BIOMOLECULES PRACTICE PAPER

Carbohydrates (hydrates of carbon) are naturally occuring compounds having general fomula Cx(H2O)y,
which are constantly produced in nature & participate in many important bio-chemical reactions.
Ex. - Glucose C6H12O6 C6 (H2O)6
Fructose C6H12O6 C6 (H2O)6
Cellulose and Satrch (C6H10O5)n
Sucrose (Cane suger) - C12H22O11, and Maltose (Malt Suger) C12(H2O)11
But some compounds which have formula according to Cx(H2O)y are not known as carbohydrate
Ex. CH2O Formaldehyde
C2(H2O)2 Acetic acid
C3(H2O)3 lactic acid

There are many compounds, which shows chemical behaviour of carbohydrate but do not confirm the
general formula Cx(H2O)y such as - C5H10O4 (2–deoxyribose), C6H12O5 (Rahmnose)
C7H14O6 (Rahmnohexose)
Modern defination of carbohydrate: Carbohydrates are polyhydroxy aldehyde or ketone
or
Substances which yield these (polyhydroxy aldehyde
or ketone) on hydrolysis

Carbohydrates H/ H 2O
 Poly hydroxy aldehyde or ketone
Carbohydrates are also known as Saccharides.
Classification of carbohydrates :

Carbohydrates

Sugar Non Sugar (Polysacch.)

Sweet in taste, crystalline & soluble in water taste less, amorphous solid
(non crystalline) insoluble in
water
Monosaccharides Oligosaccharides These yields many sweet
monosacch. on acidic hydrolysis
Tetra (C6H12O5)n+nH2O  nC6H12O6
Aldose Ketose Disacch. Trisacch.
Sucrose, maltose Refinose
lactose

Monosaccharides : (simple sugars)

These are the sugars which cannot be hydrolysed into smaller molecules. General formula is CnH2nOn .
Ex. - Glucose, Fructose, Ribose
Oligosaccharides :-
These are the sugars which yeilds 2–10 monosaccharides units on hydrolysis. such as.
(a) Disaccharides :- Two monosaccharide unit on hydrolysis (may or may not be same).
Ex. - Sucrose, Maltose
(b) Trisaccharides :- Three monosaccharide unit on hydrolysis.
Polysaccharides :- These are the non sugars which yeild a large no of monosaccharide units on
hydrolysis General formula - (C6H10O5)n. Ex.- Starch, Cellulose.
Note :- A group of polysaccharides which are not so widely used in nature is pentosans (C5H8O4)n
Monosaccharides, General formula Cx(H2O)y x = 3 – 8. Nomenclature of monosaccharides are given
according to the no. of carbons present in them.
If –CHO group is present in monosaccharide, then it is known as aldose.
If  C  group is present in monosaccharide , then it is known as ketose.
||
O

Aldoses Ketoses
3C Tropose or Triose Aldotriose Ketotriose
4C Tetrose Aldotetrose Ketotetrose
5C Pentose Aldopentose Ketopentose
H C O

5C including –CHO Aldopentose (Ribose) H—C—OH

H—C—OH
H—C—OH
CH2 OH

5C including  C  Ketopentose (Ributose)

||
O

6C Hexose Aldohexose Ketohexose

CHO
|
H  C  OH
|
HO  C  H
|
6C including –CHO Aldohexose (Glucose) H  C  OH
|
H  C  OH
|
CH 2OH

D  glucose

CH 2OH
|
CO
|
HO  C  H
|
H  C  OH
6C including  C  Ketohexose (Fructose) |
|| H  C  OH
O |
CH 2OH

D  fructose
D & L-Sugars : The series of aldoses or ketoses in which the configuration of the penultimate C-atom
(C-next to CH2–OH group) is described as D-sugars if –OH is towards RHS & L-sugars if it is towards
LHS.

Smallest carbohydrate Aldotriose CHO Glyceraldehyde

|
CH  OH
|
CH 2 OH

Fischer projection

Fisher formula of compounds containing many asymmetric carbon.

a5 b5 a3 b3 CHO
a2 b2
CHO b3 a3
a4 b4
CH2OH b5 a5
a4 b4 a 2 b2
CH2OH

Classification of Aldotetros : (i) Erythrose

(ii) Threoese
C-4

C-5

No. of C* = 3 (in Aldopentose)

No. of optical isomers 23 = 8
No. of D Sugars 4
No. of L Sugars 4
D-Aldopentose :

All Isomeric D-sugars are diastereomers.

 Aldohexose :

No. of C* = 4
No. of stereoisomers = 24 = 16
No. of D-sugars = 8
No. of L-sugars = 8

The D–family aldoses

CHO
|
H  C  OH
|
CH 2OH

D-glyceraldehyde
CHO CHO
| |
H  C  OH HO C  H
| |
H  C  OH H  C  OH
| |
CH 2OH CH2OH

D-erythrose D-threose

CHO CHO CHO CHO

| | | |
H  C  OH HO C  H H  C  OH HO  C  H
| | | |
H  C  OH H  C  OH HO C  H HO  C  H
| | | |
H  C  OH H  C  OH H  C  OH H  C  OH
| | | |
CH 2OH CH2OH CH2OH CH 2OH

D-ribose D-arabinose D-xylose D-lyxose

CHO CHO CHO CHO CHO CHO

| | | | | | CHO CHO
H  C  OH HO  C  H H  C  OH HO  C  H H  C  OH HO  C  H | |
| | | | | | H  C  OH HO  C  H
| |
H  C  OH H  C  OH HO  C  H HO  C  H H  C  OH H  C  OH HO  C  H HO  C  H
| | | | | | | |
H  C  OH H  C  OH H  C  OH H  C  OH HO  C  H 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 H  C  OH
| | | | | | | |
CH 2OH CH 2OH CH 2OH CH 2 OH CH 2OH CH 2OH CH 2 OH CH 2 OH

D( ) allose D() altrose D( ) glucose D( )mannose D()gulose D()idose D(  ) galactose D() talose
Epimers: A pair of diastereomers that differ only in the configuration about of a single carbon atom are
said to be epimers. D(+)-glucose is epimeric with D(+)- mannose and D(+)-galactose as shown below.

Epimers Epimers

CHO CHO CHO

| | |
H – C – OH H – C – OH HO – C – H
| | |
HO – C – H HO – C – H HO – C – H
| | |
HO – C – H H – C – OH H – C – OH
| | |
H – C – OH H – C – OH H – C – OH
| | |
CH2OH CH2OH CH2OH

D(+) galactose D(+) glucose D(+) mannose

C4 is epimeric carbon C2 is epimeric carbon

Another example with C2 epimeric carbon is

&

Anomers: Anomers are the stereoisomers which differs at a single chiral centre out of many & are ring
chain tautomer of the same open chain compound.
The two sugars that differs in configuration only on the carbon that was the carbonyl carbon in the open
c h a i n f o r  glucose and glucose are known as anomers their equilibrium mixture
m i s c a l l e d a s a n o m e r s

contains 36 % –D–glucose , 63.8 %
-D-glucose and 0.2 % open chain form.

C1 Carbon is known as anomeric carbon.

Haworth suggested to write  glucose and glucose in pyran structure

O
CHO
HO H OH HO H
H
H HO HO H H OH
H O H OH HO H O
HO
OH H OH H OH
H
H
H CH2OH
CH2OH CH2OH
 –D–glucose D–glucose (open structure)  –D–glucose
(sp rotation 112°) (sp rotation 19°)
 CH2OH CH2OH
O O
H H H H H OH
HO OH H OH HO OH H H
H OH H OH
Haworth formula Haworth formula
 –D–glucopyranose  –D–glucopyranose

Anomers are epimers but epimers may not be anomers.

Cyclic structures of monosaccharides
Many five membered and six membered monosaccharides occur in cyclic form. Cyclic structures of
monosaccharides are established by many experiments. The cyclic structure is due to intramolecular
hemiacetal formation between aldo / keto group and OH of any one carbon. The ring formed are
generally six membered (pyranose) or five membered (furanose). Each cyclization results in creation of
a new asymmetric centre apart from the existing ones. The isomers resulting from cyclizations are called
anomers. example, when D-glucose (open structure) cyclise, it gives -D-glucose and
-D-glucose.
Haworth projection :-
Many of monosaccharides form cyclic structures. The actual structure is almost planer and be represented
by Haworth projection, which is a way of depicting three - dimensional cyclic structure.
Rule -1 :- In a Haworth projection draw a fisher projection in which ring oxygen is in a down
position.
Rule -2 :- Imagine that carbon chain of fisher projection is folded around a barrel or drum, which
provide a ring lies in a plane  to the page.
Rule -3 :- Now plane of ring is turned 90° so that anomeric carbon is on the right and the ring
oxygen is in the rear. Obtained projection is a Haworth projection.
Example : (D-glucose)

OH H OH H
OH HO
OH H H H
CHO H OH
CHO CH2OH
CH2 OH H OH
CH2OH CHO
H OH H OH
OH H
OH H OH H

Projection :
H CH2OH H CH2OH
HO HO

H OH H O

H H
CHO
OH
OH OH
H
OH H OH H

Hawarth projection
 Chair conformation of D–glucose
CH2OH
O H
H H
CH2OH H HO OH
HO H
H
O
H HO H
HO HO H
H OH
OH
Chair forms of (conformation) α and β D-Glucose :
CH2OH
CH2OH O OH
OH OH
HO anomeric O
OH carbon
HO OH
OH
-D-Glucose (most stable glucose form) all groups are equatorial.
CH2OH
CH2OH O OH OH
HO anomeric O
OH
HO carbon
HO OH
OH
-D-Glucose –OH group at anomeric carbon is axial.

Mutarotation

Specific rotation of  glucose + 112° Specific rotation of glucose + 19°

Equilibrium mixture []D = 52.5 degree mL g–1 dm–1
Fresh α-glucose  52.5 
Fresh β-glucose
112° 36 %  glucose 19°
63.8 % glucose
When pure -D glucose is dissolved in water its specific rotation is found to be +112° with time,
however the specific rotation of the solution decreases ultimately reaches stable value of +52.5°. When
D-glucose is dissolved in water, it has a specific rotation of 19°. The specific rotation of this solution
increases with time also to + 52.5 °.
This change of optical rotation with time is called mutarotation. It is caused by the conversion of  and
glucopyranose anomers into an equilibrium mixture of both. Mutarotation is catalyzed by both acid
and base, but also occurs is even in pure water. Mutarotation is characteristic of the cyclic hemiacetal
form of glucose.
Mutarotation occurs first by opening of the pyranose ring to the free aldehyde form.
 

120°

Structure of fructose

CH2–OH HO–C–CH2OH
HOH2C–C–OH
C=O HO–C–H
HO–C–H O
O HO–C–H H–C–OH
H–C–OH l H–C–OH
l
H–C–OH
H–C–OH
H–C–OH H–C
H–C
CH2–OH H
H
 fructose  fructose (more stable)

Ring structure of fructose C, Pyranose structures 6 membered ring, C2 – C6 linkage

H
H C O CH2 OH
H
C H OH
OH C C OH
OH H
Chair conformations

-D-fructopyranose -D-fructopyranose

Furanose structure (5 membered ring)

 Mutarotation: Fructose undergo complex mutarotation. The structure of the cyclic hemiacetal form of
d-fructose can be derived from it’s carbonyl (Ketone) form using the methods described as follows.
CH2OH
C=O
HO H CH2OH
H OH C=O
H OH HO H
CH2OH H OH
H OH This oxygen is
This structure is involved in
CH2OH
involved in pyranose furanose formation
formation
It happens that the crystalline form of D-fructose is -D-Fructopyranose. When crystals of this form are
dissolved in water, it equilibrates to both pyranose & furanose forms.

HO
HOH2C O OH
H2C–C–OH anomeric HOH2C O OH
carbon HOH2C–C–OH anomeric
HO–C–H O H HO carbon
H CH2OH HO–C–H O H HO
H–C–OH H CH2OH
H–C–OH
H–C OH H
H–C OH H
CH2OH  -D-fructofuranose
CH2OH  -D-fructofuranose
 -D-fructose

* All monosaccharides are reducing sugars, hence they show mutarotation.

* Starch, cellulose are Polymers of Glucose
* Lactose and sucrose are disaccharides
* Sucrose is a non reducing sugar, gives negative test for Benedict and tollen's reagent, they do not form
osazone and do not show mutarotation.
* Acetals of carbohydrates are called as GLYCOSIDE

Formation of Glycosides
Glucose reacts with methyl alcohol in presence of dry HCl to form  and -methyl glycoside of glucose.
The reaction takes place only on OH of hemi-acetylic carbon. Other hydroxyl groups are unreactive.
H OCH3 CH3O H
C C
(CHOH)3 O (CHOH)3 O
CH CH
CH2OH CH2OH
 –Methyl glucose  –Methyl glucose

To methylate all the OH groups, methylating agent used is dimethyl sulphate.

 + CH3OH HCl


Such compounds are called glucoside (cyclic acetals). They are special type of acetals in which one of
the oxygen of the acetal linkage is the ring oxygen of the pyranose or furanose. H–C
H–C–OH
Ring structure of glucose :
HO–C–H O
(i) Glucose does not give pink colour with schief reagent.
(ii) Does not form adduct with NaHSO3, NH3 H–C–OH
(iii) Glucose exist in two isomeric form H–C
(iv) It show mutarotation CH2OH
Since there is no free aldehyde group, so it does not react with weak reagent (NH3, NaHSO3) but
strong reagent (HCN, NH2OH, C6H5NH – NH2) break up ring

Hydrolysis
  H 2O


REACTIONS OF GLUCOSE
COOH
(CHOH)4 Br2 / H2O Red P / HI n Hexane
CH2OH or Alkaline
Gluconic acid solution of I2 CH3COCl
Pentaacetate
+
COONH4 (5-OH group)
(CHOH)4 Tollen’s NH2–OH (1 eq)
C6H12O6 Oxime
Salts of CH2OH
gluconic acids HCN
COONa Glucose cyanohydrin
Fehling’s
(CHOH)4
5HIO 4
CH 2OH HCHO + 5HCOOH

COOH Schief's reagent, NaHSO 3

HNO3 No reaction
(CHOH)4 NH3
COOH
(Saccharic acid)
These reactions indicate that glucose has 6-C straight chain with one –CHO group & 5-OH group.
 General reaction of monosaccharies

1. Acetylation :

+ 5CH3COCl / (CH3CO)2O  + 5HCl

This reaction suggests presence of 5( OH ) group.

Q. The penta acetate of glucose give –ve test with Tollen’s reagent & Fehling solution, explain?
No hemiacetal linkage, So no
CHO group in aq.
alkane solution –ve test
Tollen's –ve
Sol. 5
AcCl
 Fehling
–ve
Benedict
–ve
Shifts –ve
NaHSO3
–ve

2. Red by HI / Red P:

Re
dP / HI
 n-Hexane Re
dP / HI
 n-Hexane

3. Reaction with HCN:

+ HCN  + HCN


4. Reaction with NH2–OH (hydroxyl amine):

+ NH2–OH
 + NH2OH



5. Reaction with phenyl hydrazine: Both glucose and fructose give “osazone”.
Reaction with glucose :

+ 2 C6H5 NH – NH2  Osazone

Mechanism :

O O H
+ HC– N–NHPh
HC HC–NH2NHPh HC= N–NHPh

HC–NH–NH–Ph
NH2NHPh IMPE
CHOH CH–OH CH–OH C – OH + +
–H2O –H C O H
R R R R
R
+
–H

HC=NNHPh HC=NH
2PhNHNH2
C NNHPh –NH3 C O

R R

Propose Mechanism for :

CHOH CH=NNHPh
PhNHNH 2
C=O C=NNHPh + PhNH2 + NH3
(Excess)

R R
(Osazone)
Reaction with fructose :
Fructose + 2 NH2–NH–C6H5  Osazone
Step (i) + H2N – NH – C6H5 

Step (ii) + H2N – NH – C6H5 

Step (iii) + H2N – NH – C6H5 

Both compounds give same product because structure of last four C is same in both (glucose & fructose)

Only C-1 and C-2 in glucose and fructose are involved in osazone formation addition reaction do not
run through out the chain.The failure to undergo further reaction has been explained by stabilization of the
osazone by chelation.
 Osazone :
NHPh
HOH2C O Chelation
N
H
N
HO N Ph
OH

or

So we do not get hexaphenyl hydrazone.

6. Catalytic reduction:
CH2OH CH2–OH

(CHOH)4 HO–C–H
H 2 / Ni or H 2 / Ni or
    D-sorbital + (CHOH)3
NaBH 4 CH2OH NaBH 4
D-sorbital CH2OH
D-Manitol

7. Oxidation:
Glucose Tollen
 's , Fehling
 Glucose dil
.HNO
3 
Br2 water

Glucose HIO
4  5HCOOH + HCHO + 5HIO3

Oxidation of fructose :
Fructose Tollen
 's,Fehling
 
Fructose reduces tollen’s & fehling reagent because in basic medium fructose isomerises to glucose.
Fructose Br  No reaction
2water


Fructose HNO
3  + +

8. Reaction with enzyme:

Yeast
Glucose or Fructose   2C2H5OH + 2CO2
Zymase enzyme
9. Reaction with dil NaOH / Ca(OH)2
Glucose or fructose   Glucose + Fructose + Mannose
dil NaOH

H–C–OH

C–OH l

Base-catalyzed isomerisation of aldoses and Ketoses:

Although glucose in solution exists mostly in its cyclic hemiacetal forms it is also in equilibrium
with a small amount of it’s acyclic aldehyde form.

0.02 M
Ca
( OH
 )2
 + + + Traces of other compound

Mechanism : Like other aldehyde with -hydrogen, glucose ionise to give small amount of its inolate
ion in base. Protonation of this enolate ion at one face of the double bond gives back glucose & protonation
at the other face gives mannose.
CH=O
CH–O–
CHO CH= O
HO–C–H
H OH  OH C–OH
OH— H2O HO–C–H
HO H HO H HO H
H OH H OH H OH H–C–OH
H OH H OH H OH H–C–OH
CH2OH CH2 OH CH2OH CH2 OH
D-mannose
The enolate ion can also be protonated on oxygen to give a new enol called enediol this enediol
converts to fructose as follows.


H–C=O H–C=O H–C–O
H OH OH – C–OH C–OH
 H–OH

CH2–OH
H–C–OH H–C–OH H–C–OH

C=O HO–H C=O C–O
 OH–
C–OH
Fructose
enediol
Method of ascending the sugar series: An aldose may be converted into it’s next higher aldose
eg. an aldopentose into an aldohexose.
By Kiliani Fischer upgradation:

HCN
 (
i ) H 2 Pd
  
aq. ( ii ) BaSO 4 H
3O


Theoretically two lactones are possible, since two cyanohydrin may be formed when hydrogen
cyanide adds on to the aldopentose (a new assymetrical carbon is produced)

+ HCN  +

By Sowden reaction:

H
2SO 4


Mech.
H

 CH3OH + CH2 – NO2 C=O 

CH 2 NO 2
| H 2SO 4
CH
3OH
 H  C  OH 



 




|
Sowden has also stepped up an aldose to a ketose containing two additional carbon atoms using the
above method except that 2-nitroethanol is used instead of nitromethane.
CH 2 NO 2
+ | CH
3ONa
 H
2SO 4
 +
CH 2OH

Wolfrom reaction : Wolfrom have stepped up an aldose to a ketose with one more carbon
atom by a modified Arndt-Eistere reaction.

Br
2  (
i ) Ac 2O
 CH
2N2
 (
i ) AcOH

H 2O ( ii ) SOCl 2 ( ii ) Ba ( OH ) 2
Kochetkov reaction :
R
|
R CHOH
| |
CHOH Ph
3PCH COOEt
   CH OsO
4   Na
/Hg

HClO 4 
 HCl
|  Ph 3P O ||
CHO CH
|
COOEt

Method of descending the sugar series :

Wohl’s method:
CHO CH=N–OH CN
CH–O–C–CH3 CHO
CHOH CHOH
NH2OH (CH3CO)2O O AgOH
(CHOH)3 + AgCN
(CHOH)3 (CHOH)3 (CH–O–C–CH3)3
CH2OH
CH2OH CH2OH O
CH2 –O–C–CH3
O
Ruff’s method:

Br
2 /
H 2O
 Ca
salt
 + CO2
H 2O 2 / Fe 2 
Fenton 's
reagent

Conversion of an aldose into a ketose :

3
C6H5 NHNH2
  HCl

 Zn
/ 
CH3COOH
 

An aldehyde group is reduced more readily than a ketonic group.

Conversion of a Ketose into an aldose :

H 2 / Ni [
O]
 Na
/Hg

  HNO3 
 HCl
 SOME IMPORTANT CARBOHYDRATES
1. Sucrose (C12H22O11) :  It is white, crystalline & sweet substance soluble in water obtained from the
sugar cane. When heated above its melting point, it forms a brown substance known as caramel.
It's aqueous solution is dextrorotatory []D = 66.5°
dextro laevo
H+
C12H22 O11 + H2O C6H12O6 + C6H12O6
D-Glucose D-Frucose
 [D] = 52.7° []D = –92.4°

mixture is laevorotatory
(–)
Thus hydrolysis of sucrose brings about a change in the sign of rotation, from dextrol (+) to leavo (–) &
such a change is known as inversion of cane sugar and the mixture is known as invert sugar.

The inversion of cane-sugar may also be effected by the enzyme invertase which is found in yeast.

Sucrose is non-reducing sugar because it has stable acetal linkage & in aq. solution it can not give free
carbonyl group and so it does not reduces Tollen's & Fehling's solution.

This indicates that neither the aldehyde group of glucose nor the ketonic group of fructose is free in
sucrose.

anomeric C
6 1
CH2OH CH2OH
O O
5 H
H H H 2 5
4 1 CH2OH
HO OH H O H HO
3 2 3 4
H OH HO H
–D–Glucopyranose –D–Fructofuranose

Structure of sucrose
(–D–glucopyranosyl– –D–fructofuranoside)

All non reducing sugars do not show mutarotation .

There is no free carbonyl group so it is nonreducing sugar

-glycoside bond to anometric

O
O CH2OH
position of D-glucose H
HOH2C
OH  linkage
HO HO
OH HO CH2OH O at glucose
HO OH O
HO O linkage
O CH2OH H HO
b-Glycoside at fructose
bond to anomeric H CH2OH
positon of D-fructose CH2OH OH H
2. Maltose:
It is obtained by partial hydrolysis of starch by the enzyme diastase present in malt i.e., sprouted barely
seeds.
2 (C6H10O5)n + n H2O Diastase
  n C12H22O11
Starch Maltose
As stated above, hydrolysis of one mole of maltose yields two moles of D-glucose. maltose is a reducing
sugar since it forms an osazone, undergoes mutarotation and also reduces Tollen’s and Fehling’s solutions,
Methylation studies have revealed that
(i) both glucose units are present in the pyranose form.
(ii) C1 of one glucose unit is linked to C4 of the other
Further since maltose is hydrolysed by the enzyme maltose which specifically hydrolyses -glycosidic
linkage, therefore, the non-reducing glucose unit in maltose must be present in the -form. In other
words, C1 –  of non-reducing glucose unit is attached to C4 of the reducing glucose unit as shown in the
figure on next page.

3. Lactose (Milk sugar) C12H22O11

Lactose occurs in milk and that is why it is called milk sugar.
Lactose on hydrolysis with dilute acid or by enzyme lactase, yields an equimolar mixture of D-glucose
and D-galactose. It is a reducing sugar since it forms an osazone, undergoes mutarotation and also
reduces Tollen’s or Fehling’s solution. Methylation studies have revealed that
(i) both glucose and galactose are present in the pyranose form.
(ii) glucose is the reducing half while -galactose is the non-reducing half.
(iii) C1 of galactose unit is connected to C4 of glucose unit.
Further since emulsin, as, enzyme which specifically hydrolyses -glycosidic linkages also hydrolyses
lactose, therefore, galactose must be present in the -form. In other words, in lactose, C1 – of galactose
is attached to C4 of glucose as shown in figure.
6
CH2OH
H 5
O OH
6 4 H 1
CH2OH OH H
HO O O 3 2
H
5

H H OH
4
OH H
H GLUCOSE
3 H (Reducing half)
2

H OH
GALACTOSE
(Non-reducing half)
4. Starch Amylum, (C6H10O5)n
Occurrence : The value of n (100 – 3000) varies from source to source. It is the chief food reserve
material or storage polysaccharide of plants and is found mainly in seeds, roots tubers etc. Wheat,
maize, rice, potatoes, barley, bananas and sorghum are the main sources of starch. Starch occurs in the
form of granules, which vary in shape and size depending upon their plant source.
Occurs in all green plants.Starch consists of two fractions, one being known as –amylose, which gives
blue colour with iodine. This blue colour is believed to be due to the formation of an inclusion complex.
An aqueous solution of –amylose slowly forms a precipitate, since -amylose has a strong tendency to
'revert' to the insoluble state in solution. Amylopectin is insoluble in water and is stable towards both
hydrolysis to maltose by the enzyme diastase and to D(+)-glucose by dilute acids (amylopectin gives
about 50 percent of maltose).
6 6
CH2OH 6
CH2OH CH2OH
5 O O
H H H H 5 5 O
H H H H H
4 1 4 1
HO O O
4 1
OH H OH H
3 2 2
OH H OH
3 3 2
H OH H OH n–2 H OH
 –D–glucopyranose  –D–glucopyranose

Structure of Starch ( –D–glucoamylose)

–amylose consists of an unbranched chain, with a molecular weight varying between 10,000(n  60)
and 10,00,000(n  6,000), The value of n depends on the source and treatment of –amylose.
Properties : (i) Starch is a white amorphous powder sparingly soluble in water. Its aqueous solution
gives a blue colour with iodine solution due to the formation of an inclusion complex. The blue pears on
cooling.
(ii) On hydrolysis with dilute mineral acids or enzymes, starch beaks down first to smaller molecules (n >
n’), then to maltose and finally to D-glucose.

H / H 2O
(C6H10O5)n H 
 (C6H10O5)n’ H/
/ H 2O 
 C12H22O11 orMaltase
H 2O  C6H12O6
Starch Maltose D-Glucose
(iii) Starch is a non-reducing saccharide. It neither reduces Tollen’s reagent or Fehling’s solution nor
forms an osazone. This suggests that all hemiacetal OH groups of glucose units at C1 are not free but are
involved in glycosidic linkages.
Composition : Starch is not a single compound but is a mixture of two components–a water soluble
component called amylose (10-20%) and a water insoluble component called amylopectin (80-90%).
Both amylose and amylopectin are polymers of -D-glucose.
Structure of amylose : Amylose is water soluble and gives blue colour with iodine solution. It may have
100-3000 glucose units, i.e., its molecular mass can vary from 10,000 to 500,000. It is a linear polymer
of -D-glucose in which C1 of one glucose unit is attached to C4 of the other through -glycosidic
linkage as shown in figure.
 Pectins
Pectins are found in plant and fruit juices. Their characteristic property is the ability of their solutions to
gelate, i.e. form jellies. They have a high molecular weight and are polygalacturonic acid (linked 1,4) with
the carboxyl groups partially esterified with methanol.
Glycogen (C6H10O5)n :
Glycogen is found in nearly all animals cells, occurring mainly in liver. It is the reserve carbohydrate of
animals and so is often known as 'animal starch'. It has also been isolated from plant sources.
Glycogen is a white powder, soluble in water, the solution giving a purplish-red colour with iodine. On
hydrolysis with dilute acid, glycogen gives D(+)-glucose. The molecular weight of glycogen has been
given as 10,00,000 to 50,00,000 and glycogen contains highly branched chains. Glycogen has a structure
similar to amylopectin, except that it has more cross-linking.

5. Cellulose:
Cellulose is colourless, solid which is insoluble in water & organic solvents. But it is soluble in ammonical
cupric hydroxide (Schweizer's reagent) or in conc. HCl cellulose is a regular polymer of d-glucopyranose
residues connected by -1,4 glycosidic linkages. It is straight chain polymer.
6 6
CH2OH CH2OH
5 O H 5 O
H H H
4 1 O 4 1 O
O
OH H OH H
3 2 H 3 2 H
H OH H OH
–D–glucopyranose –D–glucopyranose

Structure of Cellulose

Some points about cellulose :

1. General empirical formula (C6H10O5)
Cellulose + H2O H

2.  96% of crystalline D-glucose
3. No. of monomer units in cellulose are 1000 – 1500 in one molecule.
4. Cellulose doesn't show mutarotation (like starch)
5. It is non reducing sugar because there is no hemiacetal linkage.
6. Acetylations nitration & methylation of cellulose give trisubstituted cellulose which suggest that only three
– OH groups are free.
 Tests for carbohydrates:
(i) When heated in a dry test tube, it melts, turns brown and finally black, giving a characteristic smell of
burning sugar.

(ii) When warmed with a little concentrated H2SO4, it leaves a charred reside of carbon.

(iii) Molisch’s Test (named after Austrian botanist Hans Molisch) is a sensitive chemical test for the presence
o f c a , based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde,
r b o h y d r a t e s

which condenses with two molecules of phenol (usually α-naphthol, though other phenols (e.g. resorcinol,
thymol) also give colored products) resulting in a red- or purple-colored compound.
The test solution is combined with a small amount of Molisch’s reagent (α-naphthol dissolved in ethanol)
in a test tube. After mixing, a small amount of concentrated sulfuric acid is slowly added down the sides
of the sloping test-tube, without mixing, to form a bottom layer. A positive reaction is indicated by
appearance of a purple ring at the interface between the acid and test layers.
All carbohydrates — monosaccharides, disaccharides, and polysaccharides — should give a positive
reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are
eventually hydrolyzed to monosacharides by strong mineral acids. Pentoses are then dehydrated to
furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural. Either of these aldehydes, if present,
will condense with two molecules of naphthol to form a purple-colored product, as illustrated below by
the example of glucose:
 AMINO ACIDS AND PROTEINS
NH 2
|
Bifunctional compounds R  CH  COOH having an acidic corboxylic group & a basic amino group.
There are 20 amino acids commonly found is proteins and are standard amino acids. All are  amino
acids. Most of them have 1° amino group.(– NH2). However proline is a 2° amino

CH2—COOH Me—CH—COOH Ph—CH2—CH—COOH N COOH

NH2 NH2 NH2 H

Proline [P]
(Gly) Glycine [G] Alanine [A] Phenyl alanine [F] Proline [P] (-imino acid)
All amino acids are chiral molecules with atleast one chiral carbon except glycine, H3NCH2COO¯.
Except Glycine all other amino acids are optically active & can be assigned D & L configuration.

\\\\\\\\\\\\\\\\\\\\
COOH COOH
H NH2 H2N H
CH3 CH3
D-Alanine L-Alanine
Classification of Amino acids

Occurance Requirement Chemical Nature

Natural Essential Neutral
Synthetic Non Essential Acidic

Semi Essential Basic

Based on requirement.
1. Essential amino acids can not be synthesized in human body so dietary intake is required. For any human
being 1 gm a day is required.
2. Semi essential amino acids can be synthesized in human body but dietary intake is required during
growing stages (when more of cell division is required).
For example : Early childhood, pregrancy and lactating mother.
3. Non essential amino acid - Body can synthesize them.
Chemical classification
Neutral - Amino acid having equal number of NH2 and COOH.
Neutral amino acids are further classified as polar and nonpolar depending on whether their side chains
have polar substituents (for example, asparagine with an NH2CO group) or are completely hydrocarbon
in nature (for example alanine, valine etc.).
Acidic - Amino acid having more COOH than NH2
Aspartic acid and glutamic acids, each with a second CO2H in their side chain are acidic amino acids.
Basic - Amino acid having more NH2 than COOH
Lysine, arginine and histidine, each with a basic site in their side chain are basic amino acids.
Proteins: The name protein is taken from the Greek word "proteios", which means "first". Of all
chemical compounds, proteins must almost certainly be ranked first, for they are the substance of life.
Proteins make up a large part of the animal body, they hold it together and they run it. They are found in
all living cells.
 Chemically, proteins are high polymers. They are polyamides and the monomers from which they are
derived are the  - amino carboxylic acids. A single protein molecule contains hundreds or even thousands
of amino acid units. These units can be of twenty-odd different kinds. The number of different combinations,
i.e., the number of different protein molecules that are possible, is almost infinite.

1. Neutral amino acids (with nonpolar side chains)

ISOELECTRIC
NAME ABBREVIATIONS STRUCTURAL FORMULAE
POINT[pI]

@Glycine Gly(G) 6.0

Alanine Ala(A) 6.0

Valine* Val(V)
6.0

Leucine* Leu(L) 6.0


CH 3 N H 3
| |
Isoleucine* Ile(I) CH 3CH 2 CH  CHCO 2 6.0


N H3
|
Methionine* Met(M) CH 3SCH 2 CH  CHCO 2 5.7

@@Proline Pro(P) 6.3

Phenylalanine* Phe(F) 5.5

Tryptophan* Trp(W) 5.9

 2. Neutral amino acids (with polar, but nonionized side chains)

NAME ABBREVIATIONS STRUCTURAL FORMULAE ISOELECTRIC

POINT[pI]

Asparagine Asn(N) 5.4

Glutamine Gln(Q) 5.7

Serine Ser(S) 5.7

3. Neutral amino acids (with polar, but nonionized side chains)

NAME ABBREVIATIONS STRUCTURAL FORMULAE ISOELECTRIC
POINT[pI]
Threonine* Thr 
OH N H 5.6
3
| |
CH 3CH  CHCO 2
Tyrosine Tyr(Y) 5.7


N H3
Cysteine Cys | 5.1
HSCH 2  CHCO 2
 
N H3 N H3
Cys–Cys | |
| Cystine


OOCCHCH 2S  SCH 2 CHCOO 

4. Acidic amino acids (side chain with carboxylic acid group)

NAME ABBREVIATIONS STRUCTURAL FORMULAE ISOELECTRIC

POINT[pI]
Aspartic acid Asp(D)
2.8

Glutamic Acid Glu(E)

3.2
 5. Basic amino acids (side chain with nitrogenous basic group)
NAME ABBREVIATIONS STRUCTURAL FORMULAE ISOELECTRIC
POINT[pI]

Lystine* Lys(K)
9.7

Arginine* Arg(R)
10.8

Histidine* His(H)
7.6

Note:
* Amino acids with an asterisk are essential amino acids, that must be supplemented through diet.
| At pH = 7, Asp and Glu have a net negative charge and exist as anions. At pH = 7, Lys and Arg have a
net positive charge and exist as cations. Rest of the amino acids at this pH exist in the neutral form.
|
 Structurally, in cystine, the two cysteine molecules are joined through sulfur (disulfide linkage).
@@ Proline is an -imino acid, all amino acids are primary amines except proline and 4-hydroxyproline,
which are 2° amines.
@ Except Glycine all other amino acids are optically active.

Preparation of amino acids

(a) Gabriel Phthalimide synthesis

Better yields are generally obtained by the gabriel phthalimide synthesis ; the  - halo esters are used
instead of  - halo acids .

O O
C C
+ – KCl
N¯ K + Cl–CH2COOC2H5 N– CH2COOC2H5
C C
O O
Potasium
phthalimide HCl , H2O

+
Cl¯ H3N – CH2COOH + phthalic acid
Glycine hydrochloride
(b) Amination of α - Halo acids
Sometimes an  - chloro or  - bromo acid is subjected to direct ammonolysis with a large excess of
concentrated ammonia. For example.
Br2, P CH CHCOOH NH3(excess) CH CHCOO¯
CH3CH2COOH 3 3
+
Br NH3
-Bromopropionic acid Alanine

(c) From diethyl malonate

COOC2H 5 COOC2H 5 COOH


C6H 5 CH2Cl H3O
Na+
CH HC– CH 2C 6H 5 H–C– CH 2C6H 5

COOC2H 5 COOC2H 5 COOH

Sodiomalonic ester Ethyl benzylmalonate Benzylmalonic acid

Br2/ OH

COOH
NH 3(excess) heat Br–C– CH C H
C6H 5CH 2CHCOOH C 6H 5CH 2CHCOOH 2 6 5
–CO 2
NH 3+ Br COOH
Phenylalanine
35% overall yield

(d) Strecker's synthesis

Strecker's synthesis is also used for preparing  - amino acids


+
O N–H N–H NH2 NH2
+
–H2O CN¯ H2 O H3 O
R–C–H + NH3 R–C–H R–C–H R–C–H R–C–H
Imine
CN CN COO¯
 - amino nitrile  - amino acid
In this reaction general aldehyde is treated with mixture of ammonium chloride and KCN in aqueous
solution which forms NH3 and HCN, NH4Cl + KCN aqueous NH4CN + KCl,
NH4CN aqueous NH3 + HCN

(e) Using KOOP synthesis

Br NH NH2
O
HVZ DMSO NH3 SBH
CH3 COOH CH2—COOH CH—COONa CH—COONa CH2
NaHCO3 Xs

COONa
 Properties of Amino acids
Although the amino acids are commonly shown as containing an amino group and a carboxyl group,
H2NCHRCOOH, certain properties, (both physical and chemical) are not consistent with this structure
I. Physical properties
In contrast to amines and carboxylic acids, the amino acids are nonvolatile crystalline solids, which melt
with decomposition at fairly high temperatures.
They are insoluble in non-polar solvents like petroleum ether, benzene or ether and are appreciably
soluble in water.
Their aqueous solutions behave like solutions of substances of high dipole moment.
Amino acids as dipolar ions
Acidity and basicity constant are ridiculously low for –COOH and –NH2 groups. Glycine, for example,
–10 and K = 2.5 × 10–12, whereas most carboxylic acids have K values of about
h a

a = 1.6 × 10
s K

b a
10–5 and most aliphatic amines have Kb values of about 10–4.
All these properties are quite consistent with a dipolar ion structure for the amino acids (I)
+
H3N–CH–COO¯
R
Amino acids : dipolar ions (Zwitter ion)
Physical properties - melting point, solubility, high dipole moment - are just what would be expected of
such a salt. The acid-base properties also become understandable when it is realized that the measured
Ka actually refers to the acidity of an ammonium ion, RNH3+,
+ +
H3NCHCOO¯ + H2O H3O + H2NCHCOO¯
R R
Acid
+
[H3O ] [H2NCHRCOO ]
Ka = +
[H3NCHRCOO¯]
When the solution of an amino acid is made alkaline, the dipolar ion (I) is converted into the anion(II).
The stronger base, hydroxide ion, removes a proton from the ammonium ion and displaces the weaker
base, the amine.
+
H3NCHCOO¯ + OH¯ H2NCHCOO¯ + H2O
R (I) R (II)
Stronger Weaker Weaker
Stronger base base acid
acid
+
H
+
+ H +
H2NCHCOO¯ H3NCHCOO¯ H3NCHCOOH
OH¯ OH¯
R R R
(II) (I) (III)
Anion Ampholyte Cation

Wherever feasible, we can speed up a desired reaction by adjusting the acidity or basicity of the solution
in such a way as to increase the concentration of the reactive species.
 Isoelectric point of amino acids
What happens when a solution of an amino acid is placed in an electric field depends upon the acidity or
basicity of the solution.
+ +
H  H 
H2NCHCOO¯ H NCHCOO¯ H 3NCHCOOH
3
OH¯ OH¯
R R R
Ka 2 Ka 1
(II) A 
(I) DI (III) C
In quite alkaline solution, anions (II) exceed cations (III), and there is a net migration of amino acid
toward the anode. In quite acidic solution, cations (III) are in excess, and there is a net migration of
amino acid toward the cathode. If (II) and (III) are exactly balanced, there is no net migration ; under
such conditions any one molecule exists as a positive ion and as a negative ion for exactly the same
amount of time, and any small movement in the direction of one electrode is subsequently cancelled by
an equal movement back towards the other electrode. The hydrogen ion concentration of the solution
in which a particular amino acid does not migrate under the influence of an electric field is
called the isoelectric point (pI) of that amino acid. The isoelectric point (pI) is the pH at which
the amino acid exists only as a dipolar ion with net charge zero.
For glycine, for example, the isoelectric point is at pH 6.1.
An amino acid usually shows its lower solubility in a solution at the isoelectric point, since here there is
the highest concentration of the dipolar ion. As the solution is made more alkaline or more acidic, the
concentration of one of the more soluble ions, (II) or (III) increases.
[DI] [H  ] [A  ] [H  ]
Ka1 = Ka2 = at pI [ A ] = [C ]
[C  ] [DI]

[DI] [H  ] Ka 2 [DI]
Ka1
=
[H  ] [H  ]2 = Ka1 & Ka2

P Ka1  P Ka 2
on taking antilog pI =
2
An amino acid having more COOH than NH2 or more acidic COOH will have pI less than 7.
An amino acid having more – NH2 than COOH or more basic – NH2 will have pI more than 7.

Q. Write the structure of alanine at pH 2.5, 10.5 and 6.

Electrophoresis
The movement of charged molecules (like amino acid) under the influence of an electric field is called
electrophoresis. Electrophoresis separates amino acids on the basis of their pI values.
Amino acid is positively charged (moves towards cathode) if pH of the solution < pI
Amino acid is negatively charged (moves towards anode) if pH of the solution > pI

Q. How will you separate a ternary mixture of arginine, alanine & aspartic acid?
Ans. A few drops of a solution of an amino acid mixture are applied to the middle of a piece of filter paper.
When the paper is placed in a buffer solution (pH = 5) between the two electrodes and an electric field
is applied then arginine & alanine with pI > pH move towards the cathode and aspartic acid with pI < pH
moves towards the anode. Out of arginine & alanine, alanine will move slowly towards the cathode due
to lesser positive charge.
 Before electrophoresis After electrophoresis

– + – +

Buffer solution Buffer solution

(pH = 5) A B C
(pH = 5)


Mixture of arginine NH2 O
||
+ alanine + aspartic acid 
A = arginine (pI = 10.76) H2NCNHCH2CH2CH2CHCO
Figure (i) |
NH3
O
||

B = alanine (pI = 6.02) CH3CHCO
|
NH3
O O
|| ||

C = aspartic acid (pI = 2.98) OCCH2CHCO
|
NH3
Figure (ii)
General reactions of amino acids

(1) Reactions due to – NH2 group

H H
HBr COOH
COOH
 
NH3Cl
NH2 H CH2—COOH
MeCOCl PhCOCl
COOH CH2—COOH
NHCOMe NH2
H NH—C—Ph
NaNO2
COOH
HCl O
OH Hipuric acid
H
NOCl COOH
Cl
H
NOBr COOH
Br

H H
HCHO
COOH COOH Sorison's titration method.

NH2 NH = CH2
Reactions is used to block – NH2 group during volumetric analysis in.
(2) Reactions due to – COOH group.

H
H
COOH ROH COOR
H 
NH3
NH2 H
NaOH
CaO/
NH2
NaHCO3
Na salt

H
LAH CH2OH
NH2
H
NH3 CONH2

NH2

(3) Heating Effect

(i) Heating of amino acids leads to intermolecular dehydration to form cyclic diamides.

CH2 –C–OH O
H
NH –2H2 O
NH NH N–H
H

CH2
HO –C O

(ii) When alanine is heated, then two diastereomers are obtained. One of them (trans) is not resolvable.

O O
Me NH Me H NH Me


2MeCH(NH2)COOH –2H O +
2
NH H NH H
H Me
O O
Cis (Racemic) Trans (Meso)

(iii) When - amino acids are heated, , - unsaturated salt are formed.

RCHCH2COOH RCH = CH–COONH 4

NH2
(iv)   - amino acids when heated alone gives 
- lactam and polymer respectively. The reason for the
formation of polymer is that when - amino cyclises intramolecularly, it leads to large angle strain within
the compound
O O
O
O
OH Heat C– OH Heat
NH2 N–H + H2O N–H H2O N–H

- lactam H - lactam



NH3(CH2)5COO –NH(CH2)5–C–NH(CH2)5–C–NH(CH2)5C–

O O O
Nylon 6
(4) Peptide
A peptide is an amide formed by intermolecular reaction of the amino group of one amino acid and the
carboxyl group of a second amino acid. Dipeptides are made from two amino acids, tripeptides from
three amino acids, etc, which may be the same or different. If there are four to ten amino acid residues,
the peptide is called an oligopeptide.A polypeptide is a chain made upto of many amino acids.
If they give 3 to 10 amino acid they are oligopeptide
If they give 11 to 100 amino acid they are Polypeptide
For more than 100 it is Macropeptide

O O O O

H2N–CH2–C–OH + H2N–CH2–C–OH – NH–CH2–C–NH–CH2–C –

n
+ peptide linkage
PhCOCl ROH/H
O O O O O


Ph–C–NH–CH2–C–OH H2N–CH2–C–OR Ph–O–C–NH–CH2–C–NH–CH2–C–OR

These type polyamide can be made only using part blocking technique.
Polypeptide on hydrolysis give two amino acid are known as dipeptide
Firstly, the amino and carboxyl groups that are not to be linked in peptide bonds must be blocked so as
to be unreactive. Then all other reactive functional groups in the R's must also be protected, to prevent
their participating in the coupling procedure. The coupling must be effected by a method that does not
cause racemization or chemical alteration of the side chains.
O
HOOC –CH –NH–C–CH2–NH2 Dipeptide
Me
Gly Ala [Glycine Alanine]
Abbreviated name of amion acid with free NH2 is written first.
By convention, the amino acid with the free amino group (N-terminus) is written at the left end and the
one with the unreacted carboxyl group (C-terminus) at the right end. The suffix -ine is replaced by -yl for
each amino acid in the chain reading from left to right, followed by the full name of the C-terminal amino
acid.
 O O
H2N–CH–C–NH–CH2–C–OH Ala - Gly [Alanine-Glycine ]
Me
Total number of polypeptide possible = Xn [X = type of amino acid interacting,
n = number of amino acid molecule are interacting.]
Q. Glycine can form how many Dipeptide ? [Ans. One]
Q. Glycine can form how many Tripeptide ? [Ans. One]
Q. Glycine and Ala can form how many Dipeptide ? [Ans. Four]
Q. Gly, Ala, and Phenyl Ala can form how many Dipeptide ? [Ans. Nine]
Q. Gly, Ala, can form how many Tripeptide ? [Ans. Eight]

When Macropeptide takes different shape due to intramolecular H - bonding between different layers is
known as proteins

Proteins
You have already read that proteins are the polymers of -amino acids and they are connected to each
other by peptide bond or peptide linkage. Chemically, peptide linkage is an amide formed between —
COOH group and — NH2 group. The reaction between two molecules of similar or different amino
acids, proceeds through the combination of the amino group of one molecule with the carboxy1 group of
the other. This results in the elimination of a water molecule and formation of a peptide bond — CO —
NH —. The product of the reaction is called a dipeptide because it is made up of two amino acids. For
example, when carboxy1 group of glycine combines with the amino group of alanine we get a dipeptide,
glycylalanine.

H2N — CH2 — COOH + H2N — CH — COOH

– H2O CH3

H2N — CH2 — CO — NH — CH — COOH

CH3
Peptide linkage

Glycylalanine (Gly-Ala)

If a third amino acid combines to a dipeptide, the product is called a tripeptide. A tripeptide contains
three amino acids linked by two peptide linkages. Similarly when four, five or six amino acids are linked,
the respective products are known as tetrapeptide, pentapeptide or hexapeptide, respectively. When
the number of such amino acids is more than ten, then the products are called polypeptides. A polypeptide
with more than hundred amino acid residues, having molecular mass higher than 10,000u is called a
protein. However, the distinction between a polypeptide and a protein is not very sharp. Polypeptides
with fewer amino acids are likely to be called proteins they ordinarily have a well defined conformation
of a protein such as insulin which contains 51 amino acids.
Proteins can be classified into two types on the basis of their molecular shape.
(a) Fibrous proteins
When the polypeptide chains run parallel and are held together by hydrogen and disulphide bonds, then
fibre-like structure is formed. Such proteins are generally insoluble in water. Some common examples
are keratin (present in hair, wool, silk) and myosin (present in muscles), etc.
(b) Globular proteins
This structure results when the chains of polypeptides coil around to give a spherical shape. These are
usually soluble in water. Insulin and albumins are the common examples of globular proteins.
Structure of Proteins
Structure and shape of proteins can be studied at four different levels, .i.e., primary, secondary, tertiary
and quaternary, each level being more complex than the previous one.
(i) Primary structure of proteins : Proteins may have one or more polypeptide chains. Each polypeptide
in a protein has amino acids linked with each other in a specific sequence and it is this sequence of amino
acids that is said to be the primary structure of the protein. Any change in this primary structure i.e., the
sequence of amino acids creates a different protein.
(ii) Secondary structure of proteins : The secondary structure of protein refers to the shape in which a
long polypeptide chain can exist. They are found to exist in two different types of structures viz. -helix
and -pleated sheet structure. These structures arise due to the regular folding of the backbone of the
O
polypeptide chain due to hydrogen bonding between — C — and — NH — groups of the peptide
bond.

O
C
C
O
H O
H
O
HO
C
H C N
O
O
H
H
H N
N

-Helix structure of proteins

-Helix is one of the most common ways in which a polypeptide chain forms all possible hydrogen
bonds by twisting into a right handed screw (helix) with the — NH group of each amino acid residue
hydrogen bonded to the C = O of an adjacent turn of the helix as shown in figure.
In -structure all peptide chains are stretched out to nearly maximum extension and then laid side by side
which are held together, by intermolecular hydrogen bonds. The structure resembles the pleated folds of
drapery and therefore is known as -pleated sheet.
 N N N
RCH RCH RCH
C C
H C O H N O HN O
N HCR
HCR HCR
C
O N H O C H O CN H
N
RCH RCH RCH
C C C O
H N O
H N O HN
HCR HCR HCR
C C C
-Pleated sheet structure of proteins
(i) Ionic bonding : between COO¯ and NH3+ at different sites.
(ii) H-bonding : mainly between side-chain NH2 and COOH, also involving OH's (Of serine, for example)
and the N–H of tryptophan.
(iii) Weakly hydrophobic Van der Waal's attractive forces engendered by side-chain R groups and
(iv) Disulfide cross linkages between loops of the polypeptide chain.
The same kind of attractive and repulsive forces responsible for the tertiary structure operate to hold
together and stabilize the subunits of the quaternary structure.
(iii) Tertiary structure of proteins : The tertiary structure of proteins represents overall folding of the
polypeptide chains i.e., further folding of the secondary structure. It gives rise to two major molecular
shapes viz. fibrous and globular. The main forces which stabilise the 2° and 3° structures of proteins are
hydrogen bonds, disulphide linkages, van der walls and electrostatic forces of attraction.
(iv) Quaternary structure of proteins : Some of the proteins are composed of two or more polypeptide
chains referred to as sub-units. The spatial arrangement of these subunits with respect to each other is
known as quaternary structure.
According the their biological action, they are classified as enzymes, hormones, antibodies, etc.
Protein found in living system with definite configuration and biological activity is termed as native protein.
If a native protein is subjected to physical or chemical treatment, which may disrupt its higher structures
(conformations) without affecting its primary structure, the protein is said to be denatured. During
denaturation, the protein molecule uncoils form an ordered and specific conformation into a more random
conformation leading to precipitation. Thus denaturation leads to increase in entropy and loss of biological
activity of the protein. The denaturation may be reversible or reversible. Thus, the coagulation of egg
white on boiling of egg protein is an example of irreversible protein denaturation. However, in certain
cases it is found that if the disruptive agent is removed the protein recovers its original physical and
chemical properties and biological activity the reverse of denaturation is known as renaturation.
Tests of Proteins:
Biuret test : Addition of a very dilute solution of CuSO4 to an alkaline solution of a protein is done. A
positive test is indicated by the formation of a pink violet to purple violet color.
The name of test is derived from a specific compound, biuret, which gives a positive test with this reagent
O O
H2N—C—NH—C—NH2
biuret
When a protein reacts with copper (II) sulfate (blue), the positive test is the formation of a violet colored
complex.
H
O H
N N
R O
CH—C—N— + Cu+2 Cu
H (Blue)
N N
O H
H
(Violet)
The biuret test works for any compound containing two or more of the following groups.
O O NH S
—C—NH— —C—NH2 —CH2—NH2 —C—NH2 —C—NH2

Ninhydrin Test : The ninhydrin test is a test for amino acids and proteins with a free –NH2 group.
Amino acids are detected by ninhydrin test. All amino acids give violet - coloured product with ninhydrin
(triketo hydroindene hydrate) except proline and 4 - hydroxy proline, which gives yellow colour with it.

When such an –NH2 group reacts with ninhydrin, a purple-blue complex is formed.
O O
O
R O
OH
H2N—CH—C—OH + 2 N
OH
O O
O
(Purple-Blue)
The same violet coloured dye forms from all  - AA's with 1° amino groups because only their nitrogen
is incorporated into it. The 2° amines proline and 4 - hydroxyproline give different adducts that absorb
light at a different and thus have a different yellow colour.