CHAPTER 1: BIOLOGICAL MOLECULES

1.1 Water
 makes up 60-65% of the fresh mass of an organism
 forms saliva
 83% of blood and 75% of brain
 Converts food to components needed for survival (Reactant in a metabolism)
 Keeps mucosal membranes moist
 Allows body cells to grow, reproduce and survive
 Flushes body wastes
 Lubricates joints
 Needed by brain to manufacture hormones and neurotransmitters
 Regulates body temperature
 Acts as a shock absorber for brain and spinal cords
 Helps deliver oxygen
 Accounts for 22% of bones
 Makes up 75% of muscles
 Provides aqueous environment for many organisms

PHYSICAL PROPERTIES
1. Liquid at S.T.P.
- Under room temperature, about 20% hydrogen bonds exist in water.
2. Tasteless, odourless
3. Low electrical conductivity
4. Boiling point is dependent on the barometric pressure
5. Density:
- As water cools, the movement of water molecules slow down and the molecules are further apart. (Low
kinetic energy)
- The lower the temperature of water, the more hydrogen bonds are formed.
- Highest at 4⁰C, as water molecules are most compact with the shortest intermolecular bonds
- When water is below 0⁰C, water molecules are further apart and form the maximum 4 hydrogen bonds
with other molecules (Hydrogen bonds occupy more space). The lattice crystals occupy a bigger space
and fewer molecules compared to an equal volume of liquid and floats in the liquid water.
- At 0⁰C, water freezes where all the molecules are involved in forming the 3D structure of ice.
- ρ <ρ
6. Viscosity
- Low viscosity as hydrogen bonds between H2O molecules are being continually broken and reformed.
- Less resistance to flow, less friction (Flow faster)
- Blood easy to flow throughout our body.
- Moist surface
7. Cohesion
- Force of mutual attraction between similar molecules.
- Water molecules shows strong attraction to each other.
8. Surface tension
- Difficulty to stretch or break the surface of a liquid.
- Molecules in the bulk of the liquid are affected by various intermolecular forces of attraction.
- Molecules in the surface are not affected by molecules above them, therefore ‘pull’ together more
strongly, effectively resembling a stretched membrane (Cohesive force)
9. Adhesion
- Force of mutual attraction between dissimilar molecules
10. Thermal properties
samstudynotes
 - Water has a high specific heat capacity (4.2J is required to raise the temperature of 1g of water by 1⁰C)
→ Highest for all common solids and liquids
→ Extensive hydrogen bonds. Large amount of heat energy is required to break the hydrogen
bonds before the temperature can be raised.
→ Prevents large fluctuations in body temperature.
- Water has high boiling point and latent heat of vaporisation (2260kJ is required to change 1kg of water
from liquid to gas or vapour).
→ Highest of all common substances
→ Vaporisation always results in cooling of the surroundings
→ As water evaporates, it removes a lot of heat with it (cooling effect)
→ Large amount of heat is lost with minimal loss of water.
→ Types of vaporisation:
Evaporation (Slow) Boiling (Fast)
= occurs at water surface = occurs under the surface of water
= provides maximum cooling effect = at the boiling point
= minimum loss of water
- Water has a high latent heat of fusion of 334J/g.
- Therefore, temperature of water remains relatively stable.

CHEMICAL PROPERTIES
1. In a water molecule, 2 H atoms form a single polar covalent bond with an O atom.
2. pH 7 (amphoteric)
- has the potential to act as an acid and base
3. Dipolar (uneven charge distribution within the molecule)
- Since O is more electronegative, the region around O has a partial negative charge (2 )
- O is larger than H, hence spend more time around O atom than H.
- The region near the 2 H atoms has a partial positive charge ( )
4. Forms weak hydrogen bonds between the positively charged H atom and negatively charged O atom from
another water molecule.
- Forms, breaks and reform with great frequency which holds water molecules in place
- A water molecule can form 4 hydrogen bonds with other molecules
5. Universal solvent for ionic substances which contains charged ions, and some non-ionic substances which
contain polar groups.
- When such substances dissolve in water, the ions and polar groups are surrounded by water molecules
which separates the ions or molecules from each other.
- When ionic compounds are dissolved in water, the negative ends of O atoms are attracted to cation,
while the positive ends of H atoms are attracted to anion.
The electrostatic attractions are greater than those between cation and anion hence water molecules
gather around cation and anion.
- When polar molecules are added to water, hydrogen bonds are formed between the slightly charged
hydroxyl groups and the polar water molecules.

HYDROPHILIC MOLECULES
 Substances that dissolve readily in water
 Composed of ions or polar molecules that attract water molecules through electrical charge effects
 Water molecules surround each ion or polar molecule on the surface of a solid substance and carry it into
solution
HYDROPHOBIC MOLECULES
 Substances that do not interact with water

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1.2 Carbohydrates
 Organic compounds containing C, H and O elements
 C:H:O=1:2:1
 General formula: ( )
 Energy production= 4kcal/g
 Excess carbohydrate is converted to fats
 Functions:
1. Main source of energy in cells. Brain cells and RBCs are almost wholly dependent on carbohydrates
as the energy source
2. Food storage compound
3. Forms structural components in plant cells
 Four types: Monosaccharides, disaccharides, polysaccharides, oligosaccharides

STRUCTURAL AND STEREOISOMERISM

Structural isomerism: due to different attachments of the atoms or groups of atoms in the molecules
Example: Glucose, fructose, galactose
Stereoisomerism: occurs when the same atoms or groups are joined together but are arranged differently in the
molecule
Optical isomerism: property of any compound which can exist in 2 forms whose structures are mirrored structures
Example:
D-isomer: asymmetric atom furthest from aldehyde or ketone
group (i.e. C atom 5) has its hydroxyl group as in D-
glyceraldehyde
L-isomer: asymmetric atom furthest from aldehyde or ketone
group (i.e. C atom 5) has its hydroxyl group as in L-
glyceraldehyde

 D and L isomers of the same substances have the same physical and chemical properties except for physical
properties associated with asymmetry
 Naturally occurring sugars occur largely in D-forms An α-glycosidic bond is formed
when both carbons have the
 All amino acids are in L-forms in living organisms
same stereochemistry,
When a beam of polarised light passes through a solution, the compound is said to be:
whereas a β-glycosidic bond
 Dextro-rotatory (+) if it turns polarised light to the right
occurs when the two carbons
 Laevo-rotatory (-) if it turns polarised light to the left
have different
REDUCING AND NON-REDUCING SUGARS
Reducing sugars Non-reducing sugars
Bear a free aldehyde (−CHO) or ketone (−CO) group Do not have such groups
Have the capacity to reduce cupric ions of Benedict’s or Fail to reduce the cupric ions of Benedict’s solution to
Fehling’s solution to cuprous ions cuprous ions
Melibiose, Cellobiose, Gentiobiose Sucrose, Trehalose

MONOSACCHARIDES
 One sugar unit
 Simple sugars (monomers): cannot be broken into smaller molecules by hydrolysis

}
 Physical properties:
֎ Sweet
Result of hydroxyl groups of the carbon
֎ Soluble in water
chains with readily form hydrogen bonds
֎ Can be crystallised
with water molecules
֎ Polar
 Chemical properties:
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 ֎ Contains carbonyl groups, remaining C atoms that do not have carbonyl groups will have hydroxyl
groups
֎ Are either aldehydes (aldoses) which contain −CHO groups or ketones (ketoses) which contain C=O
[R C(=O) R’]
1. Aldoses: Glyceraldehyde, ribose, glucose, galactose
2. Ketoses: Dihydroxyacetone, ribulose, fructose
֎ Reducing sugars, can carry out reduction since carbonyl group donates electrons
֎ React with Benedict’s or Fehling solution to give a brick-red precipitate.
Reduce alkaline copper (II) sulphate into insoluble copper (I) oxide.
+ →
Blue solution Brick-red precipitate
֎ During reduction, they themselves become oxidised
H +O
| or – CHO → −COOH
−C=O Oxidation
Aldehyde Carboxylic acid
֎ Can be reduced in cells, forming alcohols
H +2
| → −
−C=O Reduction
Aldehyde Primary alcohol
+2 |
| → −CHOH
−C=O Reduction
Ketone Secondary alcohol
 Can be classified into trioses, tetroses, pentoses, hexoses, heptoses according to the number of C atoms
 Pentose and hexose can either exist in open chain form or ring structure:
֎ When dissolved in water, may change from straight chain form to ring form which is more stable
֎ Only the ring structure can be used to make disaccharides and polysaccharides
 Functional groups: Aldehyde (-CHO) or keto (-CO) groups
 Trioses (3C) -
֎ Glyceraldehyde ֎ Dihydroxyacetone

֎ Intermediate products of metabolism during the breakdown of glucose (glycolysis) or synthesis

(photosynthesis) of it
֎ Glyceraldehyde exists as phosphoglyceraldehyde and dihydroxyacetone exists as
phosphodihydroxyacetone during glycolysis and carbon fixation during photosynthesis
֎ Seldom exist long in the free state of cells
 Pentoses (5C) -
֎ Ribulose ֎ Ribose ֎ Deoxyribose

Pentose ring form

Open chain form Pentose ring form Open chain form samstudynotes
 ֎ Can either exist in an open chain form or a ring structure
֎ Physiological roles:
Krebs/citric acid cycle: 1. Synthesis of nucleotides, ribose is for the formation of ribonucleotides in RNA and
series of chemical deoxyribose is for the formation of deoxynucleotides of DNA
reactions used by all 2. Synthesis of coenzymes such as NAD (Nicotinamide adenine dinucleotide), NADP
aerobic organisms to (Nicotinamide adenine dinucleotide phosphate), FAD (flavin adenine dinucleotide) and
release stored energy coenzyme A
through the oxidation 3. Synthesis of polysaccharides
of acetyl-CoA derived 4. Ribulose biphosphate is used as a receptor for the fixation of CO2 at the beginning of the
from carbohydrates, light independent stage of photosynthesis
fats 5. Intermediate biological molecules of many important metabolic pathways such as glycolysis,
Krebs cycle and Calvin cycle
 Hexoses (6C) -
֎ Glucose ֎ Galactose ֎ Fructose

֎ All of them are isomers

֎ Structures of glucose:
1. Straight chain structure: polyhydroxyaldehyde//aldohexose
2. Self-reaction in which the linear chain of glucose bends, hydroxyl group attached to carbon
atom 5 forms a more stable ring-shaped molecule.
3. Hexose//pyranose ring structure (flat//planar ring) of glucose can exist as:
i. α-glucose: OH group of C atom 1 is below the plane
ii. β-glucose: OH group of C atom 1 is above the plane
4. Chair conformation
* D-glucose and L-glucose are optical isomers or stereoisomers
1. 3.(i.) (ii.) 4.

α-D-glucose β-D-glucose α-D-glucose

(Haworth projection)
D-glucose
(Fischer projection)

֎ Physiological roles:
1. A source of ATP in respiration (provide short term energy)
2. Balance water potential between inside and outside of cells, so as to maintain the shape of
cells
3. Synthesis of disaccharides and polysaccharides

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 4. Synthesis of other substances (fats, amino acids) when required
DISACCHARIDES
 Two sugar units//residues
 Formed by the condensation reaction with two units of monosaccharides with the removal of a water
molecule (dehydration synthesis)
 The bond linking the monosaccharides is known as a glycosidic bond (can be α or β)
o Formed between the hemiacetal group of a saccharide (or its derivative molecule) and the hydroxyl
group of an alcohol, which may be another carbohydrate
 Physical properties:
o Sweet
o Soluble in water
o White crystalline substances
o Too large to pass through cell membranes
 Chemical properties:
o Reducing sugars except sucrose
o Can be hydrolysed to form monosaccharides by heating in dilute acid or by specific enzymes
 Maltose:
o Glucrose + Glucose, bonded by α-1,4-glycosidic bond

o Respiratory substance
o Condensation reaction is catalysed by synthetase (ligase) enzyme or maltase
o Occurs in the stroma of chloroplast, in amyloplast of parenchyma cells and cytoplastin of liver and
muscle cells
o Functions:
 intermediate in the synthesis of polysaccharides and hydrolysis of starch (by amylase) or
glycogen
 acts as a control in both directions Synthetase: an enzyme
 Lactose: that catalyses the
o Galactose + Glucose, bonded by β-1,4-glycosidic bond linking together of two
molecules especially by
using the energy
derived from the
concurrent splitting off
of a pyrophosphate
group from a
triphosphate (as ATP)
o Source of energy
o Functions:
 Control of glucose and galactose release
 Food
 Sucrose:
o Glucose + Fructose, bonded by α-1,2-glycosidic bond

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 o Non-reducing sugar
o Most abundant disaccharide in nature
o Main form transported in plants
o Condensation reaction is catalysed by synthase
o Occurs in stroma of chloroplasts, cytoplasm of plant cells, and in mesophyll cells
o Transported away by phloem
o Very soluble, unreactive and can be transported efficiently in high concentrations
o Functions:
 Medium to transport carbohydrate in phloem
 It can lower water potential and create hydrostatic flow in sieve tubes
 Can be stored in plant cells as food reserve
 Found in fruits and nectar to attract animals

POLYSACCHARIDES
 Complex carbohydrates (biopolymer)
 Long chains of molecules made of repeating units of monosaccharides
 Monosaccharides are linked together by glycosidic bonds and the chains may be
branched//unbranched
 Hexosan: monosaccharides in polysaccharides may be all hexoses, common formula ( )
 Pentosan: monosaccharides in polysaccharides may be all pentoses, common formula ( )
 Long term energy sources
 Physical properties:
 Not sweet
 Insoluble in water, if dissolved in hot water, they form colloids as they are large molecules
 Cannot be crystallised but form amorphous mass desiccated
 Compact and not osmotically active in cells
 Can be extracted and purified to form white powder
 Chemical properties:
 Have no reducing power
 Starch and glycogen are easily hydrolysed into maltose by amylase
 Cellulose can be hydrolysed by cellulase (only produced by microbes and snails)
 Starch:
 Formed from the condensation of α-glucose (polymerisation catalysed by 2 synthetase)
 Made up of two components:
When amylopectin is  Amylose, a linear unbranched polymer of 200 to 1500 α-glucose units in a repeating
added to iodine- sequence of α-1,4-glycosidic linkage
potassium iodide Amylose chain coils into a helix, held by hydrogen bonds formed between hydroxyl groups
solution, a red-violet Amylose is easily hydrolysed by amylase to form maltose
colourisation results.
When hydrolysed,
amylopectin is broken
down into dextrin.
Amylase will break
Amylose chain
down α-1,4-glycosidic
bonds only. Dextrin samstudynotes
forms bright red colour
in iodine.
  Amylopectin, a branched polymer of 2000 to 200000 α-glucose units per starch molecule
The linear chains of α-glucose units are held together by α-1,4-glycosidic linkages
Branches occur at intervals of approximately 25 to 30 where α-1,6-glycosidic linkages occur
It needs isomaltase to hydrolyse the α-1,6-glycosidic bonds causing the release dependent
on the control of another enzymes

 Amylose helices are entangled in the branches of amylopectin to form a complex, compact, three-
dimensional starch molecule.
 Insoluble, compact and not osmotically active, making it a good food reserve
 Interconversion of starch and maltose maintains water potential in cells especially in the guard cells
for the opening and closure of stomata
 Glycogen:
 Branched biopolymer with α-1,4-glycosidic and α-1,6-glycosidic bonds
 Formed in the smooth endoplasmic reticulum of both liver and muscle cells, by polymerisation
catalysed by 2 synthetases
 After forming a straight chain of more than 12 units, a short chain is cut by hydrolase
 The chain is transferred to another chain to form a branch forming α-1,6-glycosidic bonds catalysed
by another synthetase

 “animal starch”, found in liver and muscles as food reserve

 Can be broken down to glucose phosphate by phosphorylase. Glucose phosphate can be used
directly for respiration
 Controls blood sugar level
 Cellulose:
 Structural polysaccharide in plant cell wall
 Cannot be hydrolysed by enzymes in the human digestive system
 Composed of unbranched chains up to 10000 β-glucose units linked by β-1,4-glycosidic bonds
 β-glucose units are catalysed by synthetase to form long chains at the plasma membrane
 enzymes involved are synthesised in the rough endoplasmic reticulum packaged into vesicles
 enzymes are released at the plasma membranes to form cellulose molecules and then
microfilaments to form new layer of cell wall
 after plant cells divide, vesicles gather in the central plate and fuse, enzymes in the vesicles form
cellulose molecules and microfilaments form the new cell wall
 each β-glucose unit is related to the next by a rotation of 180⁰ with hydroxyl groups projecting
outwards on either side of the chain
 cellulose chains run parallel to each other
 cross-linked hydrogen bonds to form microfibrils (about 80 molecules)
 fibrils give plant cells high tensile strength and rigidity

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  Chitin:
 Component of some fungal walls and the chitinous exoskeleton of arthropods
 Structure is similar to cellulose except hydroxyl of C atom 3 is replaced by — . (amino
sugar combined with an acetyl group)

 Inulin:
 Used as a food store
 Murein (peptidoglycan):
 Component of bacterial cell walls
 Consists of polysaccharides cross-linked with amino acids

OLIGOSACCHARIDES
 Small chains of carbohydrates formed by the linking of 3-14 monosaccharides
 Can be found attached to proteins and lipids forming glycoprotein and glycolipids of plasma membrane

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1.3 Lipids
o Hydrophobic
o Consist of C, H and O, may contain P and N
o Contain fewer polar O-H bonds and more non-polar C-H bonds than carbohydrates (example: olive oil
(triolein), )
o Includes fats and oils, waxes, phospholipids, steroids and some other related compounds
o Excess glucose, lipids and proteins are stored in adipose cells
o Functions:
Primary energy sources and structural compounds
signalling molecules
Insulate and protect internal organs
Fats in animals absorb and sequester nonpolar contaminants such as DDT, PCBs, organomercury
o Contains large numbers of bonded hydrogens (C-H)
Release a larger amount of energy (9kcal/gm)
o General formula: ( )
o Physical properties:
Insoluble in water (hydrophobic, when placed together, water forms hydrogen bonds with each
other excluding lipids) and polar organic solvent
Soluble in non-polar organic solvent such as chloroform, alcohol, ether or benzene
Densities are less than water
High viscosity
Greasy, leave grease spots on paper
o Solvents for fat-soluble vitamins
o Constituent of myelin sheath, insulates the axon and speeds up nerve transmission

TRIGLYCERIDES
→ Fats or oils formed by the ester linkage of one molecule of glycerol and three molecules of fatty acids
(esterification)
→ Esterification/lipogenesis:
1. Glycerol/glycerine with 3 C atoms, each bearing a hydroxyl group, neutralise with 3 molecules of
fatty acids (RCOOH), each bearing a carboxyl functional group (−COOH).
R = H or alkyl groups
2. Long and hydrophobic alkyl group forms the long chain of fatty acids
Ester bond
Stearic acid forms tristearin
{

+ H−O−H Palmitic acid forms tripalmitin

+ H−O−H Oleic acid forms triolein

+ H−O−H

water

→ Fatty acids:
 Consists of long unbranched hydrocarbon chains with carboxyl groups at one end.
 C−H bonds in the hydrocarbon chains of fatty acids are the reason fats are hydrophobic.
 Amphipathic:
 Hydrocarbon chain: hydrophobic
 Carboxylic acid group: hydrophilic
 Saturated fatty acids:

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  hydrocarbon chain has maximum hydrogen atoms bound to it and has no double bonds

 example: stearic acid,

 at room temperature, the molecules are packed closely together, forming a solid (fat)
 Unsaturated fatty acids:
 Hydrocarbon chains which contain one or more double bonds between carbon atoms with
one fewer hydrogen atom on each double-bonded carbon.
 Nearly every double bond in naturally occurring fatty acids is a cis double bond, which
creates a kink in the hydrocarbon chain wherever it occurs.
Same atoms repel,  May be arranged in the trans-form: when oils are heated and exposed to hydrogen gas
causing a kink. (hydrogenation), the hydrogen exerts a slight ‘pull’ that rotates the fatty acid chain,
changing the configuration from “cis” to “trans” (straightened)

 Example: oleic acid, (monounsaturated), linoleic acid,

(polyunsaturated)
 At room temperature, the molecules cannot pack together closely enough to solidify due to
the kinks in some of their fatty acid hydrocarbon chains, hence forming a liquid (oil).
 When triglycerides compose of more unsaturated trans-fatty acids, it tends to be solid.
 Monounsaturated: One double bond only
 Polyunsaturated: Two or more double bonds
 Essential fatty acids:
 Cannot be synthesised in the body
 Main components of phospholipids
 Need to make other fatty acids and synthesise prostaglandin (for immune system)
 Essential for normal development of brain and growth
 Obtain form daily diet
 Example: linoleic acid, alpha linoleic acid and arachidonic acid
 Non-essential fatty acids:
 Not necessary to obtain them from daily diet
 Can be synthesised during metabolic activities involving carbohydrates and proteins
→ Physical properties:
 Insoluble in water
 Less dense than water
 Soluble in non-polar organic solvents
 Form emulsion if shaken with alcohol
 Leave grease spots on paper
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 → Chemical properties:
 React with atmospheric oxygen and become rancid if kept for too long
 React with Sudan III reagent to form a dark red solution
 Can be hydrolysed by lipase or boiling with dilute alkali to form glycerol and fatty acids
→ Stored as oil droplets in the cytoplasm of adipose cells in animals. (Adipose cells are found underneath the
skin, in the mesentery or surrounding the intestines, kidney, heart and between muscle fibres.)
→ Found in seeds and fruit walls such as in oil palm fruit, seeds of groundnuts, rapeseeds, sunflower seeds and
kernels of all cereals.
→ Function:
 Energy sources, yield 38 of energy (Carbohydrates 17 )
 Energy reserves in the adipose tissues of animals and seeds of many plants.
Have more quantity of energy, less space consumption, insolubility in water and easily respire
 Used for formation of other chemicals including glucose to maintain blood glucose level and amino
acids to make proteins
 Used to form all other chemicals especially in plants and herbivores after dormancy during winter
 Insulate the animal body form cold in the form of subcutaneous fat layer (bad conductor of heat)
 Protects organs form damage
 Waterproof animals and plants, skin is covered with oil from sebaceous glands, fur and feather of
animals are covered with oil to make better insulators, plants and other animals have cuticles of fat
to reduce excessive evaporation
 Form waxy cuticles covering on the epidermis of plants to reduce transpiration and entry of
pathogens
 Certain desert animals use metabolic water from the metabolism of fats and carbohydrates
 Energy storage to reduce body weight and to make locomotion easier, suitable for motile animals
and plant structures involved in dispersal

PHOSPHOLIPIDS
 Derivative of triglyceride where one of its fatty acid chains is replaced by phosphoric acid.
 Formed by the condensation of one glycerol and two hydrocarbon fatty acids, and one phosphate group.
 Phosphate is a charged ion, this part of the molecule becomes hydrophilic.
 Hydrocarbon from the remaining fatty acid groups are hydrophobic.
 The arrangement of molecules gives the phospholipid an amphipathic property by having a hydrophilic head
and a hydrophobic tail.
Choline, C5H14NO, Choline is
an essential nutrient for
humans and many other
animals.

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  Lecithin:
Most common phospholipid which has a positive charge choline group bound to the phosphate
group
Important component of the cell membrane and membranes of organelles
Give the membranes their fluidity and allow lipid-soluble substances and small non-polar substances
to pass through
In an aqueous medium (intracellular and extracellular environment), lecithin molecules
spontaneously arrange themselves to form a bilayer with the hydrophobic tails facing the inside and
the hydrophilic heads facing the outside
This makes lecithin molecules the most suitable material for cell membranes which keeps the cell
contents inside the cells and at the same time, permits the exchange of materials with its aqueous
environment
Phospholipid bilayer forms a closed spherical vesicle in water (micelle)

Planar phospholipid bilayer with edges

exposed to water

Sealed compartment formed by

phospholipid bilayer

 Functions:
Ensure the stability in the membrane. Bilayer can easily reform even if the structure is temporarily
broken.
Bimolecular nature of phospholipid allows reorientation of molecules to form vesicles or vesicles
fuse with the membrane become part of it. Thus, endocytosis and exocytosis can easily take place.
Allows globular proteins (intrinsic proteins) with corresponding hydrophobic and hydrophilic
surfaces to span across the membrane. Bilayer with intrinsic proteins such as enzymes and transport
proteins can respectively perform enzymatic functions and allow the passage of hydrophilic
substances to cross the membrane.
Some extrinsic proteins that are attached to the outer or inner surface of the bilayer can perform
structural enzymatic functions
Short polysaccharides can bind with phospholipids or proteins. They serve as receptors or cementing
substances. They also stabilise the membrane structure by forming hydrogen bonds with water
molecules both outside and within the cell.

STEROIDS
characterized by a carbon skeleton consisting of four fused rings, consisting of 3 cyclohexane rings and 1
cyclopentane ring
have a basic four-ring hydrocarbon structure with different double bonds and functional side chains
Polycyclic, usually all trans
Common structural features:
=O or −OH at C 3
Side chain at C 17
Double bond from C 5 to either C 4 or C 6
Attachment of different groups to the core steroid structure leads to a wide variety of steroid compounds,
including cholesterol, bile salts and steroid hormones.
Properties:

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 Insoluble in water (hydrophobic)
Soluble in organic solvents such as petroleum, ether and acetone
Can be solidified at low temperature
Can form micelles with phospholipids and can be soluble in water or alcohol as emulsion
Oestradiol: one of the androstane cholesterol
types of oestrogen

testosterone progesterone
oestradiol

aldosterone
cortisol

Cholesterol:
Precursor of steroid hormones such as testosterone and oestrogen, bile acids and Vitamin D
Saturated fats that originated from animals contain more cholesterol than that found in
polyunsaturated fats or oils of plants
Found abundantly in humans, a large portion is obtained from the diet and the rest is synthesised by
the liver
Most of the cholesterol in the blood are surrounded by a layer of phospholipids in which proteins
are embedded to form low density lipoprotein (LDL)
High density lipoproteins (HDL) tend to carry excess cholesterol to the liver to be processed
Cholesterol level is regulated by the liver. Excess cholesterol will be deposited as gallstones in the
gall bladder and bile duct.
When cholesterol level exceeds the normal level, cholesterol deposits on the arterial walls
(arteriosclerosis), causing blockage of the arteries. Blockage of the coronary artery (coronary
thrombosis) causes heart attacks (myocardial infarction). Coronary thrombosis reduces blood flow to
the heart muscles causing their death in the affected region and ultimately generally heart failure.
Functions: reduces fluidity of cell membranes
Bile acids (in salt forms): help in emulsification and digestion of fats in the intestines, due to the solubility of
the hydrocarbon rings in lipid and the ionic heads in water
Adrenocortical hormones (cortisol and aldosterone): control stress responses, maintain balance of salt in
body fluid
Oestrogen and progesterone: regulate menstrual cycle and pregnancy in females
Testosterone: regulates reproductive activities in males
Calciferol (Vitamin ): Facilitates the absorption of calcium and phosphate from the small intestines
Anabolic steroids:

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Can enter into the nucleus of cells and increase the rate of transcription of genes which encode actin
and myosin proteins, the main components of muscles.
Sportsmen and women tend to take anabolic steroids to build up muscle mass. Steroids also enable
them to undergo strenuous training by making them more aggressive and delaying the feeling of
tiredness. Athletes who use steroids will have an unfair advantage.
Banned as they are harmful to liver, heart and kidney.
Inhibit the production of natural sex hormones which may lead to sterility. Cause development of
male characteristics in female.
Illegal addition of anabolic steroids into animal feed may also have harmful effect on consumers.

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1.3 Proteins

Complex macromolecules that are formed by the elements C, H, O and N. In some proteins, S and P may be
present, together with traces of Fe, Cu, I, Mn and Zn.
May be composed of one or more polypeptide chains, each polypeptide chain is a polymer consisting of
many basic units of amino acids linked together by peptide bonds through condensation process.
Account more than 50% of dry mass of most cells (molecular weight= 5,000 to 3,000,000)
Most important of biologic substances by being the fundamental constituent of cell cytoplasm.
Supply heat and energy, and material for building and repair
Only small amounts of protein are temporarily stored in the body, which can be quickly used up upon
demand
AMINO ACIDS
Carboxylic acids which have amino groups.
Have the same basic structure but differ in their R-side chains.
Consists of 2 terminals: Acidic carboxyl terminus and the basic amino (− ) terminus which make them
amphoteric
Amino and carboxyl groups are attached by covalent bonds to the central alpha carbon together with a
hydrogen atom and a R-side chain.

Carboxyl
group

Amino
group

Classes of 20 amino acids:

Non-polar/aliphatic group Polar group (hydrophilic) Acidic group (strongly Basic group (strongly
(hydrophobic) hydrophilic) hydrophilic)
Has hydrocarbon side Has polar/ionic side Has acidic side chains. (has Has − side chains.
chains. chains. (have an uneven −COOH) (extra amino group)
Neutral charge distribution) Negatively charged Positively charged
Neutral
Most have at least 1 atom
(N, O or S) with electron
pairs available for
hydrogen bonding

Protein molecules that Presence of these Proteins derived from these are soluble in water (e.g.
consists of large amount increases the solubility of globular proteins).
of these are insoluble, protein and enables The three-dimensional structure of a globular protein is
non-reactive and play a hydrogen bonding stabilised by electrostatic interactions between the
structural role (e.g. between polypeptide charged R groups in the molecule
collagen) chains

-glycine (Gly) (R group is -serine (Ser) -aspartic acid (Asp) -lysine (Lys)
only h) -asparagine (Asn) -glutamic acid (Glu) -arginine (Arg)
-alanine (Ala) -glutamine (Gln) -histidine (His)
-valine (Val) -tyrosine (Tyr)
-leucine (Leu) -cysteine (Cys)
-isoleucine (Ile) -threonine (Thr)
-tryptophan (Trp)
-proline (Pro)
-methionine (Met)
-phenylalanine (Phe)
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 Non-polar:

Polar:

Acidic: Basic:

Aspartic acid Glutamic acid

Amino acids as zwitterions:

o Carrying a positive charge on the basic part and a negative charge on the acidic part and is therefore
bipolar.
o Internal transfer of H ion from carboxyl group to amino group to leave an ion with both a negative
and positive charge
o Form of amino acids even in solid state
o No overall electrical charge
When amino acids are dissolved in water:
o Zwitterions exists
o Amino group is ionised into −
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 o Acidic group is ionised into −
o This amphoteric property allows amino acids to function as pH buffer to maintain pH of a cell
o Amino acids can readily change from anion to cation and vice versa by mopping up any excess
and ions found in the medium of a cell

Cationic form Anionic form

Essential amino acids: must be obtained from diet as they cannot be synthesised from the body
Non-essential amino acids: can be made from other amino acids through transamination
Plants are able to make all the amino acids from simpler substances.

POLYMERISATION OF AMINO ACIDS

When two amino acids bind together through condensation, a dipeptide bond is formed which can undergo
further reaction with other amino acids since it has a free amino or carboxyl group at either one of its ends.
When 1 or more amino acids are linked to the dipeptide, a tripeptide or a polypeptide is formed.
(polypeptide backbone)
Amino acids are linked by peptide bonds, a type of covalent bond which is formed when a water molecule is
eliminated during a reaction between carboxyl group of one amino acid and amino group of another.

Once the peptide bonds have been formed, the linear polypeptide chain may coil or fold into a particular
structure as a result of other types of bonds such as hydrogen bonds, disulphide bonds, ionic bonds and
hydrophobic interactions which determine the behaviour of proteins.
 Hydrogen bonds: between the H (of hydroxyl // amino groups of polar amino acid) and the O (of the
keto group) or the slightly negative charged N (of amino group of another polar amino acid)
 Weak
 Occurs frequently
 Total effect can contribute toward the stability of a protein
 Disulphide bonds: oxidation of sulfhydryl groups (−SH) found in 2 neighbouring cysteine amino acids
between different parts of a polypeptide chain or between chains of amino acids
 Strong
 Not easily broken
 Ionic//polar bond (salt bridges): negatively charged acidic R group of an amino acid of a polypeptide
chain is attracted to the positively charged basic R group of another amino acid of the same or
different polypeptide chain

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  Weaker than covalent bond
 Ionic bond can be broken by changing the pH of the medium surrounding the protein and
disrupting the protein structure, thus denaturing it
 Hydrophobic interactions: in an aqueous medium, polypeptide chains tend to fold so that the
hydrophobic R groups come into close contact to form many interactions and exclude water.
 Hydrophobic groups tend to point inwards away from the water while the hydrophilic
groups face outwards into the aqueous environment, making the protein soluble.
 Weak
 Van der Waal’s forces: involve two side chains that are very weak opposite charges or of very weak
hydrophobic feature
Formation of a polypeptide (protein synthesis):
 Happens in cells. A gene controls the synthesis of each type of protein
 Human cells contain about 30,000 such genes
 The sequence if amino acids in each type of protein is determined by the base sequence within a
gene, a part of DNA
 The part of DNA has to be transcribed or copied into another form called mRNA before it is
translated into proteins by ribosomes
 The transcription occurs in nucleus catalysed by RNA polymerase and translation occurs in the
cytoplasm involving tRNA catalysed by ligase enzyme

STRUCTURES OF PROTEINS
Level Description Stabilised by
Primary Linear sequence of amino acids in a polypeptide chain Peptide bonds
Includes the number of amino acids, number of chains, position of
disulphide bonds that link the chains
Controlled by base sequence and gene
Determines the function of a protein as it controls higher levels of
structure
Determines the properties of a protein
Secondary α-helix: polypeptide chain coiled to form a simple α-helix maintained by Hydrogen bonds
hydrogen bonds between carbonyl and amide groups between groups
A protein made up entirely of α-helix structure is stable and fibrous along the
e.g. keratin polypeptide chains
β-pleated sheet: stretched polypeptide chains are arranged parallel to
each other and are held together by hydrogen bonds and folded
longitudinally
Has high resistance to stretching
e.g. fibroin
Tertiary Coiling and folding of secondary structures Hydrogen bonds,
Three-dimensional, precise, compact and globular shape of a polypeptide disulphide bridges,
e.g. enzymes, hormones, antibodies, plasma proteins, myoglobin ionic bonds,
Results in the solubility of proteins hydrophobic
Outer surface: hydrophilic group interactions, van
Inner surface: hydrophobic group der Waals forces
Quaternary Precise arrangement of more than one polypeptide chain which are held Hydrogen bonds,
together by hydrogen bonds, ionic bonds and hydrophobic interactions disulphide bridges,
e.g. haemoglobin ionic bonds,
Found in protein with more than one subunit, can be the same or hydrophobic
different primary structures interactions, van
der Waals forces

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 Secondary structure

Primary structure

Quaternary structure
Tertiary structure

CLASSIFICATION OF PROTEINS
According to their structures:
 Globular proteins:
 Tightly coiled and folded to form sphere
 Tertiary or quaternary structure
 Structure maintained by various bonds
 Easily soluble in water
 Hydrophilic side chains are found on the surface interacting with water
 Physiologically significant functions (roles in metabolic reactions)
 E.g. enzymes, haemoglobin, hormones, globulin, antibodies
 Fibrous proteins:
 Hair-like in shape, some are in sheet or block forms as found in horn, nail and hoof
 Secondary structure
 Helical structures or pleated sheets held by hydrogen bonds
 Stable protein structure
 Insoluble in water
 Most of them cannot be digested by proteinase
 Form structural components in cells
 E.g. collagen (found in skin, tendons, cartilage, bones, teeth and blood vessel walls), fibroin
(found in silk), keratin (found in hair), actin and myosin (found in muscles)
 Intermediate proteins:
 Soluble in water
 Fibrous
 Physiological functions
 E.g. fibrinogen (used for blood clotting)
According to their composition:
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  Simple protein:
 Pure proteins, formed only from amino acids and no other non-protein component
 Albumin: (e.g. egg albumin)
- Soluble in water
- Neutral
 Globulins: (e.g. serum globulins)
- Neutral
- Insoluble in water
 Histones: (e.g. chromatin)
- Basic
- Soluble in water
 Scleroproteins (keratin, collagen)
- Neutral
- Insoluble in water
 E.g. insulin, enzymes like pepsin, fibrous proteins like myosin and collagen
 Conjugated protein:
 Formed from amino acids and a non-protein component called prosthetic group/cofactor.
 Most of them form globular proteins
 Soluble in water
Prosthetic group Conjugated protein Example
Phosphate Phosphoprotein Casein (in milk)
Carbohydrate Glycoprotein Mucus (of respiratory and digestive tracts),
(mucin) antibodies, cell membrane
Lipid Lipoprotein Component of cell membrane
Metal ion Metalloprotein Haemoglobin, myoglobin, cytochromes
(haemoprotein, haem group contains
ion.)
Flavin Flavoprotein Dehydrogenase with FAD
Nucleic acid Nucleoprotein Nucleoproteins in chromosomes and
ribosomes
Pigment molecule Chromoprotein Haemoglobin and myoglobin (haem group is
the pigment molecule)
According to their functions:
Types of protein Function Example
Enzymatic protein Nutrition (catalyses metabolic reactions) Digestive enzymes such as
Has an active site to bind to a substrate carbohydrase, protease, lipase
which is then converted into a product
Hormonal protein Coordination of metabolic processes and Non-steroid hormones such as insulin,
the activation of enzymes glucagon, growth hormone
Transport protein Transport of respiratory gases Haemoglobin
Transport of ions or molecules across cell Membrane transport protein such as
membrane channel proteins and carrier proteins
Complement protein Immunity, produced by white blood cells Antibodies (immunoglobulin),
interleukins, histamine, perforin
Structural protein Structure of cells and tissues (support) Soluble globular proteins (in
cytoplasm), collagen, keratin, elastin
Contractile protein Movement (allows muscles to contract) Actin and myosin
Storage protein Storage of amino acids Casein, albumin, gluten (in aleurone
found in cereal grains)
Receptor protein Response to chemical stimuli Receptors on nerve cell membrane

PROPERTIES IF PROTEINS
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 1. Colloid formation:
→ Globular proteins may be soluble in water, but as macromolecules, these proteins are bound by a
layer of similar charges, preventing them from settling
→ They disperse evenly throughout water forming colloid
→ Colloid:
- If added to water, form sols
- When the amount of water is reduced by heating, form gel
- Exhibits Tyndall effect i.e. if shone with light, cloudiness is observed (due to the reflection of
light by macromolecules)
- Exhibits Brownian movement i.e. if observed under microscope, vibrations can be seen as
protein molecules obtain kinetic energy
2. Amphoteric in water
→ Can behave like acid and alkali
→ Proteins containing carboxylic side chains behave like an acid
→ Proteins containing amino side chains behave like an alkali
→ Zwitterions in water
3. At isoelectric point, when total amount of positive charge is equal to the total amount of negative charge at
a certain pH, proteins are least soluble in water or become insoluble precipitate.
→ Caused by the intermolecular bonding of the 2 charges
4. Could be charged.
→ Can be separated and identified through electrophoresis
5. Can act as buffers i.e. can resist pH changes when a small amount of acid or alkali is added
→ Amino side chains neutralise acids
→ Carboxylic side chains neutralise alkali

DENATURATION AND RENATURATION OF PROTEINS

֎ Denaturation involves transformation of a well-defined folded structure of a protein formed under
physiological conditions, to an unfolded state under non-physiological conditions.
 a process in which a protein loses its native shape due to the disruption of weak chemical bonds and
interactions, thereby becoming biologically inactive.
 due to the alteration of the three-dimensional shape of proteins and is usually irreversible.
 Caused by the breakage of the cross linkages when proteins are exposed to certain extreme
conditions but their amino sequence remain unchanged
 Globular shape can unwind and bond with one another, becoming more insoluble or precipitate.

 Loses its biological function.

 Factors:
i. Heat (thermal denaturation) (>40°C): increases the kinetic energy of protein molecule and
causes weak bonds (hydrogen bonds, disulphide bonds, hydrophobic interactions, Van der
Waals’ forces) to break, peptide bonds does not break.
ii. Cold (cold denaturation)
iii. pH (strong acids and alkalis): too high concentration of and ions disrupts the ionic
bonds and cause the protein to coagulate. Under prolonged condition, peptide bonds may
break.

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 iv. Heavy metallic ions (inhibitors). E.g. mercury and silver. Ionic bonds are disrupted when
metallic ions bind with negatively charged carboxyl groups. Reduce polarity and solubility of
proteins and cause it to precipitate.
v. Organic solvents. E.g. benzene and petroleum. Form bonds with non-polar groups in protein
or disrupt hydrophobic interactions.
vi. Radiation. Kinetic energy of radiation causes atoms of protein to vibrate strongly and disrupt
covalent, ionic and hydrogen bonds. The protein then coagulates.
֎ Renaturation is possible when the denaturation does not occur beyond the critical stage and under
favourable conditions.

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1.5 Nucleic acids
 Polynucleotides, biopolymers of nucleotides that include the DNA and RNA
 Found mainly in the nucleus of the cells
 Elements C, H, O, P and N are bound up to form different components which are then linked up to produce
different types of nucleotides
 Macromolecules consist of one or two polynucleotide chains
 Exist in two major forms, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
NUCLEOTIDES
Formed from the condensation of a pentose sugar, a phosphate group and a nitrogenous base by removing
two water molecules.
Phosphoester
bond
Nitrogenous
base

Phosphate N-glycosidic
group Pentose bond
sugar
Pentose: Ribose, Deoxyribose
Nitrogenous base:
Double-ringed purine bases

Adenine (A) Guanine(G)

Single-ringed pyrimidine bases

Thymine (T) Uracil (U) Cytosine(C)

(DNA only) (RNA only)
Phosphate group is derived from phosphoric acid (gives acidic properties to nucleic acid)
May be 1, 2 or 3 phosphate groups. Therefore, nucleotides can exist as nucleotide monophosphate
e.g. AMP, nucleotide diphosphate e.g. ADP and nucleotide triphosphate e.g. ATP
Soluble in water
Bond between the base and the sugar is N-glycosidic bond
Bond between the pentose and phosphate is phosphodiester bond
Can be hydrolysed to form phosphates and nucleosides by phosphatase or nucleotidase
Can be hydrolysed to form bases and pentoses by nucleosidase

POLYNUCLEOTIDE
Polymerisation of nucleotides with the formation of phosphodiester bonds to form polynucleotides.
The phosphodiester bond is formed between the hydroxyl group on C 3 of pentose sugar of one nucleotide and
the phosphate group of another.
Phosphodiester bond is a strong bond which binds nucleotides together to form a chain of sugar-phosphate
backbone of DNA or RNA
The backbone of polynucleotide chain consists of the repeating sequence of phosphate-sugar of the nucleotides
with all bases pointing to one side of it.

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DEOXYRIBONUCLEIC ACID (DNA)
Double-stranded polynucleotide held antiparallel to each other by weak hydrogen bonds between the bases.
One strand runs from 5’ end to 3’ end, the other runs from 3’ end to 5’end.
Hydrogen bonds are formed when the purine bases on one strand paired with the pyrimidine bases on the
other strand.
Adenine always pairs up with thymine and is held by 2 hydrogen bonds
Guanine always pairs up with cytosine and is held by 3 hydrogen bonds
The structure is based on the Watson and Crick model
3’ end
5’ end

Nucleotide

5’ end
3’ end

There are hydrophobic interactions between the bases in the centre aided by water molecules surrounding
it. (contributes to the stability of the molecule.)
Weak hydrogen bonds between the complementary bases are easily broken by heat to separate the 2 DNA
strands
The strong covalent phosphodiester bonds cannot be easily disrupted, keeping the DNA intact and making it
a suitable molecule which is suitable as an inheritable genetic material.
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RIBONUCLEIC ACID (RNA)
Single polynucleotide chain strand
Made up of adenine, cytosine, guanine and uracil bases, and ribose as the pentose sugar.

Three types of RNA:

֎ Ribosomal RNA (rRNA)
→ Synthesised in the nucleolar organiser of the nucleolus
→ One of the structural components of the ribosome
→ Consists about 80% of the total RNA found in the cells
→ Have long lifespan of several days before they are hydrolysed
→ Various sizes (no fixed shape), some with more than 1000 nucleotides
→ Consist of intra-chain single-coiled sections and double-coiled sections that are bound to
protein molecules, forming subunits of ribosomes
→ Bigger eukaryotic (80S) ribosome
→ Smaller prokaryotic (70S) ribosome: found in bacteria and inside the mitochondria and
chloroplasts
→ Ribosomes consist of two subunits:
Eukaryotes: 80S → 60S + 40S
Prokaryotes: 70S → 50S + 30S
→ Bigger ribosomal units of eukaryotes contain about 3 types of rRNA
→ The smaller subunits contain only 1 type of rRNA
→ In prokaryotes, 50S contains 3 types of rRNA and only 1 in 30S
→ Transcribed from specific genes (become the organisers for the nucleolus)
→ Ribosomes in eukaryotes are formed from in the nucleolus
→ Role:
To form ribosome
Enable ribosome to combine with mRNA, to ‘read’ the codons in the mRNA and
‘translate’ the codons into sequences of amino acids in the primary structure of
protein with the help of tRNA.
rRNA forms ribosomes that provide 2 sites for tRNA to bind, each tRNA can carry 1
specific amino to the site.
rRNA in the ribosome functions as enzyme (ribozyme), catalyses the formation of
peptide bond between 2 amino acids brought to the ribosome
֎ Transfer RNA (tRNA)
→ Smallest among the 3 RNA molecules with about 80 nucleotides
→ Has 20 groups of RNA, each is specific for 1 kind of amino acid charged to it during protein
synthesis
Amino acids attach
here

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 → Transcribed from genes in the DNA in nucleus and are moved to the cytoplasm
→ Make up 10-15% of the total RNA in the cell
→ Have a shorter lifespan of several hours
→ Have a specific shape like a clover leaf, formed by the intra-chain coiling to form double
strands, aided by hydrogen bonds where adenine pairs with uracil and cytosine pairs with
guanine.
→ Have 61 types of tRNA
→ Each type has 3 bases at one loop called anticodon which is complementary to the codon of
the amino acid it carries.
→ At its 3’ end, there’s a triplet sequence of CCA that can be bonded with a specific amino acid
to the ribosome.
→ Transfers a specific amino acid to the ribosome, according to the codon in the mRNA,
complementary to its anticodon to form a protein.
→ Can be reused to bind to amino acid and transfer it to the ribosome.
֎ Messenger (mRNA)
→ Transcribed from DNA and make up about 5% of the total RNA in the cell
→ “copies” the information of at least one gene in the nucleus
→ Sequence is complementary to one of the strands of DNA (coding/sense strand) which is
exactly the same as the other strand
→ Moves across the nuclear pore into the cytoplasm where it combines with a ribosome
→ Ribosome ‘read’ the information that the mRNA carries with the help of tRNA and translates
it into a protein
→ Can be the longest RNA as it can copy several genes, each with about 300 bases
→ Does not have specific shape, may coil or fold
→ Has the shortest life span in the cell
→ After several minutes, when enough protein is synthesised, mRNA is hydrolysed, although
some may last longer.
→ A transcript of a gene base sequence
→ Acts as a template in which amino acid are joined into a protein of specific kind

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1.6 Analytical Technique

PAPER CHROMATOGRAPHY

 Technique used to separate mixtures of chemicals of similar nature by allowing their common solvent to
flow over them in a solid medium such as paper
 Ideal to separate a mixture of macromolecules
 Mixture have to be dissolved in a suitable solvent (mobile phase)
 Principles in pigment separation:
The solid medium used is a piece of paper cut to fit in a boiling tube. The paper of cellulose fibres is
porous and absorptive of liquid.
A small but concentrated amount of leaf extract is applied on one end of the paper.
The paper is hung touching a common solvent such as petroleum ether.
Solvent moves up, separating the solute molecules as indicated by different coloured spots.
Pigment molecules separate due to their differential absorptions of the medium used. Reasons for
different speeds of component to move through the pores of the paper:
Solubilities: the more soluble the pigment in the solvent, the faster it can move.
Molecular size: the smaller the size of the pigment, the faster it can move.
Charges (adhesion): if the pigment has similar charges as the medium, the faster it can
move. If the pigment is of the opposite charge with the medium, there would be an
attraction between them thus slowing down its movement in the medium.
Components with higher affinity towards the solvent will move further up the paper.
 Steps to separate different colour pigment found in chlorophyll complex in green leaves:
1. Chlorophyll is first extracted from leaves using acetone.
2. A strip of absorptive paper is used as the stationary phase.
3. The chlorophyll extract is then applied repeatedly for 20 times to form a fine concentrated spot on
the starting line drawn earlier on one edge of the paper.
4. The spot is then air-dried. Steps 3 and 4 is repeated for 20 times.
5. The chlorophyll-spotted end of the chromatography paper is then dipped into a suitable solvent such
as petroleum ether.
6. The concentrated spot of chlorophyll should stay above the surface of the solvent.
7. A boiling tube with cork stopper is used as the chromatography chamber to trap the solvent vapour.
8. The solvent moves up the chromatography paper by capillary action and passes through the
chlorophyll spot.
9. The chlorophyll dissolves in the solvent.
10. Before the solvent front reaches the top end of the chromatography paper, the process is stopped
and the chromatogram is air-dried.
11. The locations of colour pigments are then determined and marked.

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 The line where
the solvent ends
on the paper

Extract is
applied here
Apply 20 drops
and dry.
Repeat for 20
times.

 is the ratio of the distance travelled by the substance and the distance travelled by the solvent front.
To identify a pigment when a certain solvent is used
is a constant, when of an unknown pigment spot is compared with of a standard known
pigment, its identity can be determined.
 Limitations:
Only small amount of pigments can be separated at one time
Certain pigments are too similar that they tend to overlap and are not easily separated by this
technique.

ELECTROPHORESIS

Used to separate a mixture of charged molecules (amino acids, proteins and fragments of DNA) by using an
electric field.
Medium used is usually agarose gel layer/ gel in a column even though paper can be used.

Electrodes are placed on both ends of a box of cooled gel.

Small wells are cut at one end and filled with mixtures to be separated.
Acidic buffer solution is added to change the mixtures of protein or nucleic acids to positive charge.
Dye is added for identification.
When electricity is switched on, the charged molecules move to the electrode of opposite charge.
Uses:
o Separate proteins, enzymes separated are still active.

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 o Diagnose diseases, when blood plasma proteins are separated, extra proteins found could be
antibodies formed to combat certain pathogens. Antibodies are compared with the standard ones
and extracted to the determine the actual type.
o In forensic science, used for DNA-fingerprinting to identify individuals. Long DNA molecule is cut into
different length and is separated to form a specific pattern according to the enzyme used to cut it.
o Used in DNA sequencing, DNA is cut into short fragments added with a special dye that colours
specific bases at one end. From the pattern formed, the sequence of DNA/gene can be deciphered.
Limitations:
o Only a small amount of substances can be separated
o Substances without charges or too similar in charge cannot be separated.
Gel electrophoresis for DNA separation:
o Technique used to separate DNA fragments according to their size.
o DNA samples we want to examine is carefully transferred into wells at one end of the agarose gel.
o Next, the power to the gel box is turned on, and current begins to flow through the gel.
o The DNA molecules have a negative charge because of the phosphate groups in their sugar-
phosphate backbone, so they start moving through the matrix of the gel towards the positive pole.
o Since all DNA fragments have the same charge per mass, small fragments move through the tiny
pores of the gel faster than the large fragments.
o After the gel has run for a while, the shortest pieces of DNA will be close to the positive end of the
gel, while the longest pieces of DNA will remain near the wells.
o Once the fragments have been separated, the gel can be examined.
o When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each
representing a group of same-sized DNA fragments.

ISOELECTRIC POINT

- The pH at which an amino acid is electrically neutral.

- At a certain pH, the overall charge of the amino acids is 0 and the molecule concerned neither moves toward
the cathode or anode (depends on the structure of the side chain)
- All amino acids and protein have their own specific isoelectric point
- When the pH of a solution is above the isoelectric point, the amino acid/protein molecule will be negatively
charged and will move towards the anode.
- When the pH of a solution is below the isoelectric point, the amino acid/protein molecule will be positively
charged and will move towards the cathode.

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