MOLECULAR ORGANIZATION

OF THE LIVING CELLS AND

SAMPLE QUESTIONSAs 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

x g

crude microsones cofactorribosones 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 kineticsRate of mixture of zero phase IIreaction V/2 and 1st order kinetics

1st order kineticsPhase I

KMSw

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)Thermaldenaturation

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( o c)

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

COOHHO 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 H2C CH2

CH C COO- H2O + 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 GlutamineSulfhydryl

H HCH2 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 +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

COO-

-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.

9 pk2=9.78 [H3N+ - CH2 – CO-

2] = 50 [H2N - CH2 – CO-

2] = 506

4 Isoelectric point [H3N+ - CH2 – CO-2] 100%

pH. 6.073

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

50 500

.5 1 1.5 2.0Moles 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 + pk2

1) 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.

10

pH

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

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

H+

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 OOxidized ninhydrin Reduced ninhydrin

O O C

C C N = C +3H2O

C O

C 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.

H2N 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

OH Blue product

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 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.

H HO

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 ON+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 HTetrahedral 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

H2O 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

H2O 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 pH 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 [H2PO4

2-]/[ 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- pH

10, 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 HPO42- 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 OHD-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 OHD-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.

6CH2OH

H 1C O

H 2C OH H H5 OH

HO 3C H O OH4 1 H

H 4C OH OH H2

H 5C OH H3 OH β-D-glucopyranose 6CH2OH

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

ChairThe 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 CH2OHCis 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 C6H5

H C OH C=N NH C6H5

HO C H + 3C6H5NHNH2 HO C H

H C OH H C OH

H C OH H C OH

CH2OH CH2OH

Sugar alcohol

Monosaccharide can be reduced under mild condition with sodium

borohydride to give polyhydric alcohol. In this reaction, the aldehyde or

Phenyl osazone Glucose

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

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

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

Monosaccharide undergo a variety of oxidation reaction to form sugar

acids. Among these are three important types, aldonic, aldaric and uronic

D-glucitol Glucose

D-Mannitol Mannose

OH

(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

(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

(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.

Gluconic acid Glucose

Glucaric acid D-Glucose

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

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.

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

L-Ascorbic acid D-glucuronic acid

OH

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

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

D-galactosamine D-glucosamine

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