other cellular compartments.

20
Factors required for endocytosis: Endocytosis requires the following:
• Energy: Usually derived from ATP hydrolysis.
• Ca++
• Contractile element in the cell-probably the microfilament system.
Fate: Most endocytic vesicles fuse with primary lysosomes to form secondary lysosomes
which contain hydrolyic enzymes, and are, therefore, specialised organelles for
intracellular disposal. Vesicular contents are digested liberating simple sugars, amino
acids, etc. which diffuse out of the vesicles to be reutilised in the cytoplasm.

Types of endocytosis: The endocytosis is of following types:

 Phagocytosis: Phagocytosis (Greek word-Phagein-to eat) is the engulfment

of large particles like viruses, bacteria, cells, or debris by macrophages and
granulocytes. They extend pseudopodia and surround the particles to form
phagosomes, which later fuse with lysosomes to form Phagolysosomes in
which the particles are digested. Biochemical mechanism is called respiratory
burst, in which O2 consumption is increased and lead to formation of
superoxide ion O2.–

21
 CLINICAL ASPECT:
Chronic granulomatous disease has been recently implicated due to defective
phagocytosis and respiratory burst. The disease is characterised by:
• Recurrent infections
• Widespread granuloma formation in various tissues like lungs,
lymph nodes, skin, etc.
Defect: The disorder is attributed to mutations in the genes encoding the four
polypeptides that constitute the active NADPH oxidase system. The granulomas are
formed as attempts to wall off bacteria that have not been killed due to genetic
deficiencies in the NADPH oxidase system.

 Pinocytosis: It is a property of all cells and leads to the cellular uptake of

fluid and fluid contents.

a) Fluid phase pinocytosis: It is a nonselective process in which

uptake of a solute by formation of small vesicles is simply
proportionate to its concentration in the surrounding
extracellular fluid (ECF). The formation of these vesicles is an
extremely active process.

b) Receptor mediated absorptive pinocytosis:

 Approximately 2 per cent of the external surface of plasma
membrane is covered with receptors and characteristic coated
pits.
 The membrane bound receptors combine with ligands and move
laterally into clathrin (unusual peripheral protein) “coated
pits”.
 These coated pits are rapidly pinched off and are internalized as
clathrin coated vesicles.
 The protein dynamin which binds and hydrolyses GTP, is
necessary for the pinching off of clathrin-coated vesicles from
the cell surface.
 Near the periphery of the cell’s interior, there is another
structure called endosome, which do not contain hydrolytic
enzymes, is less dense than lysosomes and have an internal pH
of 5.0.
 The internalised coated vesicles fuse with the endosomes and
discharge their macromolecules into the interior of the
endosomes.
 The low pH breaks the linkage between receptor-ligand, with a
simultaneous release of clathrin, ligand, free receptors and
membrane fragments, most of which recycle back to the
plasma membrane to replenish the population of receptors
and coated pits.

22
  The ligand containing endosomes now move, by the help of
microtubule to further interior of the cells where they fuse
with lysosomes or become associated with vesicles derived
from the Golgi apparatus .
Example: The low density lipoproteins (LDL) molecule bound to
receptors are internalised by means of coated pits.

CLINICAL ASPECT:
Receptor mediated endocytosis with viruses are responsible for many
diseases, viz.
• Hepatitis virus affecting liver cells
• Poliovirus affecting motor neurons
• AIDS affecting T cells.
Iron toxicity also occurs with excessive uptake due to endocytosis.

OSMOSIS: (Greek : push)

It refers to the movement of solvent (most frequently water) through a semipermeable
memhrane. The flow of solvent occurs from a solution of low concentration to a solution of high
concentration, when both are separated by a semipermeable membrane. ln a strict sense, the
semipermeable membrane is expected to be permeable to the solvent and not to the solute.
Osmotic pressure
Osmotic pressure may be defined as the excess pressure that must be applied to a
solution to prevent the passage of solvent into the solution, when both are separated by a
semipermeable membrane.
Osmosis is a colligative property i.e. a character which depends on the number of solute
particles and not their nature. Osmotic pressure is directly proportional to the concentration
(number) of the solute molecules or ions. Low molecular weight substances (e.g. NaCl, glucose)
will have more number of molecules compared to high molecular weight substances (albumin,
globulin) for unit mass. Therefore, the substances with low molecular weight, in general, exhibit
greater osmotic pressure. Further for ionizable compounds, the total osmotic pressure is

23
equivalent to the sum of the individual pressures exerted by each ion. For instance, one molar
solution o NaCl will exert double the osmotic pressure of one molar solution of glucose. This is
because NaCl ionizes to Na+ and Cl- while glucose is non-ionizable.
The solutions that exert the same osmotic pressure are said to be iso-osmofic. The term
isotonic is used when a cell is in direct contact with an iso osmotic solution (0.9%NaCl ) which
does not change the cell volume and, thus, the cell tone is maintained. A solution with relatively
greater osmotic pressure is referred to as hypertonic. On the other hand, a solution with
relatively lower pressure is hypotonic. The term oncotic pressure is commonly used to represent
the osmotic pressure of colloidal substances (e.g. albumin, globulin).

Units of osmotic pressure:

Osmole is the unit of osmotic pressure. One osmole is the number of molecules in gram
molecular weight of undissociated solute. One gram molecular weight of glucose (180 g) is one
osmole. However, one gram molecular weight of NaCl (58.5g ) is equivalent to 2 osmoles, since
NaCl ionizes to give two particles ( Na+, Cl-). Osmotic pressure of biological fluids is frequently
expressed as milliosmoles. The osmotic pressure of plasma is 280-300 milliosmoles /L.

Applications of osmosis
1. Fluid balance and blood volume: The fluid balance of the different compartments of the
body is maintained due to osmosis. Further, osmosis significantly contributes to the
regulation of blood volume and urine excretion.

2. Red blood cells and fragility: When RBCs are suspended in an isotonic (O.95% NaCl)
solution, the cell volume remains unchanged and they are intact. In hypertonic solution
(say 1.5% NaCl), water flows out of RBC and the cytoplasm shrinks, a phenomenon
referred to as crenation.
On the other hand, when the RBC are kept in hypotonic solution (say O.4 % NaCl), the
cells bulge due to entry of water which often causes rupture of plasma membrane of
RBCs (hemolysis).
Osmotic fragility test for RBC is employed in laboratories for diagnostic purposes. For a
normal human blood, RBC begin to hemolyse in 0.45% NaCl and the hemolysis is almost
complete in 0.33% NaCl. Increased fragility of RBCs is observed in hemolytic jaundice
while it is decreased in certain anemias.

3. Transfusion: lsotonic solutions of NaCl (O.9%) or glucose ( 5%) or a suitable

combination of these two are commonly used in transfusion in hospitals for the
treatment of dehydration, burns etc.

4. Action of purgatives: The mechanism of action of purgatives is mainly due to osmotic

phenomenon. For instance, epson (MgSO₄ 7H2O) or Glauber's (Na₂SO₄ 10H2O) salts
withdraw water from the body, besides preventing the intestinal water absorption.

5. Osmotic diuresis: The high blood glucose concentration causes osmotic diuresis
resulting in the loss of water, electrolytes and glucose in the urine. This is the basis of

24
 polyuria observed in diabetes mellitus. Diuresis can be produced by administering
compounds (e.g. mannitol) which are filtered but not reabsorbed by renal tubules.

6. Edema due to hypoalbuminemia: Disorders such as kwashiorkor and

glomerulonephritis are associated with lowered plasma albumin concentration and
edema. Edema is caused by reduced oncotic pressure of plasma, leading to the
accumulation of excess fluid in tissue spaces.
7. Cerebral edema: Hypertonic solutions of salts (NaCl, MgSO₄) are in use to reduce the
volume of the brain or the pressure of cerebrospinal fluid.

8. lrrigation of wounds: lsotonic solutions are used for washing wounds. The pain
experienced by the direct addition of salt or sugar to wounds is due to osmotic removal of
water.

DONNAN MEMBRANE EQUILIBRIUM

When membrane is freely permeable to ions (say Na+, Cl-) and if the concentration of
ions on both the sides is different, the ions freely diffuse to attain equal concentration. Gibbs-
Donnan observed that the presence of a non-diffusible ion on one side of the membrane alters
the diffusion of diffusible ions.

Diagrammatic representation of Donnan membrane equilibrium

In the molecule sodium proteinate (Na+Pr-), the protein (Pr) ion is non-diffusable through
the membrane. Let us consider two sides of a compartment separated by a membrane. Initially,
sodium proteinate is on side I while sodium chloride is on side ll. Diffusible ions (Na+, Cl-) can
freely pass through the membrane. On side l, Na+ ions will balance the incoming Cl- ions besides
Pr ions, while on side ll Na+ ions have to balance only Cl- ions. Therefore, the concentration of
Na+ on side I is greater than on side ll, However, from the thermo dynamical point of view, at
equilibrium, the concentration of Na+ Cl- on both the sides should be the same:

Thus Na+ Cl- ( l ) = Na+ Cl- ( l l )

Since Na+ ( l ) > Na+ ( l l )

Cl- (I) < Cl- (I l)

Consequently, the concentration of Cl- ions should be greater on side ll. Further, the total
concentration of ions on side I is higher than on side l .

25
The salient features of Donnan membrane equilibrium are listed next.
1. The presence of a non-diffusible ions influences the concentration of diffusible ions
across the membrane.
2. The concentration of oppositely charged ions (Na+), is greater on the side of the
membrane containing non-diffusible ions (Pr).
3. The concentration of similarly charged ions (Cl-) is higher on the side of the membrane
not containing non-diffusible ions (Pr).
4. The net concentration of total ions will be greater on the side of the membrane
containing non-diffusible ions. This leads to a difference in the osmotic pressure on either
side of the membrane.

APPLICATIONS OF DONNAN MEMBRANE EQUILIBRIUM

1. Difference in the ionic concentrations of biological fluids: The lymph and interstitial
fluids have lower concentration of inorganic cations (Na+, K+ ) and higher concentration
of anions (Cl-) compared to plasma. This is attributed to the higher protein ( Pr) content in
the plasma.

2. Membrane hydrolysis: The relative strength of H+ and OH- ions and, therefore, the
acidic or alkaline nature on either side of a membrane, is influenced by the presence of
non-diffusible ions. This phenomenon is referred to as membrane hydrolysis. Donnan
membrane equilibrium explains the greater concentration of H+ ions in the gastric juice.

3. Lower pH in RBCs: The hemoglobin of RBCs is negatively charged and, therefore, causes
the accumulation of positively charged ions including H+. Therefore, the pH of RBCs is
slightly lower ( 7.25) than that of plasma (7.4).

4. Osmotic imbalance: Donnan membrane equilibrium-which results in the differential

distribution of ions in different compartments of the body, partly explains the osmotic
pressure difference.

26
 5. SIGNAL TRANSDUCTION ACROSS CELL MEMBRANE
CELL SIGNALING

In multicellular organisms there is a need for the cells to communicate with one another
in order to coordinate their growth and metabolism. The principal way by which cells
communicate with each other is by means of extracellular signalling molecules or hormones.
These molecules are synthesized and secreted by signalling cells and produce a specific response
in target cells that have specific receptors for the signalling molecule. Different cells can respond
differently to the same signalling molecule depending on the type of receptor and the
intracellular reactions initiated.
Cell signalling can be classified into three distinct types based on the distance over which
the signalling molecule acts.

 In endocrine signalling, the signalling molecule (e.g. insulin) acts on target cells
distant from its site of synthesis in cells of an endocrine organ (e.g. the pancreas).
The endocrine cells secrete the signalling molecule into the bloodstream (if an
animal) or the sap (if a plant) which carries it to the target cells elsewhere in the
organism.

 In paracrine signaling, the signalling molecule affects only target cells close to the
cell from which it was secreted. The communication from one nerve cell to another
by chemical neurotransmitters is an example of paracrine signaling.

 The third type of cell signaling is autocrine signaling, where a cell responds to a
molecule that it has produced itself.

27
THE SIGNALLING MOLECULES:

The signaling molecules, ligands or hormones can be classified based on their solubility and
the location of their receptor.
 Lipophilic hormones with intracellular receptors:
Small lipophilic (lipid-soluble) hormones diffuse across the plasma membrane and then
interact with intracellular receptors in the cytosol or nucleus. The resulting hormone–
receptor complex often binds to regions of the DNA and affects the transcription of
certain genes.
Examples:
 Steroid hormones e.g, the female sex hormones estrogen and
progesterone)
 Thyroxine
 Retinoic acid which is derived from vitamin A
 Vitamin D
 Lipophilic hormones with cell-surface receptors:
The principal lipophilic (lipid-soluble) hormones that bind to receptors located in
the plasma membrane are the prostaglandins.
 Hydrophilic hormones with cell-surface receptors:
All hydrophilic (water-soluble) molecules (which cannot diffuse across the
hydrophobic interior of the lipid bilayer) bind to receptors in the plasma membrane.
There are two subclasses of hydrophilic hormones:
(1) Peptide hormones such as insulin and glucagon;
(2) Small charged molecules, often biogenic amines, such as epinephrine
(adrenalin) and histamine.

CELL-SURFACE RECEPTORS

Hydrophilic and some lipophilic hormones bind to cell-surface receptors. These are
integral membrane proteins situated in the plasma membrane that bind the signaling molecule
(ligand) with high affinity. The ligand binds to a specific site on the receptor in much the same
way as a substrate binds to an enzyme. Binding of the ligand to the receptor causes a
conformational change in the receptor that initiates a sequence of reactions in the target cell
(often referred to as signal transduction) leading to a change in cellular function.
Cell-surface receptors can be classified into three classes depending on how they transfer
the information from the ligand to the interior of the cell:

 Enzyme-linked receptors
On binding of the ligand to its extracellular face, the cell-surface receptor undergoes a
conformational change and activates an intrinsic enzyme activity. Example, insulin
receptor.

 Ion channel-linked receptors

Here binding of the ligand again causes a conformational change in the protein but this
time such that a specific ion channel is opened. This allows a certain ion to flow through
that subsequently alters the electric potential across the membrane. For example, at
the nerve–muscle junction the neurotransmitter acetylcholine binds to specific
receptors that allow Na+ ions to flow into and K+ ions out of the target cell.

28
 G protein-linked receptors
G proteins are associated with hormone receptors on the cytosolic side of the cell
membrane
 Nomenclature: The G protein is so named because it binds guanine nucleotide.
Either GTP or GDP may be bound to the G protein.
 Structure: G proteins consist of three subunits: α,β and γ.The α subunit binds GTP
or GDP. The β and γ subunits do not bind nucleotides.
a. The hormone-receptor complex catalyzes the exchange of GDP for GTP
by the G protein. The receptor alone does not catalyze this exchange.
b.When GDP is exchanged for GTP on the α subunit Gα-GTP dissociates
from
Gβγ. Gα-GTP is the active form.

• Types: The effect of the active form (Gα-GTP) depends upon the specific type of G
protein. There are several different types of G proteins:
 Gs stimulates the enzymes Adenylate cyclase
 Gi inhibits the enzyme adenylate cyclase
 GpLc stimulates the enzyme phospholipase C.

• Function: The Gα subunit of all G proteins is a GTPase. It slowly hydrolyzes its bound GTP
to GDP and thereby returns to its inactive, GDP bound state. Gα then reassociates with
Gβγ, where it remains until it is reactivated by a hormone- receptor complex.

ADENYLATE CYCLASE: is a large integral membrane bound enzyme.

Function: Adenylate catalyses the formation of cAMP from ATP.
Hormones that activate adenylate cyclase include:
 Glucagon, which is a peptide
 Epinephrine, which is an amino acid derivative
This activation occurs at sub type of epinephrine receptors β1 and β2
adrenergic receptors.
Cholera toxin is an enzyme produced by bacterium vibrio cholerae
Action: Cholera toxin modifies the α subunits of Gs which blocks the hydrolysis of
GTP to GDP. This prevents the inactivation of Gs by this mechanism.
Result: Persistently high level of29
cyclic AMP which causes the epithelial cells of the
intestine to transport Na+ and water into the intestinal lumen which results in severe
diarrhea.
THE Gi PROTEINS:
The active Gα-GTP subunits of Gi proteins result in inhibition of adenylate cyclase.
Structure: The β and γ subunits of Gi and Gs proteins are identical. The α subunits differ.
Function: The Gα-GTP or the Gi protein inhibits adenylate cyclase also the Gβγ that is
released when the Gα-GTP dissociates from the Gi protein binds to the Gα-GTP of a Gs
protein and blocks the activation of adenylate cyclase.
Examples: An example of a hormone that inhibits adenylate cyclase is epinephrine at
the α2 receptor subtype.
Pertussis toxin is an enzyme that modifies the α subunit of Gi. The modification prevents
Gi from exchanging GDP to GTP therefore the modified Gi protein is unable to block the
activation of adenylate cyclase. Pertussis toxin is produced by Bordetella pertussis the
bacterium that causes whooping cough.

THE GPLC PROTEINS

When activated GPLC proteins stimulate phospholipase C.
 Phospholipase C is a membrane bound enzyme that hydrolyses phosphotidyl inositol
4, 5- bisphosphate (PIP2) which is a membrane phospholipid.
 Products of hydrolysis includes inositol 1,4,5- triphosphate (IP3) and diacylglycerol
(DAG) both of which are second messengers. In many cells IP3 and DAG work
together to activate protein kinase C.
 Example: A hormone that activates phospholipase C is epinephrine at the α1
adrenergic receptor subtype.

SECOND MESSENGERS:

 The binding of ligands to many G protein-linked receptors leads to a short lived increase
in the concentration of certain intracellular signaling molecules called second
messengers. (The hormone/ligand can be considered as the first messenger.)
 The major second messengers are:
 3 ,5-cyclic AMP (cAMP)
 3,5-cyclic GMP (cGMP)
 Inositol 1,4,5-trisphosphate (IP3)
 1,2-diacylglycerol (DAG)
 Ca2+.

30
 The elevation in the level of one or other of these second messengers then leads to a
rapid alteration in cellular function. cAMP and cGMP are derived from ATP and GTP by
the actions of adenylate cyclase and guanylate cyclase, respectively.
 cAMP affects cellular function by activating a protein kinase, which phosphorylates
specific cellular enzymes. Cyclic AMP is hydrolysed to AMP by a cytoplasmic enzyme
cyclic nucleotide phosphodiesterase.
 IP3 and DAG are derived from the membrane lipid phosphatidylinositol 4,5- bisphosphate
by the action of phospholipase C.
 One of the main actions of the polar IP3 is to diffuse through the cytosol and interact with
Ca2+ channels in the membrane of the Endoplasmic Reticulum (ER) , causing the release
of stored Ca2+ ions which in turn mediate various cellular responses.
 The DAG produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate, along with
Ca2+ ions released from the ER, activates protein kinase C, a membrane-bound enzyme
that phosphorylates various target proteins, again leading to alterations in a variety of
cellular processes.

31
 6. BASIC METHODS TO STUDY CELL BIOCHEMISTRY

CENTRIFUGATION
This is the process of using centrifugal force to separate the lighter portion of solution, mixture or
suspension from the heavier portions.

Applications:

In laboratory centrifuge is used to:

 Remove cellular debris from blood to separate cell free plasma or serum.

 Concentrate cellular elements and other components for microscopic analysis or

chemical analysis.

 Separate protein bound or antibody bound ligand from free ligand in

immunological assay.

 Extract solutes from aqueous or organic solvents.

 Separate lipid components like chylomicrons from other components of plasma.

ULTRACENTIFUGATION
Ultracentrifugation is an indispensable tool for the isolation of subcellular organelles,
proteins and nucleic acids. ln addition, this technique is also employed in the determination of
molecular weights of macromolecules.

The rate a t which the sedimentation occurs in ultracentrifugation primarily depends on the size
and shape of the particles or macromolecules (i.e. on the molecular weight). lt is expressed in
terms of sedimentation coefficient (s) and is given by the formula.
32
Where

v = Migration (sedimentation) of the molecule

ω = Rotation of the centrifuge rotor in radians/sec

The sedimentation coefficient has the units of seconds. lt was usually expressed in units of 10-13s
(since several biological macromolecules occur in this range), which is designated as one Svedberg
unit.

CHROMATOGRAPHY
Chromatography is an analytical technique dealing with the separation of closely related
compounds from a mixture. These include proteins, peptides, amino acids, lipids, carbohydrates,
vitamins and drugs.

PRINCIPAL

Chromatography usually consists of a mobile phase and a stationary phase. The mobile phase
refers to the mixture of substances (to be separated), dissolved in a liquid or a gas. The stationary
phase is a porous solid matrix through which the sample contained in the mobile phase
percolates.

The interaction between the mobile and stationary phases results in the separation of the
compounds from the mixture. These interactions include the physicochemical principles such as
adsorption, partition, ion-exchange, molecular sieving and affinity.

1. Paper chromatography: This technique is commonly used for the separation of amino
acids, sugars, sugar derivatives and peptides. In paper chromatography, a few drops of
solution containing a mixture of the compounds to be separated is applied (spotted) at
one end, usually -2 cm above, a strip of filter paper. The paper is dried and dipped into a
solvent mixture consisting of butanol, acetic acid and water in 4 : 1 : 5 ratio (for the
separation of amino acids). The aqueous component of the solvent system binds to the
paper and forms a stationary phase. The organic component that migrates on the paper is
the mobile phase. When the migration of the solvent is upwards, it is referred to as
ascending chromatography. Ln descending chromatography, the solvent moves
downwards.

33
 PAPER CHROMATOGRAPHY

As the solvent flows, it takes along with it the unknown substances. The rate of migration
of the molecules depends on the relative solubilities in the stationary phase (aqueous)
and mobile phase (organic). After a sufficient migration of the solvent front, the paper
(chromatogram) is removed, dried and developed for the identification of the specific
spots. Ninhydrin, which forms purple complex with c'-amino acids is frequently used as a
colouring reagent. The chemical nature of the individual spots can be identified by
running known standards with the unknown mixture.

The migration of a substance is frequently expressed as Rᶠ value (ratio of fronts)

Rᶠ value = Distance travelled by the substance

Distance travelled by solvent front

The Rᶠ value of each substance, characteristic of a given solvent system and paper, often
helps for the identification of unknown. Sometimes, it is rather difficult to separate a complex
mixture of substances b y a single run with one solvent system. ln such a case, a second run is
carried out by a different solvent system, in a direction perpendicular to the first run. This is
referred to as two dimensional chromatography which enhances the separation of a mixture into
the individual components.

2. Thin layer chromatography (TLC): ln this, in place of a paper, an inert substance, such as
cellulose, is employed as supporting material. Cellulose is spread as a thin layer on glass
or plastic plates. The chromatographic separation is comparatively rapid in TLC. In case of
adsorption thin layer chromatography, adsorbents such as activated silica gel , alumina,
kieselguhr are used.

3. Gel filtration chromatography: It separates proteins on the basis of their size and shape
using porous beads packed in a column. Large or elongated proteins cannot enter the
pores in the beads and elute (extract) from the bottom of the column first, whereas
smaller proteins can enter the beads and move through the column more slowly, eluting

34
 later. Gel filtration chromatography can be used to de-salt a protein mixture and to
estimate the molecular mass of a protein.

GEL FILTRATION CHROMATOGRAPHY

4. In ion exchange chromatography: In this proteins are separated on the basis of their net
charge. In anion exchange chromatography a column containing positively charged beads
is used to which proteins with a net negative charge will bind, whereas in cation exchange
chromatography negatively charged beads are used to which proteins with a net positive
charge will bind. The bound proteins are then eluted by adding a solution of sodium
chloride or by altering the pH of the buffer.

ION EXCHANGE CHROMATOGRAPHY

5. Affinity chromatography exploits the specific binding of a protein for another molecule,
its ligand (e.g. an enzyme for its inhibitor). The ligand is immobilized on an insoluble
support and packed in a column. On adding a mixture of proteins, only the protein of
interest binds to the ligand. All other proteins pass straight through. The bound protein is
then eluted from the immobilized ligand in a highly purified form.

35
 AFFINITY CHROMATOGRAPHY

6. High performance liquid chromatography (HPLC) : In general, the chromatographic

techniques are slow and time consuming. The separation can be greatly improved by
applying high pressure in the range of 5,000-10,000 p/s I (pounds per square inch), hence
this technique is also referred (less frequently) to as high pressure liquid chromatography.
H LC requires use of non-compressible resin materials and strong metal columns. The
eluants of the column are detected by methods such as UV absorption and fluorescence.

ELECTROPHORESIS
The movement of charged particles (ions) in an electric field resulting in their migration towards
the oppositely charged electrode is known as electrophoresis. Molecules with a net positive
charge (cations) move towards the negative cathode while those with net negative charge
(anions) migrate towards positive anode.

Electrophoresis is a widely used analytical technique for the separation of biological molecules
such as plasma proteins, lipoproteins and immunoglobulins. The rate of migration of ions in an
electric field depends on several factors that include shape, size, net charge and solvation of the
ions, viscosity of the solution and magnitude of the current employed.

Types of electrophoresis:

1. Native polyacrylamide gel electrophoresis (PAGE): In native polyacrylamide gel

electrophoresis (PAGE) proteins are applied to a porous polyacrylamide gel and separated
in an electric field on the basis of their net negative charge and their size. Small/more
negatively charged proteins migrate further through the gel than larger/less negatively
charged proteins.

36
 Native polyacrylamide gel electrophoresis (PAGE)

2. SDS-PAGE Electrophoresis: In SDS-PAGE, the protein sample is treated with a reducing

agent to break disulfide bonds and then with the anionic detergent sodium dodecyl
sulphate (SDS) which denatures the proteins and covers them with an overall negative
charge. The sample is then fractionated by electrophoresis through a polyacrylamide gel.
As all the proteins now have an identical charge to mass ratio, they are separated on the
basis of their mass. The smallest proteins move farthest. SDS-PAGE can be used to
determine the degree of purity of a protein sample, the molecular mass of a protein and
the number of polypeptide subunits in a protein.

3. Isoelectric focusing: In isoelectric focusing, proteins are separated by electrophoresis in a

pH gradient in a gel. They separate on the basis of their relative content of positively and
negatively charged residues. Each protein migrates through the gel until it reaches the
point where it has no net charge, its isoelectric point (pI).

37
PHOTOMETERY

Photometry broadly deals with the study of the phenomenon of light absorption by molecules in
solution. The beam of light consists of a stream of photons. When photons encounter solution to
be analyzed (analyte), there is a chance that some photons will be absorbed while passing
through the analyte, so that the number of photons will decrease. Spectrophotometer is used to
measure the amount of light absorbed by analyte. A spectral colour consists of a single
wavelength λ and a light of certain λ is called “MONOCHROMATIC LIGHT”.

LAWS OF LIGHT

BEER’S LAW:

Log of ratio of intensity of incident light (Io) to emergent light (Ie) is directly proportional to
the concentration of the solution (C) through which the light passes.

Log Io/ Ie ᾲ C
Log Io/ Ie = K₁C
Where

 Io =Intensity of incident light

 Ie = intensity of emergent light
 C = Concentration of solution
 K₁ = Constant
LAMBERT’S LAW:

Log of ratio of intensity of incident light (I o) to emergent light (Ie) is directly proportional to
the thickness (pathway through which the light is passing i.e of container remains constant)
provided the concentration of chromogens is kept constant.

Log Io/ Ie ᾲ t
Log Io/ Ie = K₂t
Where

 Io =Intensity of incident light

 Ie = intensity of emergent light
 t = Thickness
 K₂ = Constant
BEER LAMBERT’S LAW:

When mono chromatic light passes through a solution, the intensity of light decreases
proportionally by increasing the thickness (Lambert’s Law) and also by increasing the
concentration of solution(Beer’sLaw).

38
 Log Io/ Ie ᾲ Ct
Log Io/ Ie = KCt
Where

 Io =Intensity of incident light

 Ie = intensity of emergent light
 t = Thickness
 K = Constant
 C = Concentration of solution

Transmittance:
It is the ratio of emergent light to incident light.
“It is a measure of ability of a solution to transmit the light.”
So we can write:
Log 1/T=KCt
Absorbance:
Log 1/T is also known as absorbance.
“It is the ability of solution to absorb light (A).”
So
A= log 1/T
Transmittance has logarithmic relationship with the concentration and thickness of the
solution while the absorbance has a direct relationship with the concentrated and thickness.

Therefore, “A” is used to measure the intensity of light in our calculation:

Au = KCut
Au/Cu = Kt
Where,

 Au = Absorbance of unknown
 Cu = Conc. Of unknown
As = KCst
As/Cs = Kt
 As = Absorbance of standard
 Cs = Conc. Of standard
“K” is a constant and in an experiment, t also remains constant. So

Kt= constant
Hence,

39
 Au/Cu = As/Cs
Cu = Au x Cs/As

1. COLORIMETER
Colorimeter (or photoelectric colorimeter) is the instrument used for the
measurement of coloured substances. This instrument is operative in the visible range
(400-800 nm) of the electromagnetic spectrum of light. The working of colorimeter is
based on the principle of Beer-Lambert law.
The colorimeter, in general consists of light source, filter sample holder and
detector with display (meter or digital). A filament lamp usually serves as a Iight source.
The filters allow the passage of a small range of wave length as incident light. The sample
holder is a special glass cuvette with a fixed thickness. The photoelectric selenium cells
are the most common detectors used in colorimeter.

COLORIMETER

2. SPECTROPHOTOMETER

The spectrophotometer primarily differs from colorimeter by covering the

ultraviolet region (200-400 nm) of the electromagnetic spectrum. Further, the
spectrophotometer is more sophisticated with several additional devices that ultimately
increase the sensitivity of its operation several fold when compared to a colorimeter. A
precisely selected wavelength (say 234 nm or 610 nm) in both ultraviolet and visible
range can be used for measurements. In place of glass cuvettes (in colorimeter), quartz
cells are used in a spectrophotometer. In a spectrophotometer, the monochromatic light
from its source passes through a series of slits, lenses, filters and mirrors. This optical
system selects a wavelength, concentrates light, increases the optical purity and focuses
it on the sample.

40
 3. FLAME PHOTOMETRY

Flame photometry primarily deals with the quantitative measurement of

electrolytes such as sodium, potassium and lithium. The instrument, namely flame
photometer, works on the following principle. As a solution in air is finally sprayed over a
burner, it dissociates to give neutral atoms. Some of these atoms get excited and move to
a higher energy state. When the excited atoms fall back to the ground state, they emit
light of a characteristic wavelength which can be measured. The intensity of emission
light is proportional to the concentration of the electrolyte being estimated.

RADIOIMMUNOASSAY (RIA)
This technique has revolutionized the estimation of several compounds in biological fluids
that are found in exceedingly low concentrations (nanogram or picogram). RIA is a highly
sensitive and specific analytical tool.

Principle:

Radioimmunoassay combines the principles of radioactivity of isotopes and

immunological reactions of antigen and antibody, hence the name. The principle of RIA is
primarily based on the competition between the labelled and unlabelled antigens to bind
with antibody to form antigen-antibody complexes (either labelled or unlabelled). The
unlabelled antigen is the substance (say insulin) to be determined. The antibody to it is
produced by injecting the antigen to a goat or a rabbit. The specific antibody (Ab) is then
subjected to react with unlabelled antigen in the presence of excess amounts of isotopically
labelled (1311) antigen (Ag+) with known radioactivity. There occurs a competition between
the antigens (Ag+ and Ag) to bind the antibody. Certainly, the labelled Ag+ will have an upper
hand due to its excess presence.

As the concentration of unlabelled antigen (Ag) increases the amount of labelled antigen-
antibody complex (Ag+-Ab) decreases. Thus, the concentration of Ag+-Ab is inversely related
to the concentration of unlabelled Ag i.e. the substance to be determined. This relation is
almost linear. A standard curve can be drawn by using different concentrations of unlabelled
antigen and the same quantities of antibody and labelled antigen. The labelled antigen-
antibody (Ag+-Ab) complex is separated by precipitation. The radioactivity of 1311 present is
Ag+-Ab is determined.

Applications

 RIA is no more limited to estimating of hormones and proteins that exhibit antigenic
properties.

 By the use of haptens (small molecules such as dinitrophenol, which, by themselves,

are not antigenic), several substances can be made antigenic to elicit specific
antibody responses.

41
  ln this way, a wide variety of compounds have been brought under the net of RIA
estimation. These include peptides, steroid hormones, vitamins, drugs, antibiotics,
nucleic acids, structural proteins and hormone receptor proteins.

 Radioimmunoassay has tremendous application in the diagnosis of hormonal

disorders, cancers and therapeutic monitoring of drugs, besides being useful in
biomedical research.

ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA)

Enzyme-linked immunosorbant assay (ELISA) is a non-isotopic immunoassay. An enzyme is used
as a label in ELISA.

Principle

ELISA is based on the immunochemical principles of antigen-antibody reaction. The stages

of ELISA are summarized.

1. The antibody against the protein to be determined is fixed on an inert solid such as
polystyrene.

2. The biological sample containing the protein to be estimated is applied on the

antibody coated surface.

3. The protein antibody complex is then reacted with a second protein specific
antibody to which an enzyme is covalently linked. These enzymes must be easily
assayable and produce preferably coloured products. Peroxidase, amylase and
alkaline phosphatase are commonly used.

4. After washing the unbound antibody linked enzyme, the enzyme bound to the
second antibody complex is assayed.

5. The enzyme activity is determined by its action on a substrate to form a product

(usually coloured). This is related to the concentration of the protein being
estimated. The principle for the use of the enzyme peroxidase in ELISA is illustrated
next.

42
Applications

o ELISA is widely used for the determination of small quantities of proteins

(hormones, antigens, antibodies) and other biological substances.

o The most commonly used pregnancy test for the detection of human chorionic
gonadotropin (hCG) in urine is based on ELISA. By this test, pregnancy can be
detected within few days after conception.

o ELISA is also been used for the diagnosis of AIDS.

43
 REFERENCES

a) Lippincott illustrated reviews: Biochemistry by Pamela C. Champe, Richard A.

Harvey, Denise R. Ferrier. Latest edition published by Lippincott Williams &
Wilkins.

b) Marks basic medical biochemistry—a clinical approach by Smith C, Marks AD and

Liebeman M. Latest edition published by Lippincott Williams & Wilkins

c) Textbook of Medical Biochemistry by Chatterjea MN, Shinde R.

d) Instant notes Biochemistry by Hames BD and Hooper NM.

44