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MOLECULAR ORGANIZATION

OF THE LIVING CELLS AND
SAMPLE QUESTIONS
As Written

by

TINUOYE Peter Sunday
(MBA, B.Sc, ANIMN, Cert.Comp.)
TABLE OF CONTENT
1.0 Molecular Organization of the Living Cells 1
1.2 Types of Membrane 2
1.3 Function of Membrane 3
1.4 Chemical Composition of a Cell 4
1.5 Seperation Of Materials Obtained From Cell Disruption 5
1.6 Isolation Of Membrane –Cell Disruption Techniques 7
1.7 Morphology and function of organelles 9
1.7.1 Mitochondrion – Morphology 9
1.7.2 Chloroplast 10
1.7.3 Endoplasmic Reticulum 11
1.7.4 Lysosomes 12
1.8 Enzymes 13
1.9 Classification Of Enzymes 14
1.10.1Enzyme Cofactors 15
2.0 Lipids 20
3.0 Amino Acids And Proteins 33
4.0 Importance of Water and the Concept of Ph and
Buffers 41
4.1.0 Importance of Water 41
4.1.2 Concept of Ph 42
4.1.3 Acids and Bases 52
4.1.4 Buffer: System 54
5.0 Carbohydrates 60
6.0 Chemical Reaction 70
1.0 MOLECULAR ORGANIZATION OF THE LIVING CELLS
An eucaryotic cell has a considerable degree of internal structure
unlike the prokaryotic cells; with a large number of distinctive membrane-
enclosed organells.
Membranes are those flexible structures which are important components
of the cell structure. They occur both in prokaryotic cell e.g. bacteria and
eucaryotic cells e.g. liver cells.
In some eucaryotic cells, they make up as much as 80% of total dry
mass. They also serve as permeability barriers separating different cells in
the tissues, thereby regulating the flow of subtrate into and product out of
the cells. The membranes serve as structural bases to which certain
enzymes, proteins, lipids, carbohydrates, hormone receptors and light
receptors and transport system are firmly bound.
Generally, cells, are divided into distinct components or organelles –
nucleus, mitochondria, golgibodies, lysosomes, microsomes, etc. Like
membranes, bimembranes are also responsible for signal transduction
from which information is passed from outside of the cell to inside or is
passed between cells components.

1.2 TYPES OF MEMBRANES
1. Plasma membrane – The outermost covering of the cell.
2. Nuclear envelope or nuclear membrane
3. Membrane of the golgi bodies
4. Membrane of the endoplasmic reticulum
5. Mitochondrial membrane
6. Lysosomal membrane
7. Chloroplast membrane
8. Other membrane inclusion – such as lipid droplets, pigment
granules etc.
1.3 FUNCTIONS OF SUBCELLULAR MEMBRANE
The cell membrane is one of the membrane possessed by most
cells. Several other types of sub-cellular membranes have similar and
distinct functions.
1. Nuclear membrane separates the nucleus from the rest of the
cell.
2. The inner membranes of the mitochondria contain enzymes that
catalyse the reaction of the final state of respiration.
3. The endoplasmic reticulum contain the enzymes that catalyses
the reaction of the final state of respiration.
4. the rough endoplasmic reticulum supports ribosomes and the
enzymes that catalyse the synthesis of protein from amino acids.
5. The smooth endoplasmic reticulum contains hydroxylation
enzymes, steroids, synthesis enzymes and enzymes for drug
metabolism.
6. lysosomal membrane contains enzymes that digest substance
brought into the cell.
Membranes are impervious, mechanical barriers separating the cell and
it’s organelles from the environment and therefore highly specialized
structures that perform many functions with great precision and accuracy.

1.4 CHEMICAL COMPOSITION OF CELL
Animal cell membrane consist of association of lipids and
glycoprotein. Different membrane can be characterized broadly on the type
and proportion of these components.
The chemical composition of a particular membrane is not
necessarily constant with time, but it’s distinctive identity is usually
retained. The changes in the membrane may be required to regulate final
activity of the membrane or they may represent stages in the differentiation
of the structure. The changes in composition may be reduced by changes
in temperature or nutritional status or by hormone or drug administration.
Most cell membrane contain about 60% protein and 40% lipid, but
there is a considerable variation. The lipids of the membrane are mainly
polar lipids, the predominant ones being, phospho lipids of
phosphoglycerides while sphingolips occur in smaller amounts.
Almost all polar lipids of many cells are localized on their membrane.
The endoplasmic reticulum membrane and organelles memlorane
membrane contain relatively little cholesterol or tri-acyl-glyceraol whereas
plasma membrane of some cells of higher of some animals contain much
cholesterol both free and esterified.
The ratio of the different types of lipids in membrane is characteristic
of the type of membrane, the organ and species. Each type of membrane,
contain several or many kinds of protein and polypeptides.
Membrane protein can be classified into:
(a) Extrinsic or peripheral protein
(b) Intrinsic or integral protein
The extrinsic proteins are loosely attached to the membrane surface
and can be easily removed in soluble form by a mild extraction method.
The intrinsic protein which make up about 70% or more of the total
membrane protein are very tightly bound to the lipid portion of the
membrane and be only removed by drastic treatment.
The intrinsic protein are highly insoluble in natural agnous siptoms
but can be extracted by detergent such as sodium dodacyl sulphate
hydrochloride. When 6m guanidine hydrochloride was used to extract
erytrocyte membrane 17 different polypeptide chains were obtained among
them was glycophorin, a glycoprotein that extends completely across the
membrane.
The inner mitochondrial membrane is one of the most complex
membrane, it contains over a 100 kinds of polypeptide chains. In summary,
membrane in particular the plasma membrane contains phospholipids,
cholesterol, glycolipids and glycoproteins. Because the components of the
membrane are in a fluid state, it’s able to move within the plain of the
bilayer.

1.5 SEPERATION OF MATERIALS OBTAINED FROM CELL
DISRUPTION
The techniques generally employed in separation of materials
obtained from cell disruption is centrifugation at different contrition forces.
The rational for this technique is that sedimention rate of particles of
different size and density vary. For particles of the same mass but different
density, the ones with the highest density will sediment at a faster rate than
the less dense particles. Particles having similar banding densities can
usually be efficiently separated from one another by differential
centrifugation or the rate zonal method, provided there is about a tenfold
difference in their sedimentation rate. In differential centrifugation, the
material to be separated (e.g. tissue homogenate) is centrifugally divided
into a number of fractions by increasing stepiose the applied centrifugal
field. The centrifugal field at each stage is chosen so that a particular type
of material sediments during the predetermined time of centrifugation, to
give a pellet of particles sedimented through the solution and a
supernatent solution containing unsedimented material.
Any type of particle originally present in the homogenate may be
found in the pellet or the supernatent or both factions depending upon the
time and speed of centrifugation and the size and density of the particle.
At the end of each stage, the pallet and supernatent are separated
and the pellet washed several times by resuspension in the
homogenization medium followed by recentrifugation under the same
conditions. This procedure minimizes cross contamination, improves
particle separation and eventually gives a fairly pure preparation of pellet
fraction.
The seperation achieved by differential centrifugation may be
improved by repeated (2 to 3 times) resuspention of the pellet in
homogenization medium and recentrifugation under the same conditions
are in the original pelleting, but this will inevitably reduce the yield
obtained. Further centrifugation of the supernatent in gradually increasing
centrifugal fields results in the sedimentation of the intermediate and finally
the smallest and least dense particles. Ribosones are obtained from the
crude microsones by treating it with deoxycholate detergent to break up
the lipids rich membrane and centrifuge the ribosones out of the detergent
solution. A scheme for the fractionating rat liver homogenate into
subcellular fractions is given in fig. 2.
Homogenized liver in 0.25m sucrose buffer

10 min 700 x g

pellet supernatent 10 min 7000 x
g

crude nuclei and
cell debris crude mitochondria supernatent 10 m
120 min x105,000
xg

crude microsones cofactor
ribosones soluble
enzymes
cell saps
cytoplasm
Fig. 2
1.6 ISOLATION OF MEMBRANE –CELL DISRUPTION
TECHNIQUES
Separation of membrane from one another is no simple task and has
never been accomplished completely when starting from a whole cell.
The situation is much simpler with mammalian erythrocyte where
osmotic bursting releases the entire cell contents so that what needed to
be done is simply to centrifuge and wash the strom which are infact
plasma membrane with the other cells. The procedure is usually to break
up the cell by one or more of the following techniques.
1. Application of hypotonic solution.
This affects the osmotic lysis of the tissue cell with consequent
entering of the cell content into the medium.
2. Application of mechanical devices (homogenizers).
The most commonly used homogenizer is the potter-Elvehjem
Homogenizer. It consists of a glass tube of precise bore into
which it is motor-driven at about 2000rev/mion to ensure
thorough mixing of the content.
Pestle

Tube

Potter-Elvehjem homogenizer

3. Horizontal Glass Disc Homogenizer.
This is a rotating device used with excellent result, used for thick
walled microorganisms. It is derived from a stirrer used in the
paint and enamel industries. A smooth thick glass tube is fixed at
the lower end of vertical slightly into cell suspension containing
lead free glass of appropriate size. With external cooling and
without excess of oxygen, cells are broken up in about a few
minutes.
4. Fresh press (Hughess press).
A sudden change in pressure can be applied in different
modification for bursting cells. In a French press, a frozen cell
pellet is pushed through a tiny opening in the process of which it
melts and the cells are disrupted.
5. Freezing and thawing.
Freezing and thawing reduces the cell to fragments, but these
are difficult to separate into any defined membrane types.
6. Sonication.
This will break up some cells, but here again the danger of
progressive damage to the individual membrane is high.

1.7 MORPHOLOGY AND FUNCTION OF ORGANELLES
1.7.1 MITOCHONDRION – Morphology
The rat liver cell mitochondrion is globular, other mitochondria
appear in different shapes in various cells. From electron microscope,
mitochondrion is about 2nm long, 1 nm wide in an intact cell. It consist of
an outer membrane and an inner membrane space, an inner membrane
which surrounds the inner compartment called the matrix. The inner
membrane is invaginated to forms folds called cristae. Each crista bears
several cristal nerves. The outer membrane can be removed with
detergent such as lubrol leaving behind a structure called mitoplast.
Mitochondrion resembles bacteria in that it contains DNA, small ribosones
and a protein synthesizing apparatus sensitive to chloramphenicol.
Function.
Mitochondrion is the power house of the cell. It is the site of the oxidation
of carbohydrates, fats and protein to carbon dioxide and water by
molecular oxygen. It is site of enzyme electron transport chain, kreb cycle
and oxidative phosphorylation (ATP formation). It is the site of various
transport system for anions, cations, nucleotides and organic acids
especially di and tricarboxylic acids of the kreb’s cycle.
Outer membrane
Inner membrane
Krebb’s cycle

Crista with
cistal knob

Fig. 2 A diagram of mitochondrion

1.7.2 CHLOROPLAST
MORPHOLOGY. Chloroplasts are membrane bound cell organelles of
higher plants. They contain ribosones and protein synthesizing apparatus
DNA and transfer RNA. They are enclosed in an outer chloroplast
membrane and formed from lamella vesicles called thykaloids. Chloroplast
consists of an outer and inner membrane just like the mitochondrion.

Thykaloids contain
Chloroplast which
Produce chlorophyll
Fig. 3 A diagram of chloroplast
Function.
a. Chloroplast provides chlorophyll which functions in
photosynthesis or the conversion of radiant energy of A.T.P. for
biosynthesis of glucose and other precursors. Photosynthesis
liberates oxygen as a byproduct and this is required by animal for
respiration.
b. Chloroplasts are the main source of energy of photosynthetic
cells in the light. The key component of the whole photosynthetic
process is chlorophyll of one kind or another.
c. Chloroplast also possess some transport system.

1.7.3 ENDOPLASMIC RETICULUM
MORPHOLOGY. The endoplasmic reticulum consists of flattened single
membrane residue whose inner compartments the cisterna interconnect to
form channel throughout the cytoplasm. There are two types of
endoplasmic reticulum, the rough and the smooth. The rough endoplasmic
reticulum is stubbed with ribosones. The endoplasmic membrane is the
site of many enzymes.

Fig 4 A diagram of endoplasmic reticulum
Function
1. The smooth endoplasmic reticulum functions in the synthesis of
phospholipids, cholesterol and other membrane component.
2. It’s the site of several microsomal enzymes such as Cyt C
reductase, Cyt. P450, Cyt b5 glucose-6-phosphotase, mixed
function oxidase (MFO) which functions in drug metablolism,
hence the smooth endoplasmic reticulum functions in the
detoxication of drug and other substances.
3. They are sites of ribosones especially the rough endoplasmic
reticulum. These ribosones function in protein synthesis.
4. The endoplasmic reticulum serves to channel protein products
throughout the cytoplasm.
5. The endoplasmic reticulum is directly attached to the nuclear
membrane at some parts.
1.7.4 LYSOSOMES
MORPHOLOGY. They are single membrane bound organelles that contain
roughly spherical structure particles. Lysosomal membrane

Hydrolytic
digestive enzyme

Fig. 5 A diagram of Lysosomes
Function
1. It walls hydrolytic digestive enzymes such as ribonuclease
(RNASE), phosphotase etc. Thus it acts as a special digestive
organelle of the cell.
2. It functions on the digestion of material brought into the cell either
by pinocytosis or phagocytosis.
3. It also serves for digest cell component after cell death i.e. in
damaged or drying cells; they lyses and release their sanitary
enzymes into the cytoplasm of the cell.

Model Question
1. Sketch a typical animal cell and state the biological functions of
the membrane.
2. Describe separation of materials from disrupted cells.
3. Describe techniques used in cell disruption
4. compare and contast the structure and function of
(a) chloropasts and mitochondria
(b) lysosomes and enoplasmic reticulum
References
Conn, E.E. and Stumpt, prk (1976) Outline of Biochemistry
Wiley Eastern Ltd
Datta, P and Ottaway, H (1976) – Biochemistry 4th ed
Bailliere Tindal – London
William H.B. (1986) Introduction to Organic and Biochemistry 4th ed
Brooks/Cole publishing company.

1.8 ENZYMES
Enzymes are proteins specialized in catalyzing biological reactions.
They are among the most remarkable bimolecular known because of their
extraordinary specificity and catalytic power, far greater than any man-
made catalysts. Although enzymes become intimately involved in the
reaction, they catalyze and remain essentially unchanged at the end of the
reaction. Their chemical and physical properties are similar to protein in
that they are denatured physically by heat and chemically by various
reagents. Their molecular weight can vary widely between 103 – 106.
Enzymatic activity is not disturbed over the whole of the molecule,
but localized in particular sharply delimited areas called active site or
active centers. Their activity is expressed in enzyme units. An enzyme unit
is the amount of enzyme which acts on one micromole of the substrate per
minutes under optimum conditions. The specific activity is used to define
the purity of an enzyme. It is expressed as the number of enzyme units per
milligram protein.
1.9 CLASSIFICATION OF ENZYMES
Many enzymes are named with the suffix-ase to the substrate they
catalyze, e.g urease catalyses the hydrolysis of urea to ammonia and
carbon dioxide and phosphatase, the hydrolysis of phosphate esters.
However, this nomenclature has not always been practical because many
enzymes have names which do not relate to the substrate they catalyze
e.g. pepsin, trysin and catalase. For this reason, a systematic classification
of enzymes has been adopted on the recommendation of an international
enzyme commission. The new system, thus divided enzymes into six major
classes and sets of subclasses, according to the type of reaction. Each
enzyme is assigned a recommended name usually short and appropriate
for everyday use, a systematic name which identifies the reaction it
catalyses.

International classification of enzymes class names, code number and
types of reaction.
1.0 Oxido-reductase (Oxidation – reduction reaction)
1.1 Acting on >CH – OH
1.2 Acting on >C = O
1.3 Acting on >C = CH-
1.4 Acting on >CH – NH2
1.5 Acting on >CH – NH-
1.6 Acting on NADH, NADPH.

2.0 Transferases (transfer of functional gropus)
2.1 One – carbon groups
2.2 Aldehydic or ketonic groups
2.3 Acyl groups
2.4 Glycosyl groups
2.7 Phosphate GROUPS
2.8 S – Containing groups

3.0 Hydrolases (hydrolysis reactions)
3.1 Esters
3.2 Glycosidic bonds
3.4 Peptide bonds
3.5 Other C-N bonds
3.6 Acid – anhydrides

4.00 Lyases (addition to double bonds)
1. >C=C<
2. >C=O
3. >C=N-

5.00 Isomerases (Isomerization reactions)
1. Ratemases

6.00 Ligases (Formation of bonds with ATP Cleavage)
1. C-O
2. C-S
3. C-N
4. C-C

1.10.1 ENZYME COFACTORS
Some enzymes depend for activity only on their structure as protein,
while others require one or more non protein components called cofactors.
The cofactor may be a metal ion or an organic molecule called a co-
enzyme, some enzymes require both for catalysis.
(a) Enzymes with a native protein.
Amylases, pepsin, trypsin and urease
(b) Enzymes with a protein and a cofactor
This group includes enzymes with a prosthetic (active)
group which has a low molecular weight. When the
prosthetic group can be readily separated off, it is called a
co-enzyme.
The catalytically active enzyme – cofactor complex is called the
Holoenzyme. When the cofactor is removed, the remaining protein, which
is catalytically inactive by itself is called an apoenzyme and are subdivided
according to their prosthetic group.

(a) Enzymes whose prosthetic groups contain metals –
metalloenzymes
(1) Iron (ii) ion Fe++ - peroxidase and catalase
(2) Copper (ii) ion Cu++ - Cytochromes oxidase and Tyro-Sinase
(3) Zinc (ii) ion Zn++ - Alcohol dehydrogenase and
carboxypeptidase
(4) Magnesium (ii) ion Mg++ - phosphoshydrolase and
phosphostransferases
(5) Sodium ion Na+ or Potassium ion – Plasma membrane
ATPase.
(b) Enzymes whose prosthetic groups are organic compounds
without metals. They are most often vitamin derivatives e.g. NAD,
NADP, FAD, FMN and co-enzyme A.
The co-enzymes play important chemical role in the catalytic process as
they act as donors and acceptor for electron of certain molecules. They are
however not themselves changed during the reaction and return to their
original state by a further enzymatic reaction.
FACTORS THAT AFFECT ENZYME REACTION
The rate of an enzyme catalyzed reaction depends on the following
factors.
1. Enzyme Concentration
The rate of an enzyme catalyzed reaction depends directly on
the concentration of the enzyme. The relation between the rate of a
reaction and increasing enzyme concentration in the presence of an
excess of the substrate which is being transformed is illustrated
below.

Rate of
reaction

Amount of enzyme

2. Substrate Concentration
However, with a fixed concentration of enzyme and with
increasing substrate concentration, another relationship is observed. With
fixed enzyme concentration, an increase of substrate will result at first in a
very rapid rise in velocity or rate of reaction. As the substrate concentration
continues to increase, the rate of reaction begin to slow down gradually
until with a large substrate concentration when no further change in
velocity is observed. This observation was described as dysphasic by
Michealis.
Maximum velocity (v)

Zero order kinetics
Rate of mixture of zero phase II
reaction V/2 and 1st order kinetics

1st order kinetics
Phase I

KM
Sw

3. Effects of temperature
Enzymes are very sensitive to elevated temperature. The
protein nature of an enzyme makes it possible for enzyme to undergo
thermal denaturation at elevated temperature. Increasing temperature will
decrease the effective concentration of an enzyme and consequently
decrease the reaction rate. At a temperature range of 00c – 450c, the
predominant effect will be an increase in the reaction rate as predicted by
chemical kinetic theory. At above 450c and until 550c, rapid thermal
denaturation becomes increasingly important and destroys the catalytic
function of enzyme protein.

(b)Thermal
denaturation

Rate of optimum
reaction (a) Increasing temperature
rate (a&b)

Temperature
(a) increasing rate of reaction as a function of temperature
(b) decreasing rate of reaction as a function of thermal denaturation
(c) the broken line – the combination of (a & b)
4. EFFECTS OF PH
Since enzymes are protein, any pH change will profoundly affect the
ionic character of the anion and carboxylic acid groups on the protein.
Consequently the catalytic site and the conformation of the enzyme
molecule will be affected. Low or high pH is capable of causing
considerable denaturation and hence inactivation of the enzyme activity.
The relation between pH and the rate of reaction gives a bell-shaped curve
with relatively small plateau and with sharply decreasing rates on either
side. The plateau is the optmal pH point.

Optimal pH

Rate of
reaction

pH
MODEL QUESTION
1. a) Define enzyme and enzyme active site.
b) Describe briefly, the problems that led to the systematic
classification of enzyme.
2. Classify enzymes systematically and describe any three factors that
effect enzyme activity.
3. Describe enzyme cofactors

REFERENCES
Conn, E.E. and Stumpt, P.K. (1976) Outline of Biochemistry

2.0 LIPIDS
Lipids are diverse group of compounds that are extractable from living
system by organic solvent such as chloroform, ether, benzene. Lipids have
several biological important functions namely:
1. as structural components of membrane
2. as storage and transport forms of metabolic fuel
3. as a protective coating on the surface of many organisms
4. as cell-surface components concerned in cell recognition,
species specificity and tissue immunity
1. CLASSIFICATION
Lipids have been classified in several different ways. The most satisfactory
classification is based on their backbone structure.
(a) Fatty acids. This includes
(1) saturated fatty acids
(2) unsaturated fatty acids
(3) cyclopropane and
(4) branched fatty acids
(b) Glycerol derived lipids. This includes
(1) mono, di and triglycerides
(2) glycerol ether
(3) phosphatides
(c) Sphingosine derived lipids. This includes
(1) sphingomyelin
(2) cerebrosides
(3) ganglosides
(4) ceramides
(d) Steriods and their derivatives
(e) Terpenes
Thus lipids can be broadly classified into two namely simple and complex
lipids. The complex lipids contain as components acylglycerol, the
phosphoglycerides, the sphingolipids and the waxes. They are saponifiable
lipids because they yield soaps (salts of fatty acids) on alkaline hydrolysis.
The simple lipids contain fatty acids hence are non-saponifiable –
Terpenes, steroids and protaglandins.

2. FATTY ACIDS
Fatty acids are monocarboxylic acids obtained from the hydrolysis of
triglycerides. The most fatty acids in nature are straight chain, saturated or
unsaturated compound. They contain a number of even and odd carbon
atom. The common and structural formulars for some fatty acids are
presented in table 1.
Table I Some natural fatty acids
Carbon atoms Structural formula Common name mp(oc)
Saturated fatty acids.
12 CH3(CH2)10COOH lauric acid 44
14 CH3(CH2)12COOH myristic acid 58
16 CH3(CH2)14COOH palmitic acid 63
18 CH3(CH2)16COOH stearic acid 70
20 CH3(CH2)18COOH arachidic acid 77
Unsaturated fatty acids
16 CH3(CH2)5CH=CH(CH2)7COOH palmitoeleic - 1
18 CH3(CH2)7CH=CH(CH2)7COOH Oleic acid - 16
18 CH3(CH2)4(CH=CHCH2)2(CH2)6COOH linoleic acid - 5
18 CH3CH2(CH=CHCH2)3(CH2)6COOH linolenic acid - 11
20 CH3(CH2)4(CH=CHCH2)4(CH2)2COOH arachidomic acid - 49
Branched fatty acids are known to exist in fewer natural substances
as milk. Fatty acids contain that cyclopropane ring is known to exist in
same organism e.g. of cyclopropane ring.
(CH2)5CH3 (CH2)7CH3

(CH2)7COOH
(CH2)9COOH
Lactobacellic acid Stercullic acid
Fatty acids occur in diets only to a minor extent. The major fraction of the
ingested fatty acid occurs in more complex lipids. e.g. triglycerol and
phosphatides. The fatty acid composition in mammal is a function of four
variables:
(1) species of mammals
(2) the tissue where it is found
(3) class of complex lipids
(4) the type of diet the mammal eat.
Fatty acids are amphipathic molecules i.e. they possess both
hydrophobic and amphipathic properties. The long chain of methylated
group is hydrophobic while the changed group is hydrophilic.
Unsaturated fatty acids are characterized by having one to six
reactive double bonds in the molecule.

3. GLYCERIDES
These are lipids which are derived from trihydiric alcohols.
O
CH2OH CH2 O C R1
O
CHOH + 3RCOOH CH O C R2
O
CH2OH CH2 O C R3
Glycerol fatty acid triglyceride
Tri esters are the most abundant lipids in animals and plants. Glycerol is a
sweet viscous liquid miserable with water and ethanol. Each of the OH
group of the glycerol may be esterified with fatty acid. When one is
esterified, it is known as monoglyceride while when two OHs and three
OHs are esterified they are known as di and triglycerides respectively.
O O
CH2 O C R1 CH2 O C R1
O
CH OH CH O C R2
CH OH CH2 OH
Monoglyceride diglyceride

Of all, the triglycerides are the most abundant, while mono and
diglycerides are only found in trace amount in neutral lipids. The glycerides
comprise two types of lipids – fats and oil. The difference between fats and
oil is their condition at different temperature. Fat is solid while oil is liquid at
room temperature.
The melting point of triglyceride is determined by the nature of the
fatty acid composition. In general the higher the proportion of short chain,
and unsaturated fatty acid, the lower the melting point e.g. tributerene
melts at 71oC. Both triolein and tristerene contain 18 carbon atoms but the
trilein melts at 17oC while the tristerene melts at 88oc. This emphasizes the
importance of unsaturation in determining the physical properties of
triglycerides.
Generally, triglycerides are insoluble in water, but soluble in organic
solvent. e.g. benzene, ether, chloroform etc. they can be degraded in
hydrolysis in air and acidic medium to give glycerol and the salts of fatty
acid. The product obtained by the process gives soap.
Triglyceride which occurs in living system has mixed fatty acids
content e.g. 1-oleodipalmitic i.e. if the number of glycerides is in either
direction means that the 1st C of the acid that is esterified is Oleic acid. 2-
oledipalmitic as the name indicates, differ in the composition of the fatty
acid inside the group, therefore they are one isomer. They tend to have
distinct physical and chemical properties. More complex triglyceride may
contain fatty ester group. 1-palmitoyl-2-oleoyl-3-leoleoyl. Glycerol esterified
to 1st C is palmitic.
4. CHEMICAL PROPERTIES OF TRIACYLGLYCERIDES
The chemical properties of triglycerides are determined by the nature of
the fatty acid.
1. Iodine number
The degree of unsaturation in a lipid is measured by it’s iodine
number. The iodine number is the number of grams of iodine that would
add to the double bonds present in 100g of the lipid, if iodine itself could
add to the alkenes.
The reagent usually used is iodinebromide. The data are calculated
as if iodine only was used. Fatty acids without double bonds have zero
iodine number, while fatty acids with double bonds like oleic acid has
iodine number of 90, linoleic acid 181 and linolenic acid 274. Animal
fats in general have low iodine number while vegetable oils have higher
values.
2. Hydrolysis
(a) with enzymes
Biological hydrolysis is affected by enzymes. The enzymes in the
digestive tracts of mammals cleaves the ester link of triacyl
glycerates to fatty acids and glycerols.
O
RI C OCH2 HO CH2
O O
RII C OCH + 3H2O Enzymes 3R C OH + HO CH
O Lipase
RIII C OCH2 HO CH2
Fatty acid glycerol

(b) With alkali
Strong base produces glycerol and salt of fatty acid on
hydrolysis. This reaction is known as saponification.
O
RI C OCH2 HO CH2
O O
RII C OCH + 3 NaOH 3R C O Na+ +HO CH
O
RIII C OCH2 HO CH2
Fatty acid Salt glycerol

3. Hydrogenation
This is a catalytic addition of hydrogen molecules to the
double bonds in vegetable oil. Margarine is made from hydrogenated
oils.
4. Rancidity
When fats and oil are left exposed to warm moist air for any
length of time, they become rancid i.e. development of unfavourable
flavours and odors. This can be brought about by (i) hydrolysis of ester
link (ii) the oxidation of double bonds.
Enzymatic hydrolysis of e.g. butter, fat produces odorous fatty
acids while oxidative action on the unsaturated side chain produces
also volatile carboxylic acids and aldehydes that are odorous.
5. Saponfication value
Saponification value of an oil and fat is defined as the number
of milligrams of KOH required to neutralize the fatty acids, resulting
from complete hydrolysis of 1g of the sample. The saponification value
is inversely proportional to the mean of the molecular weight of the fatty
acid in the glyceride present. Express mathematically
Saponification value = A – B x 28.05
W
where, A = volume of HCl used in blank titration
B = volume of HCl used for the titration of the sample
W = weight in grams of the oil sample and 28.05 conversion
factor.
6. Free fatty acid (FFA)
This is defined as the number of milligrams of KOH required to
neutralize the free acid in 1g of the substance.

Expressed mathematically,
Acid value = titre - value x 5.61
Weight of sample
It is used to determine the amount of free fatty acid present in the lipids.
The quality of lipids depends on the above factors.
5. ESSENTIAL FATTY ACIDS
These are fatty acids that cannot be synthesized by mammals and
must be obtained from plant sources, e.g. linoleic acid and linolenic acid
Linoleic acid is an important precursor in mammals for the biosynthesis of
arachidonic acid. The essential fatty acids are precursors in the
biosynthesis of a group of fatty acid derivatives called prostaglandins.
CH3(CH2)4CH = CH CH2CH = CH (CH2)7 COOH Linoleic acid
CH3CH2CH = CHCH2CH = CHCH2CH = CH(CH2)7COOH Linolenic acid
6. PHOSPHATES
These are lipids derived from phosphatidic acid. Phosphatids are divided
broadly into two groups’ Nitrogen and Iron nitrogen RIII esterified
O
CH2 OH CH2 O C RI
O
CH OH + esterified CH2 O C RII
O O
CH2 P O H CH2 O P O RIII

OH OH
The distinguishing features of phosphatides are the composition of the RIII.

RIII CH2 CH NH2

COOH
HO CH2 CH COOH RIII containing nitrogen atom.

NH2 Serine

O
CH2 O C RI
O
CH O C RII NH2
O
CH2 O P O CH2 CH COOH

OH Phosphatidyl serine
HO CH2 CH2 NH2 ethano alanine

O
CH2 O C RI
O
CH O C RII
O
CH2 O P O CH2 CH2 NH2

OH Phosphatidyl ethanoalanine
HOCH2CH2NHCH3 N- methyl ethanoalanine
O
CH2 O C RI
O
CH O C RII
O
CH2 O P O(CH2)2.NHCH3

OH Phosphatidyl N. methyl ethanoalanine
HOCH2CH3N+(CH3)2 Choline

O
CH2 O C RI
O
CH O C RII
O
CH2 O P O(CH2)2. N+(CH3)3

OH Phosphatidyl Choline
Compound without N RIII= H or HOCH2CHOHCH2OH

O
CH2 O C RI
O
CH O C RII
O
CH2 O P O CH2CHOHCH2OH

OH Phosphatidyl glycerol
RIII may be equal to inositol
OH
OH OH

OH
OH OH
Inositol

O
CH2 O C RI
O
CH O C RII OH
O OH OH
CH2 O P O

OH OH OH
Phosphatidyl inositol
Phosphoglycerides are found in cellular membrane only in small amount
and occur in adipose tissue.
In phosphoglyceride, one of the parents’ OH – group of glycerol is
esterified to phosphoric acid. The parent compound of the series is the
phosphoric ester of glycerol. The compound has asymmetrical carbon
atom and can be designated as D-glycerol adyde. D-glycerol-1-phosphate
and α-glycerol 3-phosphate.
H
H C OH
HO C OH O
HO C O P OH

H OH
Phosphoglyceride
Because of this ambiguity, a convention has been adapted that the
stereochemistry of glycerol derivative is based on sterospecific numbering
system. Based on the sterospecific numbering α-glycerol-3-phosphate
becomes glycerol-3-phosphoric acid.
All phosphoglyceride possess a polar heald and two non-polar
hydrocarbon chains. They are amphipathic or polar lipids.

O
CH2 O C RI
O non-polar chain
CH O C RII
O
CH2 O P O RIII polar head

OH

The most abundant phosphoglycerol in higher plant and
animal are phosphatidyl ethanoalanine and phosphatidylcholine.
Phosphatidyl ethanoalanine is also called cephalin and phosphatidyl
choline – lecithin. The phosphoglycerides are white waxy solid darkens on
exposure to air. They undergo complex chemical change because of the
tendency of the unsaturated fatty acid component to be peroxided by
atmospheric oxygen.
8. HYDROLYSIS OF GLYCEROPHOSPHATIDES
1. With mild alkali
Phosphoglycerides when hydrolysed with mild alkali gives fatty
acids as soap but leaves glycerol phosphoric acid alcohol portion of the
molecule intact e.g. phosphatidylcholine on hydrolysis yields glycerol–3-
phosphorylcholine.
2. With strong alkali
Phosphoglycerides on hydrolysis with strong alkali causes the
cleavage of fatty acid and also of head alcohol.
3. Acid hydrolysis
On acid hydrolysis, phosphoglycerides yields glycerol since the linkage
between phosphoric acid and glycerol is stable to base hydrolysis.
4. Phosphoglycerides can also be hydrolyzed by specific
phospholipases, which is an important tool in the determination of
phosphoglyceride structure.
Phospholipase A, removes fatty acid from position one, phospholipase
A2 from two position. Removal of one fatty acid molecules from a
phosphoglyceride yields a lysophosphoglyceride.

REFERENCES
Brown W.H. Introduction to Organic and Biochemistry. Brook/Cole
publishing Co. 4th
Conn, E.E. and Stump P.K. Outlines of Biochemistry John Wiley &
Sons Inc.

MODEL QUESTIONS
1. Define and classify lipids based on their backbone structures
2. Describe the chemical properties of triacyl glycerides with
appropriate equations.
3. Describe rancidity in fats and oil.
4. Draw the structures of phosphatidyl serine and phosphatidyl choline
5. Describe the various products of the hydrolysis of
glyceroohosphatides with hydrolytic agents.

3.0 AMINO ACIDS AND PROTEINS
Proteins are macromolecular polymers composed of amino acids as
the basic unit. These polymers contain carbon, hydrogen, oxygen, nitrogen
and usually sulfur. The elementary composition of most proteins is very
similar, approximate % are C=50 – 55,H=6 – 8, O=20 – 28, N= 15 – 18 and
S = 0 – 4. These figures are useful for making rough estimates of protein
content of biological matter and foodstuffs. The nitrogen content of most
protein is about 16%, and as this element is easily analysed as NH3 by the
kjeldahl nitrogen procedure, the protein content can be estimated by
determining the nitrogen content and multiplying by 6.25 (100/16).
The fundamental structural unit of proteins is the amino acid as may
be easily demonstrated by hydrolyzing purified proteins by chemical or
enzymatic procedures.
1. STRUCTURES AND CLASSIFICATION
The general formular of a naturally occurring amino acid may be
represented with a modified ball and stick formula or the Fischer projection
formula.
H H
COOH
R R C COOH
NH3 NH2
Ball and Stick model Fischer projection formula

Because the NH3 group is on the carbon atom adjacent to the carboxyl
group, the amino acids having this general formula are known as alpha (α)
amino acids. If the R in the structure is not equal to H, the α carbon atom is
asymmetric. Thus, two different compounds, having the same chemical
formula may exist, one will have the general structures shown and the
other will be the mirror image isomer of the first. It is known that all the
naturally occurring amino acids found in the protein have the same
configuration.
2. CLASSIFICATION
The naturally occurring amino acids may be classified according to
the chemical nature (aliphatic, aromatic, heterocyclic) of their R group with
appropriate subclasses. The twenty commonly amino acids, obtained on
the hydrolysis of protein may be divided as:
(1) non-polar or hydrophobic
(2) polar but uncharged
(3) polar because of a negative charge at the physiological pH of 7
(4) polar because of a positive change at physiological pH.

(1) Amino acids with non-polar or hdrophobic R groups.
This group contains amino acids with both aliphatic and aromatic
residues that are hydrophobic in character.
Aliphatic
H CH3 H CH3 H

CH3 C COO- CH C COO- CH CH C COO-
+NH3 CH3 +NH3 CH3 +NH3
Alanine Valine Leucine

CH3-CH2 H H2 C CH2

CH C COO- H2 O + CH COO-
N
H2
CH3 +NH3
Isoleucine Proline

H

CH3 S CH2 CH2 C COO-

+NH3
Methionine H
Aromatic H
CH2 C COO-
CH2 C COO-
+NH3
+NH3
N
Tryptophan Phenylalanine

One of the compounds proline is unusual in that it’s nitrogen atom is
present as a secondary amine rather than as a primary amine.
2) Amino acids with polar but uncharged R group.
Most of these amino acids contain polar R residues that can
participate in hydrogen bond formation. Some have a hydroxyl group or
sulfhydrye group (cyteine) while two have amide groups, e.g. asparagine.
glycine, which lacks an R group is included in this grouping because of it’s
definite polar nature. Both aliphatic and aromatic compounds are included
in this group.
H H CH3 H
H C COO- HO – CH2 C COO- CH C COO-
+NH3 +NH3 HO NH3
Glycine Serine Threonine

H H

HS CH2 C COO- NH2 C CH2 CH2 C COO-
‫װ‬
+NH3 O +NH3
Cysteine Glutamine
Sulfhydryl

H H
CH2 C COO- NH2 C CH2 C COO-
O
+NH3 +NH3
Tyrosine Asparagine
3) Amino acids with positively charged R groups
Three amino acids are included in this group. Lysine, with it’s second
amino groups (pk = 10.5) will be more than 50% in the positivlely charged
state at any pH below the pka of that group. Arginine with a strongly basic
guanidinium function (pk = 12.5) and histidine with its weakly basic (pk =
6.0) imidazole group are included.

H H
+NH3 CH2 CH2 CH2 CH2 C COO- NH2 C NH CH2 CH2 CH2 C COO-

+NH3 +NH2 +NH3
Lysine Arginine
H
HC C CH2 C COO-
+NH3
HN+ NH
C
H Histidine
4) Amino acids with negatively charged R groups. This group includes the
two dicarboxytic amino acids, aspartic acid and glutamic acid. At neutral
pH their second carboxyl groups with pka’s of 3.9 and 4.3 respectively
dissociate, giving a net charge of -1 to these compounds.
H H
-
OOC CH2 C COO- -
OOC CH2 CH2 C COO-

+NH3 +NH3
Aspartic acid Glutamic acid
3. Properties of amino acid
1. Amino acids with certain exception, are generally soluble in water
and are quite insoluble in non-polar organic solvents such as
ether, chloroform and acetone unlike carboxylic acids and organic
amines.
2. The melting points are high, higher than solid carboxylic acids
and amines.
3. Amphoteric substance or zwitterions. It reacts with alkalis and
acids.
Titration of amino acids
Values of pka for ionizable groups of amino acids are usually
obtained by acid base titration and determining the pH of the solution as a
function of added base or acid depending on the how the titration is done.
Consider a solution containing 1.0 mol of glycine that has been
added excess strong acid, so that the carboxyl and the amino groups are
fully protonated. The solution is titrated with 1.0m NaOH, the volume of
base added and the pH of the resulting solution are recorded and then
plotted. The most acidic group and the one to react first with added NaOH
is the carboxyl group. The carboxyl group is half-neutralized when 0.5m of
NaOH has been added. At this point, the dipolar ion has a concentration
equal to that of the positively charged ion and pH equals 2.35, the pka of
the carboxyl group.
[H3N+ - CH2 – CO2H] = [H3N+ - CH2 – CO-2] where pH = pka = COOH
dipolar ion
the endpoint of the first part of the titration is reached when 10mol of
NaOH has been added. At this point, the predominant species in solution is
the dipolar ion, and the pH of the solution is 6.07. The next section of the
curve represent titration of the NH3+ group. When another 0.5mol of NaOH
is added to total 1.5mol, half –NH3+ groups are neutralized and converted
to NH2. At this point, the concentration of the dipolar ion and the negatively
charged ion are equal, the pH 9.78, the pka of the amino group of glycine.

10

9
pk2=9.7
pH 8 [H3N+ - CH2 – CO-2] = 50

[H2N - CH2 – CO-2] = 50
6

4 Isoelectric point [H3N+ - CH2 – CO-2] 100%
pH. 6.07
3

2 pk1 = 2.35 [H3N+ - CH2 – CO2H] = [H3N+ - CH2 – CO-2]
50 50
0

.5 1 1.5 2.0
Moles of NaOH per mole of glycine
The second endpoint of the titration is reached when a total of 2.00mol of
NaOH is added and glycine is converted entirely to anion.
Titration curves such as that for glycine help both to determine pka
value for the ionization groups of an amino acids and the isoelectric point.
Isoelectric point – Is the isoelectric pH (pH1) that is the arithmetic mean of
pk1 and pk2 i.e.
pH1 = (½ pk11 + pk21) in which there is no net electric charge on the
molecules, a net charge of zero.
From the titration curve, the pH1 for glycine is 6.07. Half – way
between the pka, values for the α – carboxyl and α – amino groups.
P1 = ½ (pkaCO2H + pka – NH3+)
= ½ (2.35 + 9.78)
= 6.07
Determine the p1 of
(1) aspartic acid with pk1 = 2.1, pk2 = 9.8
(2) alanine pka, 2.3 and pka 9.7
REACTIONS OF AMINO ACIDS
The properties of amino acids depends on the presence of carboxyl and
amno groups. These reactions are well known in organic reactions.
Reaction of the carboxyl groups
1. The carboxyl group may be esterified with alcohols.
O
H+
R CH COO- + C2H5OH R CH C OC2H5 + H2O
+NH3 +NH3
2. Converted into the corresponding acylchloride
PCl5
-
R CH COO R CH COCL
POCl3
+NH3 +NH3
The +NH3 in acylation reactions has to be protected to prevent it reacting
violenting with the pcl5. Such acyl chloride represent activated form of the
amino acid which in turn can be coupled with the amino group of a second
amino acid to produce dipeptide.
3. The carboxyl group of amino acids may be decarboxylated
chemically and biologically to yield the corresponding amine.

R CH CO2H R CH2 CO2-
NH2 NH2
Thus, the vasoconstrictor agent, histamine is produced from
histidine. Histamine stimulates the flow of gastric juice into the stomach
and is involved in allergic responses.

Reaction of Amino group
1. Reaction with strong oxidizing agent Nitrous acid (HNO2). The
amino group reacts with strong oxidizing agent, Nitrous acid to liberate
(N2). This reaction is important in the estimation of α amino group in amino
acids. Proline and hydroxyproline do not undergo this reaction.

R CH COOH + HNO2 R CH COOH + N2 + H2O + H+

+NH3 OH
2. Reaction with a mild oxidizing agent. The amino group of amino acid
reacts with mild oxidizing agent ninhydrin to form ammonia, CO2 and the
aldehyde.

R CH COOH + oxidized ninhydrin

NH2 R CH + NH3 + CO2 + Reduced ninhydrin

NH2
The second equivalent of ninhydrin (oxidized) then reacts with the reduced
ninhydrin and NH3 formed to produce a highly colored product, having the
following structure O
C OH HO C
C + NH3 +
C
C
C OH H O
Oxidized ninhydrin Reduced ninhydrin
O
O C
C
C N=C +3H2O
C
O
C
OH Blue product
The intense blue product is generally characteristic of those amino acids
having α – amino groups. Proline and hydroxyproline that are secondary
amines, react with ninhydrin to produce yellow products. Asparagines
produces a characteristic brown product because of it’s free amide groups.
3. Reaction with 1-fluro-2-4-dinitrobenzene (FDNB)
The intensively colored dinitrobenzene nucleus is attached to the
nitrogen atom of the amino acid to yield derivative, the 2,4-dinitrophenol or
DNP – amino acid. The compound FDNB will react with the free amino
acid group on the NH2 – terminal end of a polypeptide as well as the amino
groups of free amino acids. By reacting a protein or intact polypeptide with
FDNB, hydrolyzing and isolating the colored DNP – amino acid, one can
identify the terminal amino acids in a polypeptide chain.

H2 N CH CO2H + NO2 F
R
NO2
NO2 N COOH + HF

H R

3. Biological function of Protein
1. Proteins have many different biological functions. The enzymes are
the largest class. Nearly 2,000 different kinds of enzyme are known, each
catalyzing a different kind of chemical reaction. The hexokinase catalyzes
the transfer of a phosphate group from ATP to glucose, the first step in
glycolypsis, other enzyme dehydrogenate fuel molecules, still others e.g.
cytochromec, transfer electron toward molecular oxygen during respiration.
Each type of enzyme molecule contains an active site, to which its specific
substrate is bound during catalytic cycle.
2. Storage protein. Another major class of protein store amino acids as
nutrients and as building blocks for the growing embryo e.g. ovalbumin of
egg white, casein of milk and gliadin of wheat seeds.
3. Transport protein: Some proteins are capable of binding and
transporting specific types of molecules via the blood. Serum albumin
binds free fatty acids tightly and thus serves to transport these molecules
between adipose tissue and other tissues or organs in vertebrates. The
lipoprotein of blood plasma transports lipids between the intestine, liver
and adipose tissue. Hemoglobin of vertebrate, erythrocytes transports
oxygen in invertebrate.
4. Protective proteins: Some proteins have a protective or defensive
function. The blood proteins thrombin and fibrinogen assist in blood
clothing and thus prevent the loss of blood from vascular system of
vertebrate. The most important of these, are the antibodies or immune
globulins, which combine and thus neutralize foreign protein (body) and
other substances that happens to gain entrance into the blood or tissues of
a given vertebrate.
5. Structural proteins. Another class of protein comprises those that
serve as structural elements. In vertebrates, the fibrous protein collagen is
the major –extracellular structural protein in connective tissue and bone
Collagen fibrils, by forming a structural continuum also helps bind a group
of cells together to form a tissue. Two other fibrous proteins in vertebrate
are elastin of yellow elastic tissue and α-keratin.
6. Hormones: Among the proteins functioning as hormones are growth
hormones, or somatotropin, a hormone of the anterior pituitary gland.
Insulin secrated by certain specialized cells of the pancreas is a hormone
regulating glucose metabolism, its deficiency in man causes the disease
diabetes mellitus.
5. The structure of the protein molecules
Our knowledge of the structure of protein began with the work of Emil
Fischer, who devised methods for uniting amino acids through their amino
and carboxyl groups with the elimination of water. The union of two
molecules of glycine to form the dipeptide glycyl-glycine may be
represented as
H
CH2 NH H HO OC CH2 N C =O +H2O
COOH CH2 NH2 COOH CH2 NH2
Glycine glycine Glycylglycine
The principal linkage existing between the amino acids in the protein
molecule is through the amino group of one acid and the carboxyl group of
another. This is called peptide bond or linkage.

H
N C=O
Structures
Four basic structural levels are assigned to protein.
1. Primary Structure
This is referred to as the linear sequence of amino acid
residues making up it’s polypeptide chain. The peptide linkage
between each of the amino acids is the only link, no other
forces or bonds are indicated in the molecules.
2. Secondary Structure
The term refers generally to the structure which polypeptide or
a protein may possess resulting from hydrogen bond interaction
between amino acid residue fairly close to one another in the
primary structure. An example is a right-handed α-helical spiral
which is stablished by hydrogen bonding between the carbonyl
and the imido groups of the peptide bonds that appear in a
regular sequence along the chain.
R

α-helix
3. Tertiary Structure
This refers to the tendency of the polypeptide chain to
undergo extensive coiling or folding to produce a complex,
somewhat right structure. Folding normally occurs from
interaction between amino acid residues relatively far apart in the
sequence. The tertiary structure of many globular proteins
contain α-helix and β-pleated sheet structures, which vary widely
in number. For example, lysozyme with 129 amino acid in a
single polypeptide chain has only 25% of it’s amino acid in a-helix
regions. Cytochrome with 104 amino acids in a single polypeptide
chain has no a-helix structure, but does contain several regions
of β-pleated sheet.
The stabilization of the structure is due to the different
reactivities associated with the R-groups in the amino acid
residues. This involves folding of regular units of the secondary
structure as well as the structure of areas of the peptide chain
that are devoid of secondary structure. Disulphide linkages are
the strongest bonds, maintaining the tertiary structure of the
protein. Usually the hydrophilic amino acids tend to be folded to
the interior while most of the polar residues are on the surface.

C O
N+ H3 O H
(a) O- (b)
HC

CH2OH H
(c)
CH2OH (d)

Key:
(a) Electrostatic interaction
(b) Hydrogen bonding
(c) Interaction of nonpolar side-chain caused by mutual repulsion
of solvents.
(d) Vander Wamino acidl’s interaction
4. Quaternary Structure
This refers to the structure of protein resulting from interaction
between separate polypeptide units of a protein containing
more than one subunits. Most proteins of molecular weight
greater than 50,000 consist of two or more non-covalently
linked polypeptide chains. The arrangement of protein
monomers in an aggregation is known as quaternary
structure. A good example is hemoglobin, a protein that
consists of four separate protein monomers, two α-chains of
141 amino acids each and two β-chain of 146 amino acids
each. The chief factor stabilizing the aggregation of protein
submits is hydrophobic interaction.
Table: Quaternary Structure of selected proteins.

Protein Mol wt. Number of Subunit Biological functions
Subunits Mol. Wt.
Inslin 11,466 2 5,733 a hormone regulating
Glucose metabolism
Hemo- 64,500 4 16,100 Oxygen transport in
globin blood plasma
Alcohol 80,000 4 20,000 an enzyme of alcohol
dehydro- fermentation
genase
Iaetate 134,000 4 33,500 an enzyme of anaerobic
dehydro- glycolysis
genase
Aldolase 15,000 4 37,500 an enzyme of anaerobic
glycolysis
Fumarase 194,000 4 48,500 an enzyme of the
Tricarboxylic acid cycle
Tobacco 40,000,000 2,200 17,500 plant virus coat
Mosaic
Virus
Source: William, 1987

Model Question
1. Define protein and draw the ball and stick model of amino acids
2. (a) Classify amino acids according to the chemical nature of their
R groups.
(b) Explain why amino acid has higher melting points than solid
carboxylic acid and amines.
3. (a) Explain why amino acid is soluble in water and insoluble in
benzene or ether.
(b) Describe the titration curve of any amino acids.
4. (a) Give four biological functions of protein
(b) Describe the reaction of amino acid with
(a) alcohols (b) acylchloride

4.0 IMPORTANCE OF WATER AND THE CONCEPT OF PH AND
BUFFERS
4.1.0 IMPORTANCE OF WATER
Water is a remarkable molecule essential to life, solubilizes and
modifies the properties of bio-molecules such as nucleic acids, proteins
and carbohydrates by forming hydrogen bonds with their polar functional
groups. These interactions modifies the properties of the biomolecules and
their confirmations in solution. The accompanying changes impart
properties to these bio-molecules essential to the process of life. Bio-
molecules even relatively non-polar bio-molecules such as certain lipids
also alter the properties of water. The dissociation behaviour of the
functional; groups of the bio-molecules in aqueous solution at various
values is central to understanding their reactions and properties both in the
living cells and in the laboratory.
Water constitutes a physical end product of oxidative metabolism of
foods. The active sites of enzymes are constructed so as to either exclude
or include water depending on whether water is or is not a reactant.
Homeostasis: The maintenance of the composition of the internal
environment that is essential for health includes consideration of the
distribution of water in the body and the maintenance of appropriate pH and
electrolyte concentration. Two third (2/3) of total body water 55 – 65% of
body weight in men and about 10% less in women is intracellular fluid. The
remaining extra-cellular fluid, blood plasma constitutes approximately 25%.
Regulation of water balance. This depends on hypothslamic mechanisms
for controlling thirst, on antidiuretic hormone and one retention or excretion
of water by the kidneys and evaporative losses due to respiration and
perspiration.

PROPERTIES OF WATER
1. Slightly skewed tetrahedral molecules
The three-dimensional structure of water molecules is an irregular
tetrahedron with oxygen at it’s centre. The two bonds with hydrogen are
directed towards two corners of the tetrahedron, while the unshaved
electrons on the two sp3 hybridized orbitals occupy the two remaining
corners. The angle between the two hydrogen atoms (105 degrees) is
slightly less than the regular tetrahydral angle (107.5 degrees) forming a
slightly skewed tetrahedron. The ammonia molecules also forms a
tetrahedron one in which the bond angles between the hydrogen (107
degrees) approach the tetrahedral angle even more closely than water.

2e

2e
H
2e
N

105o

H H
Tetrahedral Structure H
Of water molecule Tetrahedral Structure of ammonia
2. Formation of dipoles
Because of it’s skewed tetrahedral structure, electrical change is not
uniformly distributed about the water molecules. The side of the oxygen
opposite to the two hydrogen is relatively rih in electron while on the other
side, the unshielded hydrogen nuclei form a region of local negative
change. The term dipole denotes molecules such as water that have
electrical change(s) unequally distributed about their structure. Ammonia
also is dipolar as are many biochemical compounds such as alcohols,
phospholipids, amino acids and nuclei acids.
H∂+ H∂+
O∂- O∂-

H∂+ H∂+
3. Formation of hydrogen bonds
Liquid water, like ice, exhibits macromolecular structure. This
structure arises as a result of the ability of water dipoles to self associate in
the solid and liquid states. The electrostatic interaction between a
hydrogen of one water dipole and the unshared electron pair of another
water dipole forms a hydrogen bond. Hydrogen bonds favour the
association of water dipoles in ordered arrays. Hydrogen bonds require
both a hydrogen donor and a hydrogen acceptor. A water dipole can serve
both as a donor and an acceptor of a hydrogen atom.
Hydrogen bond in liquid water has the following properties.
1. Hydrogen bond is relatively weak compared to covalent bonds.
2. It has a bond energy of about 4.5 kcalmol-1compared to 110 kcalmol-
for the covalent H-O bonds in the water molecules.
3. It is the strongest when the two interacting molecules are oriented to
yield high electrostatic attraction.
4. It has a characteristic bond length which differs from one type of
hydrogen bond to another.

4.1.2 CONCEPT OF PH
Because of the small mass of hydrogen and because the atoms
single electron is tightly held by the oxygen atom, there is a finite tendency
for a hydrogen ion to dissociate from the oxygen atom to which it is
covalently bound in one water molecule and joins to the oxygen atom of
the adjacent water molecule to which it is hydrogen bonded. This is only
possible provided the internal energy of each molecule is favourable.
H H H
O∂+ H + OH-

O H O H

H
2H2O H3O∂+ + OH-
Two ions are produced, the hydronium (H3O+) and hydroxide (OH-) ions.
The pH Scale:
The dissociation of water is an equilibrium process
H2 O H+ + OH-
for which it’s equilibrium constant can be written
Keg = [H+] [OH-]
[H2O]
The magnitude of the equilibrium constant at any given temperature can
be calculated from conductivity measurement on pure distilled water. Since
the concentration of water in water is very high, it is equal to the number of
grams in litre divided by molecular weight.
Cone = 1000 = 55.5M
18
H2 O H+ + OH-
1 x 10-7 + 1 x 10-7
To determine the equilibrium constant
Keg = [H+] [OH-] = [1 x 10-7] [1 x 10-7] at 25oc
[H2O] 55.5
Keg x 55.5 = 10 x 10-14
Kw = 1 x 10-14 ionic product.
In an acid solution the H+ concentration is relatively high and the OH-
concentration correspondingly low, while in a basic solution, the situation is
reversed. The ionic product of water is the bases for the p H scale, a means
of designating the actual concentration of H+ and OH- ions in any aqueous
solution in the acidity range between 1.0M H+ - 1.0M OH-
pH = log10 1/ [H+] = -log10[H+]
pH = log10 1/1 x 107 = 7.00

4.1.3 Acids and Bases
Acid is a molecular specie tending to loss an hydrogen ion while a
base is a specie that attend to gain an hydrogen ion.
Hydrogen ion dissociates from acid thus
A B + H+
As the dissociation is reversible the specie B formed when A loses a
hydrogen ion is in infact a base, when the equilibrium is displaced to the
left B adds a H+. Such a pair of specie is known as a conjugate acid-base
pair. An acid that loses a H+ to form it’s conjugate base, must always have
a change which is one unit more positive than its conjugate base.
Hcl H + cl+
CH3COOH H+ + CH3COO-
H2PO4 H+ + HPO42-
NH4+ H+ + NH3
Strong Acid
When strong mineral acids are dissolved in water, the dissociation of
the H+ may be considered to be complete. Thus Hcl, HclO4, HNO3 and the
first hydrogen of H2SO4 are completely ionized in dilute solution. i.e.
equilibrium is completely over to the right.
Weak Acid
When weak amino acidcids such as CH3COOH, H3PO4, H2PO4-,
H2PO42-, HSO4- and CH3.NH3+ are dissolved in water, they are incompletely
dissociated, that is to say both the acids and their conjugate bases are
present in the solution in similar concentration.
HA A- + H+
Where the change on the conjugate base, A- is one unit less positive than
that on the conjugate acid HA.
Acid dissociation constants
The law of mass action maybe applied to these equilibrium
KHA = [A-] [H+]
[HA]
Where KHA is the equilibrium or acid dissociation constant of the acid HA.
The constant KHA has the dimension of the concentration and also a
measure of the strength of the acid, the larger the value of KHA, the
stronger the acid. These acids are arranged according to their strength at
25oc.
H3PO4, K = 8.91 x 10-3, CH3COOH, K = 2.24 x 10-5, H2PO4-, K = 1.58 x
10-7, CH3.NH3+, K = 2.40 x10-11.
Measurement of pH
Measurement of pH is one of the most important and frequently used
analytical methods in biochemistry. This is so because, the pH determines
many important features of the structure and activity of bio-molecules,
consequently the behaviour of cells and organisms.
a) Hydrogen electrode: The primary standard for measurement of H+
concentration is the hydrogen electrode, a specially treated platinum
electrode that is immersed in the solution whose pH is to be determined.
The solution is in equilibrium with the gaseous hydrogen at a known
pressure and temperature. The electromotive force at the electrode
responds to the equilibrium
H2 2H+ + 2e-
The potential difference between the hydrogen electrode and a reference
electrode of known emf (calomel electrode) is measured and used to
calculate the H+ ion concentration.
The hydrogen electrode has been replaced with glass electrode
because of it’s cumbersomeness in use.
b) Glass electrode: If a thin bulb of a special glass is placed in a
solution it acquires a potential which depends on the pH in the same way
as does that of a hydrogen electrode. In order to measure the potential of
the glass membrane, it is necessary to have a reference electrode
(generally Ag.AgclHcl) inside the glass bulb as well as a reference
electrode connected to the test solution by a salt bridge. The potential
difference between the two reference electrode is given by the equation.
E = E’ + 2.303RT x pH
F
Where R is the gas constant. F the Faraday, T the Kelvin temperature and
E’ is a constant for the system.
In practice, it is always necessary to measure the potential of the
glass electrode system in standard buffer of known pH and then in the test
solution. If Es is the potential of the electrode system in a standard buffer
pHs, then the pH of the test solution pHs is given by
pHx = pHs + (Ex – Es) F
2.303Rt
The potential of the hydrogen saturated calome electrode system can be
measured with an ordinary potentiometer whereas the glass electrode
system which has no high resistance can be measured with a high input
impedance voltmeter usually arranged as a pH meter.
Precaution. Calibration measurement in a buffer of known pH must always
be made before measuring the pH of the test solution.
4.1.4 Buffer: System
A buffer solution is one that is capable of resisting a change in pH on
the addition of acid or alkali. It usually consists of a mixture of a weak
Bronsted pH acid and it’s conjugate base e.g. acetic acid and sodium
acetate or a weak base with it’s conjugate acid. Example ammonium
hydroxide and ammonium chloride.
The buffer solutions that give the best buffer of pH range of 4 – 10
have these properties in common.
1. The mixture with [base]/[acid] ratio of 1, is optimally buffered against
both strong acid and strong base and it’s pH equals the pka of the
acid component.
2. The mixture with [base]/[acid] ratios between 0.1m and 1.0m are
significantly buffering and their pH will fall within 1 unit of the pka
value of their acid components.
3. The pH of any mixture of this type can be calculated by applying the
Henderson-Hassel balch equation.
pH = PKa + log[base] , where pka is the value of its acid components
[acid]
Exercise
(a) Calculate the pH of a buffer solution which is 0.05m in
sodium acetate and 0.1m in acetic acid. The pka fro acetic
acid is 4.73.
(b) If the solution contained 0.1m sodium acetate instead, what
is it’s pH?
Solution
(a) pH = pka + log[salt]
[acid]
pH = 4.73 + log(0.05)
(0.1)
= 4.73 + log0.5
= 4.73 +Ī.6990
= 4.43
(b) pH = 4.73 + log(0.1)
(0.1)
= 4.73 + log1
= 4.73 + 0
= 4.73
Note: pka is the pH of the solution in which the ratio of the concentration of
the conjugate base and that of the weak acid is unity.
[A-] = [HA] i.e when it has been half converted to its salt.
Some commonly used laboratory buffers are

Compounds pka1 pka2 pka3 pka4
N-(2-acetamido)
Iminodiacetic acid (ADA) 6.6
Acetic acid 4.7
Ammonium chloride 9.3
Carbonic acid 6.1 10.30
Citric acid 3.1 4.7 5.4
Diethanolamine 8.9
Ethanolamine 9.5
Fumaric acid 3.0 4.5
Glycine 2.3 9.6
Phosphoric acid 2.1 7.2 12.3
Pyrophosphoric acid 0.9 2.0 6.7 9.4
Triethanolamine 7.8
Tris(hydroxymethyl)
Amino methane 7.8
W-Tris(hydromethyl)Methyl-
2-amino ethanesulfonic acid 7.5

Physiological buffers
The buffers important invivo are those which are effective around pH
7.4, the pH of blood. The pH of urine, however can vary between 4 and 9
bicarbonate. The pk1 of carbonic acid is 6.1. The ratio of base/acid at pH
7.4 is therefore 2o:1, which means that the bicarbonate system is a good
buffer when blood is being acidified, but very poor if it is being made
alkaline. The concentration of HCO3- ions in plasma is about 0.03m.
bicarbonate is also useful in buffering urine phosphate. The pka of the
equilibrium H2PO4 H2PO42- is 6.8 i.e. the ratio [H2PO42-]/[ H2PO4-] in
plasma is 4.1. This makes phosphate a more efficient buffer than
bicarbonate at physiological pH but it’s concentration in plasma is only
0.0002m.
In cells, the various phosphate esters which have important buffers
is the chief buffer in urine Amino acid. Most of these compounds are
dibasic i.e. in going from pH1- pH10, they lose two protons. The pks of the
COOH and NH3+ groups are not important except in buffering the Hcl
released in the gastric juice. The free amino acid is also small.
Proteins as pH buffers
In addition to the specialized functions of proteins, they contribute to
the general buffer capacity of the cellular content by the virtue of their high
content of weakly acidic and basic groups. Hemoglobin provides a good
example of a protein of a specialist function, undertaking the role of an
efficient pH buffer in an unusual manner.
The occurrence of O2 – consuming and CO2 – releasing cellular
respiration in tissues of the body far from the lungs, require that oxygen be
transported in the arterial blood supplied to these tissues and that carbon
dioxide be carried from the tissues to the lungs in the renous blood. In
man, arterial oxygen transport is accomplished by the combination of
hemoglobin with oxygen at the lungs to form oxy-hemoglobin. Arriving at
the respiring tissue, the oxy-hemoglobin delivers up it’s oxygen and reverts
to hemoglobin.
The problem of carbon dioxide transport appears less formidable
since carbon dioxide is much more soluble in aqueous media than is
oxygen. Erythrocytes are known to contain enzyme carbonic anhydrase
which promotes the rapid reaction of carbon dioxide with water to form
carbonic acid. At the pH of blood (7.4) carbonic acid will dissociate 96% into
H+ and bicarbonate ions.
CO2 CO2 + H2O H2CO3 H+ + HCO3-
Respiring tissue lungs
Thus the carriage of considerable amount of carbon dioxide in venous
blood would tend to decrease its pH. The problem becomes more evident
from the fact that quantity of carbon dioxide equivalent to between 20 and
40dm3 of 1moldm-3 monobasic acid is excreted via the lungs of man in one
day.
The fact that, the pH of the CO2- depleted arterial blood supports the
presence in the blood, concentration of bones. Sufficient to associate with
the H+ ions formed in equivalent concentration to the HCO3- ions.
Although a portion of the buffer base required for CO2 transport at pH
7.4 is supplied by plasmaphospahtes and plasma protein (as HPO 42- and
Pr-) more than three quarters is provided by haemoglobin. This reaction is
best expressed in terms of the pka values.
HHbO2 H+ + HbO2- pka = 6.62
HHb H+ + Hb-, pka = 8.18
From the pka values, at normal pH of blood (7.4), only 14% of
oxyhaemoglobin will be in its undissociated state HHbO2 but 85% of Hb will
be present in this condition (HHb). Thus at pH 7.4 oxyhaemoglobin loses
it’s oxygen and becomes converted inot haemoglobin, a quantity of H+
must be taken up
(H+)
HHbO2

(predominantly HbO2-) pH 7.4 predominantly HHb)

The O2 – consuming tissue produces CO2 which causes H+ ion to be
liberated simultaneously obtaining its oxygen from oxyhaemoglobin, it
forms sufficient base (Hb-) to associate with the majority of these H+ ions.
This phenomenon is known as isolydric exchange which differs from
normal pH buffering that relies on the buffering capacity of a single
conjugate pairof one pka value.
Exercise II: Calculate, the buffer capacity of a buffer solution containing
10ml of 0.1m sodum acetate and 10ml of 1.0m acetic acid, when 1 ml of
0.1m Hcl was added to it.
Solution
Iml of 0.1m Hcl = 0.1 x 10-3 mole Hcl.
Initial conc. of acetate ion = (0.1 x 10-3 x 10) moles acetate ion.
Initial conc. of acetic acid = (0.1 x 10-3 x 10) moles acetic acid ions.
Final conc. of acetate ion = (1 x 10-3 – 0.1 x 10-3) moles acetate acid ion
= 0.9 x 10-3 moles acetate ion.
Final conc. of acetic acid = (1 x 10-3 – 0.1 x 10-3) moles acetic acid ion
= 1.1 x 10-3 mole acetic acid
Total volume of solution = 21ml
Total conc. = (0.9/21 x 10-3 x 103) moles acetate/litres
and (1.1/21 x 10-3 x 103) moles acetic acid/litre
pH = pka + log[salt]/[acid]

Final pH of solution = 4.73 + log[0.9/21]
[1.1/21]
= 4.73 + log[0.9]
[1.1]
= 4.73 + log0.9 – log1.1
= 4.73 + Ī.9542 – 0.0414
= 4.73 – 1.0 + 0.9542 – 0.0414
= 4.6428
pH of the buffer before addition of 1ml of 0.1m Hcl
= 4,73 [CH3COO-] = [CH3COOH]
= pH change = 4.73 – 4.64
= 0.087 ~ 0.09

5.0 CARBOHYDRATES
Carbohydrates are polyhydroxy aldehydes or ketones or substances
that yield such compounds on hydrolysis. The name carbohydrate
originated from the fact that most substances of this class have empirical
formulas suggesting they are carbon ‘hydrate,’ in which the ratio of C:H:O
is 1:2:1. Example, the empirical formula of D-glucose is C6H12O6 which can
be written as (CH2O)6 or C6(H2O)6. Many carbohydrate conform to this
formula, while yet some don’t, but contain Nitrogen, phosphorous or sulfur.
1. CLASSIFICATION
Carbohydrates are polyhydroxyaldehydes or ketones or substances
that yield such compounds on hydrolysis. The name carbohydrate
originated from the fact that most substances of this class have empirical
formulas suggesting they are carbon ‘hydrate’ in which the ratio of C:H:O is
1:2:1. Example, the empirical formula of D-glucose is C6H12O6 which can
be written as (CH2O)6 or C6(H2O)6.Many carbohydrate conform to this
formula, while yet some don’t, but contain nitrogen, phosphorous or sulfur.

1. CLASSIFICATION
There are three major classes of carbohydrates namely –
Monosaccharide, Disaccharides and Polysaccharides. The word
saccharide comes from a Greek word for sugar.
2. Monosaccharide or Simple sugar consists of a single polyhydroxy
aldehyde or ketone unit. The most abundant monosaccharide in nature is
the 6-carbon sugar D-glucose. They are colourless crystalline solid, soluble
in water, but insoluble in non-polar solvents. Most monosaccharides have
sweet taste and general formula (CH2O)n, where n = 3 or some larger
number.
The backnone of monosaccharide is an unbranched single bonded
carbon atom. One of the carbon atom forms a carbonyl group by double
bonding with one atom of oxygen, while the other atom has hydroxyl
group. If the carbonyl is at the end of the carbon chain, the
monosaccharide is an aldehyde and called ALDOSE. However if the
carbonyl is at any other position, the monosaccharide is ketone and called
KETOSE.
The simplest monosaccarides are the two 3-carbon trioses
glyceraldehydes an aldose and dihydroxy acetine a ketose.
Monosaccharides with 4,5,6 and 7 carbon atoms in their backbones are
called tetrose, pentose, hexoses and heptoses respectively. Each of these
exists in two series aldotetrose and keto tetrose etc.
The hexoses, which include the aldohexose D-glucose and the
ketohexoses D-fructose are the most abundant monosaccharide in nature.
The aldopentoses D-ribose and 2-deoxy-D-ribose are components of
nucleic acids.

Two triose
H H
C=O H C OH
H C OH C=O
H C OH H C OH
H H
Glyceraldehyde an aldose Dihydroxyacetone, a ketose

Two common hexoses

H H
C=O H C OH
H C OH C=O
HO C H HO C H
H C OH H C OH
H C OH H C OH

CH2 OH CH2 OH
D-glucose an aldohexose D-fructose a keto hexose
The pentose components of nucleic acid
H H
C=O C=O
H C OH CH2
H C OH H C OH
H C OH H C OH
CH2 OH CH2 OH
D-Ribose, the sugar component D-Deoxy-D-ribose, the sugar
Of ribonucleic acid (RNA) component of deoxyribonucleic
Acid (DNA)
And various polysaccharide derivatives of trioses and heptoses are
important intermediate in carbohydrate metabolism.
3. DISACCHARIDES
This consists of two short chains of monosaccharide units joined
together by covalent bond e.g. sucrose or cane sugar, which consist of 6-
carbon sugars D-glucose and D-fructose joined in covalent bond.
CH2OH OH H
O H CH2OH
H H H OH
OH OH O H
H OH CH2OH O
α-D-Glucose unit α-D-fructose unit
Maltose is another example of disaccharide that consists of two molecules
of D-glucose joined by a glycoside bond between carbon 1 of one glucose
and carbon 4 of the second glucose.

CH2OH CH2OH
O O
OH
OH OH O OH
OH OH
α-D-Glucose unit β-D-fructose unit

4. POLYSACCHARIDES
These consist of long chains having hundreds or thousands of
monosaccharide units. Some polysaccharides such as cellulose has linear
chain whereas others such as glycogen has branched chain. The most
abundant polysaccharides are starch and cellulose which consist of
reoccurring units of D-glucose but differ on the position of the linkage
polysaccharides differ generally in the nature of their reoccurring
monomeric units, while the hetro polysaccharide consists of alternating
residue of D-glucoronic and N.acetyl-D-glucosamine. Thus
polysaccharides generally have two major functions :– storage structural
facilities.

(a) Storage: Stoarge of glucose in plants and animals is mainly in the
form of starch and glycogen respectively. Starch exists as α-amylose which
consists of long unbranched chain of D-glucose units and are bounded by
α(1 – 4) linkage as in maltose. It’s molecular weight is between (1,000 –
500,000) and are not very soluble in water. It gives a blueblack coloration
with iodine solution.

Other forms of starch are amylopectin, a highly branched starch.
The branches are about 12-glucose residue long occur at an average of
every 12 glucose residues. The backbone glycosidic linkage is α(1 -4) and
the branched parts are at α(1 – 6) linkage. Amylopectin yields a colloidal,
which gives a red and violet colour with iodine solution. It has a molecular
weight of about 100 million. The partial breakdown products of amylopectin
are large molecules called the dextrins; used to prepare mucilage, paste
and fabric sizes. The major components of starch can be enzymatically
hydrolysed into different ways.

Amylase can be hydrolysed by α-amylase (α(1 – 4)-glucn-4-
glucanohydrolase) which is found in saliva and pancreatic juice and in
digestion of starch. The enzyme α-amylase hydrolyses α1 – 4 linkage to
give a mixture of glucose and free maltose. Also the amylopectin is
attacked by α and β-amylases. The resulting polysaccharide of
intermediate chain length that are formed from starch component by the
action of amylase are referred to as dextrin. Neither α nor β amylase can
hydrolyses the α1 – 6 linkage at the branch point of amylopectin glycogen.
Liver and muscle tissue are the main sites of glycogen production and
storage in the body. The enzyme regulate glycogen to ensure a steady
supply for the body chemical energy. Glycogen differs from starch by the
absence of any molecule of unbranched amylose. It is however more
branched than the amylopectin. It’s molecular weight ranges from 300,000
– 100,000,00 corresponding to about 1,800 – 60,000 glucose units the
branches occur at the 8th – a0th of glucose residue.

(b) Structural polysaccharides

Many polysaccharides serve primarily as structural elements in
cell walls and coats intercellular spaces and connective tissue where they
give shape, elasticity or rigidity to plant and animal tissues as well as
protection and support to unicellular organisms. It is also a major organic
compounds found in the exoskeleton of insects and crustacea.

Plant Cell Walls. For plants to withstand the large osmotic-pressure
difference between extracellular and intracellular fluid, they require rigid
cell walls, to keep them from swelling. In large plants, the cell walls help in
maintaining physical strength, rigidity to stems, leaves and sustain weights.
The most abundant cell wall and structural polysaccharide in the plant
world is the cellulose, a linear polymer of D-glucose in β(1 – 4) linkage.
The methylation of cellulose not only indicates the linkage but also thwe
unbranching of cellulose. The only chemical difference between starch and
cellulose is that while starch is α(1 – 4) linkage, cellulose is β(1 – 4)
linkage. It is not attacked by either α or β-amylase like starch. It can only
be hydrolyzed by cellulose an enzyme found in ruminants which has
molecular weight of about 50,000 – 2,500,000. It is insoluble in water and
equivalent to 300 – 15,000 glucose units.

5. ASYMMERIC CARBON CENTERS OF MONOSACCHARIDES

All the monosaccharide except dihydroxy acetone contain one or
more asymmetric or crucial carbon atoms and thus occurs in optically
active isomeric forms. The simpliest aldose glyceraldehydes, contain only
onechiral centre and thus is capable of existing as two different optical
isomers that are non-super-imposable mirror images of each other. D-
glucose, the common form of glucose in nature is dextrorotatory, with a
specific rotation of [α]D20 = +52.7o, while D-fructose, the common form of
fructose is levorotatary [α]D20 = -92.4o. both sugars are of D-series, since
their absolute configuration is related to the D-glycerldelhyde and for those
sugars having two or more asymmetric carbon, a convention has been
adopted that the prefixes D and L refers to asymmetric carbon atom
fartherest removed from the carbonyl carbon atom. Aldoses and ketones of
the L series are the mirror images of their D-counter parts.

Two sugars which differ in configuration around one specific
carbon atom are called epimers, e.g. manose is the epimers of glucose,
while glucose is the epimer of gulactose with respect to carbons 2 and 4
respectively.
CHO CHO
H C OH HO C H
HO C H HO C H
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
D-glucose D-Mannose

D-Glucose and D-Mannose epimers at C2

CHO CHO
H C OH H C OH
HO C H HO C H
H C OH H C OH
H C OH CH2OH
CH2OH
D-glucose
D-Glucose and D-Galactose epimers at C4
6 RING FORMS OF COMMON MONOSACCHARIDES
Many monosaccharide behave in aqueous solution as though they
possess asymmetrical coutre, than is given by open chain structural
formula. The open chain linear structure points out several important
character. Firstly, the asymmetric carbon atom is clearly shown and
secondly steroisomers which result because of asymmetric carbon atom
are more apparent when linear form is drawn.
D-glucose and D-fructose in solution are not the open chain
structure predominantly, rather the open chain forms of glucose and
fructose can cyclic to form Hemiacetal. D-glucose may exist in two different
isomeric forms as α-D-glucose whose angle of rotation [α]D20 = 112.2oc and
β-D-glucose whose [α]D20 -+ 18.7oc.
Both have been isolated in pure form and don’t differ in elementary
composition. When the α and β glucose are dissolved in water, however,
their optical rotation changes with times and approaches [α]D20 + 52.7o in
their mixture. This change is called mutarolation. It is due to the formation
of equilibrium mixture of about 1/3 α-D-glucose and 2/3 of β-D-glucose and
a very small amount of straight chain compound at 25oc. The α and β
isomers of D-glucose are interconvertible in aqueous solution. From
various chemical consideration it has been deduced that the α and β
isomers of glucose are not open chain structure but rather six ringed
structure, which have been formed by the reaction of alcoholic hydroxyl
gropu at C5 with carbonyl group at C1. Such 6-member ring form of sugar
are called PYRANOSE. Pyranose, because they are derived from
hetrocyclic compound pyran, so that the systematic name for the ring form
of α-D-glucose pyranose.
The reaction between aldehyde and alchohol results to the formation
of hemiacetal.
OR’
R C=O + OH R’ R C OH
H H
Aldehyde Alchohol hemi acetal

O R
R C R + OH R’ R C OR’
H OH
Ketone Alchohol hemi ketal

Consider a sugar like glucose in which the carbon one (C1) has an
aldehyde functional group and the c5 with alcohol functional group. The
carbon (C1) reacts with carbon (C5) to give a ring-like compound called
hemiacetal.
6
CH2OH
1
H C O
2
H C OH H H5 OH
3
HO C H O OH4 1
H
4
H C OH OH H2
5
H C OH H3 OH
β-D-glucopyranose
6
CH2OH

CH2OH
O
H H H
OH OH
OH H
H OH
α-D-glucopyranose
When the OH is at the top of the anomeric C, the sugar is β while at the
bottom, the sugar is α-sugar. The anomeric structure is known and named
after a reference pyran α-D-glucopyranoside.
The position of the OH group at the anomeric carbon atom
determines the α or β name.
Consider another sugar fructose in which the carbon (C2) has a
ketone functional group and carbon (C5) has alcohol functional group. The
ketone group of carbon (C2) reacts with the alcohol group at carbon (C5) to
form four member compound called hemiketal.
CH2OH O CH2OH
HO C CH2OH β-D-fructofuranose
H C H H H

H C OH O H HO

H C OH H

CH2OH α-D-fructofuranoside
Similarly, when the OH is at the top of the anomeric C the sugar is
β while at the bottom it is known as α-sugar. The anomeric is named after
a reference Furan α-D-fructofuranoside.

Sugar can be represented as because

CH2OH
O
H H H
OH OH anomeric carbon atom
OH H
H OH

Of the internal hemiactal reaction between carbon
(C1) and carbon (C5), glucose gives a ring-like structure compound form
during the reaction.

CONFORMATION. Most sugars can be represented by Haworth projection
formular either in chair conformation or boat form.

O O

Boat

Chair
The chair formation is more stable than the boat because of the separation
of substituents elements.

6.0 CHEMICAL REACTION
Monosaccharides are stable in all hot dilute mineral acids (Hcl,
H2SO4,HNO3) as a result of the hydrolysis of monosaccharide, there is a
quantitative recovery of most monosaccharide present.
Conc. acid dehydrates sugar to give furfurals
CH3 O CH2OH
Glucose H+
Conc
5-hydroxy methyl furfural

Furfurals are derivatives of furan and condenses with phenol to give
characteristic colour product which are often used for colorimetric analysis
of sugar.
Dilute base. Dilute aqueous base at room temperature causes
rearrangement about the anomeric carbon atom and it’s adjacent carbon
atom without affecting substituent at other carbon atom. E.g. treatment of
D-glucose with dilute alkaline yields an equilibrium mixture of D-glucose
and D-manose. OC CH2OH
H C=O C OH C=O
H C OH OH- HO C H HO C H
HO C H H C OH H C OH
H C OH H C OH H C OH
H C OH H C OH CH2OH
CH2OH CH2OH
D-glucose Trans enediol D-fructose
HO C H H C=O
HO C HO C H
HO C H HO C H
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
Cis enediol D-manose
Derivative of Manose
Aldopyranoses readily react with alcohol in the presence of mineral
acid to form anomeric α and β glucosides.

CH2OH CH2OH
O O
+
H H H H H H H

OH OH ROH OH
OH H OH H OR
H OH
H OH
Glocoside
Glucosides are asymmetric mixed acetal. Also the glucosidic linkage
is also formed by the reaction of a monosaccharide with the OH group of
another monosaccaride. In this way disaccharides are formed that are
linked by glucosidic chain.
CH2OH CH2OH
O O
H H H H H H

OH OH + OH
OH H OH H OH

H OH H OH
CH2OH CH2OH
O O
H H H H H H

OH
OH H OH H OH
O
H OH H OH
+ H2O
Glycosidic bond
Oligosaccharides and polysaccharides are chains of
monosaccharides joined by glycosidic linkages. The glycosidic linkage is
stable to bases but hydrolysed by boiling with dilute acid to yield free
monosaccharide and free alcohol. Glycosides are also hydrolysed by
enzymes called glycosidases.
D-Acyl derivatives
The free hydroxyl groups of monosaccharide and polysaccharide
can be acylated to yield D-acyl derivatives, which are very useful for
structure determination of monosaccharide. If α-D-glucose is treated with
excess acetic anhydride yield penta-o-acetyl α-D-glucose.

CH2OCOCH3
O
H H H
OCOH3
CH3COOOCOCH3H
H OCOCH3
Penta-O-acetyl α-D-glucose
Osazones
Sugar reacts with amines e.g. the reaction with excess
phenylhydrazine to form osazone a crystalline compound. The reaction
can be utilized to determine the configuration of sugar e.g. glucose,
fructose and manose to form osazone of the same shape, this indicates
the configuration of these sugars about carbon 3, 4, and 5 must be
identical sugar alcohol.
H C=O H C=N NH C6 H5
H C OH C=N NH C6 H5
HO C H + 3C6H5NHNH2 HO C H
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
Glucose
Sugar alcohol Phenyl osazone

Monosaccharide can be reduced under mild condition with sodium
borohydride to give polyhydric alcohol. In this reaction, the aldehyde or
ketone function is reduced to the alcohol. For instance glucose is reduced
by sodium borohydride to give glucitol.

H C O CH2OH
H C OH H C OH
HO C H NaBH4 H C OH
H C OH HO C H
H C OH HO C H
CH2OH CH2OH
Glucose D-glucitol

H C=O CH2OH
HO C H HO C H
HO C H NaBH4 HO C H
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
Mannose D-Mannitol
These sugar alcohols are called pentisole when 5-carbon atom is present
in a molecule and hexitol when 6-carbon. Closely related to this substance
is cyclic hexatol e.g. inositol. It is found widely distributed in living
organism.
OH OH
OH

OH OH Inositol
Sugar Acids
OH
Monosaccharide undergo a variety of oxidation reaction to form sugar
acids. Among these are three important types, aldonic, aldaric and uronic
(a) Treatment of aldose with a mild oxidizing agent such as bromine
water, convert the aldehydric function at G to carboxylic group and
an aldonic acid formed. D-glucose yields gluconic acid.

H C=O COOH
H C OH H C OH
HO C H Br2/H2O HO C H
H C OH H C OH
H C OH H C OH
CH2OH CH2OH
Glucose Gluconic acid
(b) The use of stinger oxidizing agent e.g.dilute HNO3 will induce the
oxidation of both C1 and primary C6 alcohol group to give aldaric
acid.
D-glucose yields D-glucaric acid

H C=O COOH
H C OH H C OH
HO C H HNO3 HO C H
H C OH H C OH
H C OH H C OH
CH2OH COOH
D-Glucose Glucaric acid
(c) Selective oxidation of primary alcohol yields uronic acids only the
carbon atom bearing the primary alcohol group is oxidized to a
carboxyl group. D-glucose yields D-glucoronic acid. Both uronic and
aldonic acids occur in nature especially as intermediate of
carbohydrates metabolism. Aldonic is a constituent of
polysaccharide e.g. vitamin C.

H C=O O=C
H C OH OH C O
HO C H HO C
H C OH H C
H C OH HO CH
COOH CH2OH
D-glucuronic acid L-Ascorbic acid
The monosaccharides are also found in cell where they are also found in
cell where they are esterified with phosphoric acid e.g. glucose-1-
phosphate or glucose-6-phosphate.
O
CH2O P OH
O- O
H glucose-6-phosphate or glucose-1-
O phosphate, if the phosphate moves
OH OH O P OH from 6 to 1.

OH O-

Fructose sugar esterifies with phosphate to form Fructose-1-phosphate
and Fructose-1-6 diphosphate respectively.
Deoxy Sugar
They are monosaccharide that lack oxygen atom in their molecule.
The most abundant deoxy sugars found in nature is 2-deoxy-D-ribose, the
sugar component of deoxyribonucleic acid.
CHO
CH2
H C OH 2-Deoxy D-ribose
H C OH
CH2OH
Amino Sugar
These are monosaccharide which at least one of the OH group is
replaced by amino group. Two of these sugars are of wide distribution
namely D-glucosamine and D-galactosamine.

CHO CHO
CHNH2 H C NH2
HO C H HO C H
H C OH HO C H
H C OH H C OH
CH2OH CH2OH
D-glucosamine D-galactosamine
In both sugars, they have OH- group at C2 replaced by an amino
group. D-glucosamine occurs in polysaccharide of vertebrate tissues also
a major component of chitin, a structural polysaccharide found in
exoskeleton of insect and crustacean.
D-glactosamine is a component of glycolipids and of the major
polysaccharide of cartilage.
REACTION WITH HYDROGENCYANIDE (HCN)
Monosaccharide react with HCN to give the hydrocyanide of the
compound. This is an important method of extending the carbon chain of
sugar.

H C=O CN
H C H H C OH
HO C H + HCN HO C H
H C OH HO C H
H C OH H C OH
CH2OH H C OH
CH2OH
MODEL QUESTIONS
1. Describe the structural and storage polysaccharide
2. Describe the formation of sugar alcohols and acids.
3. Describe the