Sunny Article 3

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 membraneenclosed 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 ribosones

cofactor 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 manmade 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 coenzyme, 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

phosphoshydrolase

and

carboxypeptidase (4) Magnesium

(ii)

ion

Mg++

-

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)

Rate of reaction

mixture of zero and 1st order kinetics

V/2

Zero order kinetics phase II

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 reaction

(a) Increasing

optimum temperature

rate

(a&b)

(a)

Temperature 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 -

18

CH3(CH2)4(CH=CHCH2)2(CH2)6COOH linoleic acid -

18

CH3CH2(CH=CHCH2)3(CH2)6COOH

20

16 5

linolenic acid - 11

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

CH2OH

CH CH2

Glycerol

fatty acid

O

C O

R2

O C R3

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 CH2

O

C

O R1

CH2

O

C

R1 O

CH

OH

CH

O

C

R2

CH

OH

Monoglyceride

CH2

OH

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. 2oledipalmitic 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

O RII

C

O OCH + 3H2O

Enzymes

O RIII C

CH2

3R

C

OH + HO

CH

Lipase OCH2

HO

Fatty acid (b)

CH2

glycerol With alkali

Strong base produces glycerol and salt of fatty acid on hydrolysis. This reaction is known as saponification. O RI

C

OCH2

HO

O RII

C

CH2

O OCH + 3 NaOH

3R

C

O Na+

+HO

CH

O RIII C

OCH2

Fatty acid

3.

HO Salt

CH2

glycerol

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

RI

C O

CH

+

OH

esterified

CH2

O

O CH2

P

RII

C O

O

H

CH2

OH

O

P

O

RIII

OH

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

CH2 CH2

CH

NH2

COOH CH COOH

RIII containing nitrogen atom.

NH2 Serine CH2

O

O C

RI

O CH

O

RII

C

NH2

O CH2

O

P

O

CH2

OH HO

CH2

CH2

O

CH2 O C

CH

COOH

Phosphatidyl serine NH2

RI

ethano alanine

O CH

O

RII

C O

CH2

O

P

O

CH2

OH

CH2

O

NH2

Phosphatidyl ethanoalanine

HOCH2CH2NHCH3 O C

CH2

N- methyl ethanoalanine RI

O CH

O

RII

C O

CH2

O

P

O(CH2)2.NHCH3

OH

Phosphatidyl N. methyl ethanoalanine

HOCH2CH3N+(CH3)2 Choline

CH2

O

O C

RI

O CH

O

RII

C O

CH2

O

P

O(CH2)2. N+(CH3)3

OH

Phosphatidyl Choline

Compound without N RIII= H or HOCH2CHOHCH2OH

CH2

O

O C O

RI

CH

O

RII

C O

CH2

O

P

O

OH

CH2CHOHCH2OH Phosphatidyl glycerol

RIII may be equal to inositol OH OH

OH

OH OH

OH Inositol

CH2

O

O C

RI

O CH

O

RII

C

OH

O CH2

O

P

OH

OH

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

HO

C

O

H

O P

OH

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.

CH2

O C

O

RI

O CH

O

C

non-polar chain 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–3phosphorylcholine. 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 Ball and Stick model

NH2 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

C

CH3 COO-

H CH

C

CH3 COO-

H CH CH

C COO-

+NH3 Alanine CH3-CH2

CH3

+NH3

H CH

CH3 Leucine

Valine

C

H2 C COO-

H2 O

CH3 +NH3 Isoleucine

+NH3

CH2 + N H2

CH

COO-

Proline

H CH3

S

CH2 CH2

C COO-

+NH3 Methionine Aromatic

H 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

+NH3

COO-

+NH3

Glycine

CH HO

NH3

Serine

Threonine

H HS CH2

COO-

C

H COO-

C

NH2 C CH2 CH2 ‫װ‬ O

+NH3 Cysteine Sulfhydryl

H CH2 C COO-

+NH3 Glutamine

NH2 C CH2 O

+NH3

COO-

C

H C

COO-

+NH3 Asparagine

Tyrosine 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 +NH3 CH2 CH2 CH2

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

Lysine

+NH2

+NH3 Arginine

H HC

C CH2 C COO-

HN+

NH

+NH3 C

H COO-

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 -

OOC CH2

C

H COO-

+NH3 Aspartic acid 3.

-

OOC CH2

CH2

C

COO-

+NH3 Glutamic acid

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 pH 8

pk2=9.7 [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. R

CH

The carboxyl group may be esterified with alcohols. COO- + C2H5OH

H+

+NH3 2. R

CH

O R

CH

C

OC2H5 + H2O

+NH3 Converted into the corresponding acylchloride COO

-

PCl5 R POCl3

CH

COCL

+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

NH2

CO2-

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

+NH3 2.

COOH + N2 + H2O + H+

OH

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 NH2

COOH + oxidized ninhydrin 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

+ NH3 +

C

C C Oxidized ninhydrin

OH

H

C O Reduced ninhydrin

O C

O 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

R NO2

F

NO2

N

COOH

H

R

+ HF

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

COOH Glycine

H HO OC CH2 NH2 glycine

CH2

N C =O

+H2O

COOH CH2 NH2 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 N+ H3 (a) OHC

O

O H

(b)

CH2OH

H

(c) CH2OH

Key: (a) Electrostatic interaction (b) Hydrogen bonding

(d)

(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 Inslin

Mol wt. 11,466

Number of Subunit Subunits

Mol. Wt.

2

5,733

Biological functions a hormone regulating Glucose metabolism

Hemo-

64,500

4

16,100

globin Alcohol

Oxygen transport in blood plasma

80,000

4

20,000

dehydro-

an enzyme of alcohol fermentation

genase Iaetate dehydrogenase

134,000

4

33,500

an enzyme of anaerobic glycolysis

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.

4.

(b)

Describe the titration curve of any amino acids.

(a)

Give four biological functions of protein

(b)

Describe the reaction of amino acid with (a)

4.0

alcohols

(b)

acylchloride

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. Biomolecules 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 2e

H

N

105o H Tetrahedral Structure Of water molecule 2.

H H Tetrahedral Structure of ammonia

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∂-

H∂+ 3.

O∂-

H∂+

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 kcalmolfor 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∂+

O

H

O

H + OH-

H

H H3O∂+ + OH-

2H2O

Two ions are produced, the hydronium (H3O+) and hydroxide (OH-) ions. The pH Scale: The dissociation of water is an equilibrium process H+ + OH-

H2 O

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 H+

H2 O

+ 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]

[H2O]

at 25oc

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 OHconcentration 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 OHpH = 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 B + H+

A

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. H + cl+

Hcl CH3COOH H2PO4 NH4+

H+ + CH3COOH+ + HPO42-

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

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

Triethanolamine

7.8

Tris(hydroxymethyl)

5.4

9.4

Amino methane

7.8

W-Tris(hydromethyl)Methyl2-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 plasma is 4.1. This

H2PO42- is 6.8 i.e. the ratio [H2PO42-]/[ H2PO4-] in 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 HHb

H+ + HbO2- pka = 6.62 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

H

C

OH

H

C

OH

C

OH

C=O H

H

C

OH

H

Glyceraldehyde an aldose

Dihydroxyacetone, a ketose

Two common hexoses H

H

C=O

H

C

OH

H

C

OH

HO

C

H

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

OH

CH2 OH D-glucose an aldohexose

C=O

CH2 OH D-fructose a keto hexose

The pentose components of nucleic acid

H

H

H

C=O

C=O

C

OH

CH2

H

C

OH

H

C

OH

H H

C

OH

C

OH

CH2 OH D-Ribose, the sugar component

CH2 OH 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 6carbon sugars D-glucose and D-fructose joined in covalent bond. CH2OH

OH O

H

H H

OH OH

H CH2OH

H

OH

O

H OH α-D-Glucose unit

H CH2OH O 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 α-D-Glucose unit 4.

POLYSACCHARIDES

OH OH β-D-fructose unit

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-4glucanohydrolase) 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. Dglucose, 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

CH2OH D-glucose

H

C

OH

CH2OH D-Mannose

D-Glucose and D-Mannose epimers at C2

CHO

CHO

H

C

OH

HO

C

H

H

C

OH

H

C

OH

H

C

OH

HO

C

H

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

H

OH

H

Aldehyde

Alchohol

hemi acetal

O R

C

R

C

R + OH

H

R’

R

C

OR’

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

H

1

C

CH2OH O

H

2

C

OH

HO

3

C

H

H

4

C

OH

5

H

6

C

H5

H O

OH

OH4

1

OH H3

OH

H

H2 OH β-D-glucopyranose

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 HO

C

H

C

H

H

C

OH O

H

C

CH2OH

CH2OH β-D-fructofuranose

CH2OH

H

H H OH

HO H

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

Of

OH OH

H

H

OH

the

internal

anomeric carbon atom

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

H C=O

C

H

C

OH

HO

C

H H

HO

C

H

H

C

C

OH

H

C

C

OH

H

C

CH2OH D-glucose HO HO

OH-

C

CH2OH OH H

C

H HO

OH OH

C=O C

H

HO

C

H

HO

C

H

H

C

OH

H

C

OH

H

C

OH

H

C

OH

CH2OH

CH2OH

HO C

OH

CH2OH Trans enediol

H

C=O H H

C C

H OH OH

CH2OH D-fructose

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

H

H

O +

H

ROH

OH

H

OH

OH OH

H

H

OH

H

H

H

OH

H

H

OH

OR

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

H

H

O H

OH

OH

H +

H

H

OH

OH

H

OH

H

OH

H

H OH

OH

CH2OH

CH2OH O

H

H

O H

H

H

H

OH OH

H

H

OH

OH

H

OH

O 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

H

C

OH

HO

C

H

H

C

H

C

+ 3C6H5NHNH2

C=N

NH

C6 H5

C=N

NH

C6 H5

HO

C

H

OH

H

C

OH

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

HO

C

H

H

C

OH

H

C

OH

H NaBH4

H

OH

HO

C

H

HO

C

H

CH2OH D-glucitol

H C=O HO

C

OH

C

CH2OH Glucose

C

CH2OH H

HO

C

H

HO

C

H

H

C

OH

H

C

OH

NaBH4

HO H H

CH2OH

C

H

C

OH

C

OH

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

HO

C

H

H

C

OH

H

C

OH

CH2OH Glucose

H Br2/H2O

HO H H

C

OH

C

H

C

OH

C

OH

CH2OH 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

HO

C

H

H

C

OH

H

C

OH

H HNO3

HO H H

CH2OH

OH

C

H

C

OH

C

OH

COOH

D-Glucose (c)

C

Glucaric acid

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

HO

C

H

HO

C

H

C

OH

H

C

H

C

OH

HO

CH

COOH D-glucuronic acid

O

CH2OH 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-1phosphate or glucose-6-phosphate.

O CH2O P OH O-

O H

glucose-6-phosphate or glucose-1O

OH OH

O OH

P

phosphate, if the phosphate moves 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

H

C

OH

2-Deoxy D-ribose

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 HO

C

H

H

C

OH

H

C

NH2

HO

C

H

C

H

HO

H

C

OH

H

CH2OH

C

OH

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

HO

C

H

H

C

OH

H

C

OH

H

H

CH2OH

+

HCN

HO

C

OH

C

H

HO

C

H

H

C

OH

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