The document discusses the structure and properties of proteins and amino acids. Some key points: - Proteins are polymers of amino acids linked by peptide bonds. There are 20 standard amino acids that vary in properties based on their side chains. - Proteins have primary, secondary, tertiary, and quaternary levels of structure determined by amino acid sequence and interactions. - Amino acids form peptides and proteins through condensation reactions between carboxyl and amino groups, forming peptide bonds. - Proteins can be classified based on function, shape, nutritional value, and composition. Structural proteins like collagen resist digestion while globular proteins are soluble.

1. Dr.S.Sethupathy,M.D.,Ph.D.,
Rajah Muthiah Medical College,
Annamalai University.

2. Proteins
 Greek word – proteios which means of supreme
importance.
 ¾ of the the total dry body weight.
 Composed of mainly carbon, hydrogen, oxygen and
nitrogen
 Sulphur and phosphorus as minor constituents.
 Nitrogen content of proteins is 16% by weight
 The proteins are polymers of amino acids.

3. Amino acids
They have a carboxylic group (-COOH)
and primary amino group (-NH2)
attached to alpha carbon - carbon next
to carboxyl carbon .
 Based on R group the amino acids vary
from each other.

4. Peptide bond
Amino acids in proteins are linked
by peptide bonds.
α- carboxyl group of one amino
acid reacts with α- amino group of
another to form a peptide bond by
removal of water. ie : - CO- NH –
bridge.

5. Peptides and proteins
Two amino acids form dipeptide and
three amino acids form tripeptide.
10-50 amino acids form polypeptide
More than 50 amino acids form
proteins.
The sequence of amino acids
determine the structure of proteins .

6. Neutral amino acids
 They have one amino and one carboxylic
group.
 They may be aliphatic or aromatic amino
acids.
 Simple Aliphatic amino acids
 Glycine and alanine .
 Glycine is the smallest and optically inactive
 Branched chain amino acids are valine,
leucine, isoleucine

7. Nonpolar aliphatic , simple A.A

8. Branched chain A.A (VIL)

9. Neutral amino acids
Hydroxy amino acids are serine and
threonine
Sulphur containing amino acids are
cysteine, methionine
Aromatic amino acids are
phenylalanine, tyrosine and
tryptophan.

10. Hydroxy amino acids (Polar)

11. Sulphur A.A
Non polar - Polar

12. Aromatic non polar - polar A.A

13. Acidic amino acids and their
amides
Aspartic acid glutamic acid – they
have one amino and two carboxyl
groups
The amide derivatives of these
acidic amino acids are asparagine
and glutamine respectively.

14. Acidic amino acids

15. Amide derivatives of Asp, Glu

16. Basic amino acids are lysine
(two-NH2 group), arginine (has a
guanidino group) in the aliphatic
chain and histidine has an
imidazole group.
Imino acids: proline is an imino
acid. It has a secondary imino
group (-NH-)

17. Basic amino acids

18. Imino acid- non polar

19. Nutritionally Essential amino acids
They are methionine, tryptophan,
threonine , valine, isoleucine,
leucine, phenylalanine and lysine.
They cannot be synthesized in the
body and they have to be supplied in
the diet.
Their deficiency causes nutritional
disorders.

20. Histidine and arginine are semi
essential as their synthesis in the
body is inadequate especially
during growth and recovery from
illness and needs dietary supply
then.
Other amino acids are nutritionally
non- essential as they can be
synthesized in the body.

21. Glucogenic and ketogenic A.A
 The amino acids which can be converted to
glucose are called glucogenic amino acids. Eg.
Glycine, alanine, glutamate etc.
 The amino acids which cannot be converted to
glucose but can form acetyl units are called
ketogenic amino acids
 The only ketogenic amino acid is leucine
whereas Phenylalanine, tyrosine, tryptophan,
and lysine are both ketogonic and glucogenic.

22.  Derived amino acids present in proteins
 Hydroxy proline and hydroxy lysine do not
have genetic code .
 They are important constituents of collagen.
 Non protein amino acids
 Derived amino acids are found free in the
cells.
 They are produced during metabolism of
amino acids. Ex : Ornithine, citrulline and
homocysteine.

23.  Non alpha amino acids
 Gamma amino butyric acid is derived from
glutamic acid.
 Beta alanine is a constituent of pantothenic
acid.
 Amino acids having non polar side
chains
 Alanine , valine , isoleucine , leucine
,methionine , proline, phenyl alanine and
tryptophan .
 They have hydrophobic groups.

24. GABA β-alanine

25.  Amino acids having non-ionic polar side
chains
 Glycine, serine, threonine, cysteine,
tyrosine, glutamine and asparagine are
hydrophilic in nature.
 Amino acids having ionic polar side
chains
 Acidic amino acids are aspartic acid and
glutamic acid. Basic amino acids are lysine,
arginine and histidine. They are hydrophilic
in nature.

26.  Amino acids
 Exist as NH3
+ - cationic form at acidic pH
 Exist as COO- - anionic form at alkaline pH
 Isoelectric pH (pI)
 It is the pH at which amino acids or proteins
having equal number of positive and negative
charges – zwitterionic form
 Do not move in electric field due to zero net
charge.

27. Isoelectric pH

28.  The isoelectric point (pI) for neutral amino acids
can be calculated:
 pI = pk1 + pk2 / 2
 Glycine - pk1 – 2.34 pk2 - 9.6
 pI = 2.34+ 9.6 /2 = 5.97
 The isoelectric point (pI) for more Acidic amino
acids can be calculated:
pI = pk1 + pkR / 2
Glutamic acid – pI = acidic group and R group /2
pk1 – 2.19 pk2 – 9.67 pkR - 4.25
pI = 2.19 + 4.25 / 2 = 3.22

29. Dibasic amino acids -pI
 The isoelectric point (pI) for more amino,
monocarboxylic amino acids can be calculated:
pI = pk2 + pkR / 2
Lysine – pk1-2.2, pk2- 8.9 , pkR- 10.5
pI = 8.9+ 10.5 = 9.7
 pK1=Dissociation constant of (-COOH) group
 pK2=Association constant of (-NH2) group
 pKR=Dissociation (or) Association constant of “R”
side chain

30. Isoelectic pH (pI)
 pK 1 is due to carboxyl group and pK2 is due
to amino group.
 pI can be calculated from the pK values.
 Eg : glycine pI = pk1 + pk2 /2 ie : 2.4 +9.8
/ 2 = 6.1. Buffering capacity is minimum at
pI. The pK value of histidine is 6.4 and is an
effective buffer at physiological pH of 7.4.

31.  Optical activity
 Except glycine all amino acids have
asymmetric carbon and are optically active.
 With reference to alpha carbon, D,L isomers
are formed.
 Natural amino acids are L- isomers.
 D-amino acids are constituents of certain
antibiotics - Actinomycin-D, valinomycin
and bacterial cell wall peptidiglycans.

32. Decarboxylation
 The amino acids will undergo alpha
decarboxylation to form amines.
 Histidine Histamine + CO2
 Tyrosine tyramine + CO2
 Tryptophan tryptamine + CO2
 glutamic acid GABA + CO2
(gamma amino butyric acid)

33. Gamma Amino Butyric Acid (GABA )
from glutamate

34. Amide formation
The carboxyl group of aspartic acid and
glutamic acid (other than alpha
carboxyl group) reacts with ammonia
to form their amide derivatives.
Aspartic acid + NH3 asparagine
Glutamic acid + NH3 glutamine

35. Glutamine formation

36. Transamination
 A pair of an amino acid and α - keto acid
react to form a new pair of α -keto acid and
amino acid by transfer of amino group.
 It helps in pooling of amino groups of the
different amino acids on α keto glutarate to
form glutamate and helps in transport of
ammonia to liver.
 It also helps in the synthesis of non essential
amino acids.

37. Transamination – PLP coenzyme

38. Oxidative deamination
Glutamic amino acid undergoes
oxidative deamination and releases
ammonia and α- keto acid.

39. Oxidative deamination

41. Carbamino compound
 Carbon dioxide adds to the alpha amino
group of amino acids to form carbamino
compounds. By this mechanism,
hemoglobin transports CO2 from tissues to
lungs for elimination.
 Hb – NH2 + CO2 Hb –NH- COOH
 (Carbamino Hb)

42. Carbamino compound

43. Transmethylation
S- adenosyl methionine is the
active donor of methyl group and
after donating methyl group, it
becomes S- adenosyl
homocysteine.

44. Transmethylation

45. Ester formation
Serine ,threonine residues can form
ester linkage with phosphoric acid.
It plays a role in the regulation of many
enzyme activities by phosphorylation
and dephosphorylation of serine or
threonine residues in them.

46. Ester formation

47. Glycosidic linkage
Hydroxyl groups of serine and threnoine
can form O-glycosidic bonds with
carbohydrate residues in glycoproteins.
Amide group of asparagine and
glutamine can form N-glycosidic bonds
with carbohydrate residues in
glycoproteins.

48. Glycosidic linkages

49. Cysteine and cystine

50. Reaction of SH group of cysteine
 It can form disulphide linkage in peptides
which may be either intra chain or inter
chain .
 Two molecules of cysteine react to form
Cystine or dicysteine.

51. Ninhydrin test

52. Biuret test

53. Xanthoproteic test

54. Lead sulphide test

55. Millon’s test

57. Sakaguchi test

58. Hopkin’s cole reaction

59. Cyanide-nitroprusside test

60. Methionine - negative
 Cysteine and cystine give this test positive
but methionine gives negative because
sulphur in methionine is not released due
to methyl group attached to it.
 Casein and gelatin contains methionine and
insignificant amount of cysteine , gives the
cyanide –nitroprusside test negative.

61. Purpose of transamination

62. A.A derivatives of clinical
importance
 Gamma amino butyric acid (GABA)- inhibitory neuro
transmitter ,formed from glutamic acid by glutamate
decarboxylase using pyridoxine as coenzyme.
 Pyridoxine deficiency, GABA not formed ,
convulsions occur.
 Histamine - mediator of allergic reactions.
 Thyroxine maintains Resting Metabolic Rate (RMR).
 Cycloserine , a derivative of serine is used as
antituberculous drug. Azaserine is an anticancer drug.

63. Proteins
 Proteins are polymers of amino acids linked by
peptide bond.
 Alpha amino group of one amino acid reacts with
alpha carboxyl group of another amino acid
forming a peptide linkage with removal of water.
 Peptides have amino terminal (Free alpha amino
group) and carboxyl terminal (Free alpha
carboxyl group) .
 Biologically important peptides are glutathione,
oxytocin , antidiuretic hormone, insulin etc.

64. Peptide bond

65. Structural organization of proteins
 Primary structure: It denotes the number and sequence
of covalently linked amino acid residues in a polypeptide.
Bonds are peptide bonds and disulphide linkages.
 Peptide 1- Gly-Val-Lys
 Peptide 2 - Val – Lys –Gly
 The sequence determines structure.
 Free alpha amino group is the N terminal amino acid
 The free alpha carboxyl terminal amino acid is the last
amino acid.
 The higher levels of structural organization are dependent
upon the primary structure.

66. Structural organization of proteins

67. Bonds responsible for protein
structure
Covalent bonds are peptide and
disulphide linkages .
Non covalent bonds are Hydrogen
bonds, Hydrophobic bond,
electrostatic or ionic bonds and
weak Van der Waals’ forces.

68. Bonds

69. Structural organization of proteins

70. Secondary structure
 It is the folding and twisting of polypeptide
chain forming a two dimensional structure due
to interaction between adjacent amino acid
residues in the linear sequence of the peptide.
 Eg. Alpha – helix is a right handed helix
 Beta pleated sheet is mainly due to inter or
intra chain interactions which may be
antiparallel or parallel sheets.

71. Tertiary structure
 Peptides further fold into a three dimensional
structure due to interaction between amino
acid residues which are far away in the linear
sequence of the peptide.
 Non-covalent forces-Hydrogen bonds,
electrostatic bonds, hydrophobic bonds and
weak van der Waals forces - secondary and
tertiary structure
 Domain is the compact functional unit of a
protein. Eg : catalytic domain of an enzyme ,
regulatory domain of a protein.

72. Quaternary structure
 More than one polypeptides interact and form
a single functional assembly. Eg : Hemoglobin
is a tetramer. (α2β2 - four polypeptides) .
 Each polypeptide chain is called as subunit or
monomer.
 Creatine kinase is a dimer .If a dimer is
composed of similar copies of polypeptides, it is
called as homodimer. Eg: CK-MM.
 If both copies are different, they are called as
heterodimers Eg : CK-MB.

73. Classification of proteins
 Based on its functions
 Structural proteins Eg: collagen is the most
abundant protein in mammals.
 Pepsin as an enzyme , Transferrin as a
Transport protein, Ferritin as a Storage
protein and insulin as hormone and
immunoglobulis as the mediator of
immunity are examples.

74. Based on shape
 Globular proteins: They are oval in shape
and are easily soluble. Eg: albumin,
globulin.
 Fibrous proteins: They are elongated and
resist digestion . Their solubility is
minimum. Eg; collagen, keratin, elastin.
 Axial ratio (Length/Breadth) is more than 10
for fibrous proteins and for globular proteins
it is less than 10.

75. Based on nutritional value
 Nutritionally rich proteins: Complete
proteins or first class proteins and with all
essential amino acids in required
proportions. Eg: Egg white , Casein of milk.
 Incomplete proteins: Lack one essential
amino acid. Eg: Cereals lack lysine and
pulses lack methionine.
 Poor proteins: Lack many essential
amino acids .Eg: Zein from corn lacks
tryptophan and lysine.

76. Based on compostion
Simple proteins : They are made up of
amino acids only.
Eg . Albumin and Globulins are soluble
in water and heat coagulable proteins.
Histones,, Protamines.

77. Conjugated proteins
 They are proteins combined with the non-
protein substance called prosthetic group.
 Eg. Glycoproteins- blood group antigens,
lipoproteins- chylomicrons,
nucleoproteins – histones with DNA,
phosphoproteins – casein of milk and vitellin
of egg yolk,
metalloproteins – hemoglobin , cytochrome.

78. Derived proteins
Derived proteins: They are derived
from simple and conjugated proteins.
 Denatured or coagulated proteins are
called primary derivatives.
 Partial hydrolysis of proteins leads to
secondary derivatives - proteoses,
peptones and peptides.

79. Biologically important peptides
 Thyrotropin releasing hormone (TRH) is a tripeptide
and releases thyroid stimulating hormone.
 Glutathione is a tripeptide and helps in the maintenance
of RBC membrane integrity and keeping certain
enzymes in an active state.
 Oxytocin and antidiuretic hormone (ADH or
Vassopressin) , Angiotensin I are peptide hormones.
 Gramicidin S is an antibiotic produced by Bacillus brevis
 It is circular ,composed of 10 amino acids and has D-
phenyl alanine.

80. Estimation of proteins
 The protein sample is digested by boiling
with concentrated sulfuric acid at 360o C in
the presence of copper sulfate and sodium
sulfate as catalysts.
 Ammonia liberated is measured. Proteins
contain 16% nitrogen, it can be calculated.
 ie Weight of nitrogen x 100 /16 or nitrogen x
6.25.
 Accurate procedure but it is tedious and
takes longer time.

81. Biuret method
 Cupric ions chelate with peptide bonds in
alkaline medium to produce violet color and
measured colorimetrically.
 Compared with known concentration of
protein standard treated similarly with
Biuret reagent.
 It is simple and widely used in clinical
laboratory.
 But its sensitivity is less and not suitable for
estimation of mg quantities.

82. Lowry’s method
 Reduction of Folin - Ciocalteau reagent ( Phospho
molybdic acid and phosphotungstic acid ) by the
tyrosine and tryptophan residues of protein.
 A blue color is developed and can be compared with a
known standard.
 This is very sensitive and microgram quantity can be
measured.
 If the tryptophan and tyrosine content is different
between test protein and standard protein, the
accuracy is lost.

83. Spectrophotometric method
 Proteins absorb UV light at 280 nm.
 This is due to tyrosine and tryptophan content.
 It is compared with known standard protein
solution.
 The method is accurate, simple and sensitive up
to micrograms.
 Since reagent is not added, the sample can be
used for other tests.
 But the instrument is expensive.

84. Nephelometry
 The detection of scattered light by turbid particles in
solution.
 Antigen-antibody complexes can be detected.
 A beam of light is passed through the solution and the
scattered light detected at an angle of 30-90 o
(preferably 60 o ).
 This is rapid and suitable for automation but the
instrument is expensive.
 Microgram quantity can be measured.

85. Turbidimetry
 The proteins in biological fluids such as urine, CSF can
be estimated by adding precipitating agents such as
sulfosalicylic acid and the turbidity is measured.
 It is simple, cheaper but less accurate.
 Detector is kept at an angle of 180 o.
 RIA and ELISA: Protein of Nanogram and picogram
quantities can be estimated by Radio immuno assay
(RIA) and Enzyme linked immuno sorbent assay
(ELISA).

86. Steps for determination of
primary structure
 N-Terminal amino acid determination
 Dansyl chloride combines with N-terminal amino acid
 The tagged polypeptides are hydrolyzed by boiling at
110 o C in 6H HCl for 18- 36 hours.
 The number of dansyl amino acids indicate the
number of polypeptides.
 N- terminal amino acid can also be determined using
Sanger’s reagent –1, Fluoro 2,4-dinitro bezene(1-F, 2,4-
DNB).

87. Ends of the peptide

88. Dansyl chloride

89. Sanger’s method

90. C-terminal amino acid
 C- terminal amino acid can be determined
using carboxypeptidase A and B.
 Carboxy peptidase A will not act if the C-
terminal amino acid is Arginine, proline or
lysine
 B will act only when the penultimate amino
acid residue is proline.

92. Breaking up larger peptides into smaller
peptides
Larger peptides are broken into smaller peptides
using
1. Trypsin hydrolyzes peptide bonds formed by
alpha carboxyl group of lysine or arginine.
2. Chymotrypsin hydrolyzes peptide bonds formed
by alpha carboxyl group of Phenyl alanine, tyrosine ,
tryptophan or leucine.
3. Cyanogen bromide hydrolyzes peptide bonds
formed by alpha carboxyl of methionine .

93. Sequencing of peptides
 The peptides with a length of 10-30 amino acids can
be sequenced using Edman’s degradation technique.
 Edman’s reagent is phenyl iso thio cyanate.
 N- terminal amino acid reacts with this and phenyl
thiohydantoin derivative is formed.
 By this reaction, sequentially amino acids of a
peptide can be identified.
 The amino acids composition of the peptide
subjected to various hydrolyzing agents are arranged
 By overlapping technique ,primary structure can be
elucidated

94. Enzymatic hydrolysis
Trypsin, chymotrypsin
 B A H J U E K I D L I F T G
 B A H J U E K I D L I F T G
 B A H J U E K I D L I F T G
 By overlapping, the whole sequence is determined.

95. Overlapping technique
 Hydrolysis of peptides using different agents to
produce fragments

97. Edman degradation

98. NMR
 Nuclear magnetic resonance (NMR) Spectroscopy is
useful in determining functional group and change in
conformation after binding with ligand.
 X- ray diffraction study is useful in studying the
structure of pure , crystallized protein.
 Chemical synthesis of peptides by solid phase
technique: Here the carboxy terminal amino acid is
attached to insoluble polystyrene beads so that
washing between reactions is rapid. The other amino
acids are added sequentially.
 Insulin was first synthesized by this method.

99. Precipitation of proteins
 Salting out: When a neutral salt such as
ammonium sulphate or sodium sulphate is
added to protein solution, the shell of
hydration is removed and protein gets
precipitated.
 The globulins are precipitated by half
saturation with ammonium sulphate where
as albumin is precipitated by full saturation
because it is more hydrated than globulin.

100.  Proteins are least soluble at iso electric pH.
 Organic solvents such as alcohol cause
precipitation of proteins by removing water from
their shell.
 Precipitation by heavy metal ions
 In alkaline solution, heavy metal ions are cations
and combine with anionic proteins to form metal
proteinates and get precipitated.
 Salts of Copper, zinc, cadmium, mercury, lead are
toxic because they precipitate proteins. Based on
this principle , raw egg can be used as antidote for
mercury poisoning.

101.  Precipitation by alkaloidal agents
 Tungstic acid, phosphotungstic acid, tannic acid,
picric acid, trichloro acetic acid and sulphosalicylic
acid are powerful protein precipitating agents.
 Tannic acid is useful in tanning process in leather
industry.
 Techniques of protein separation
 Chromatography
 It is a physical method of separation in which the
components to be separated are continuously
distributed between two phases
 stationary phase and mobile phase

102. Paper chromatography
 Paper (cellulose) acts as supporting medium.
 Water adsorbed to paper acts as stationary phase and the
non polar solvent such as butanol act as mobile phase.
 When the mobile phase moves, non polar compounds are
carried by them where as polar compounds are retained by
polar stationary phase.
 Rf value (ratio of fronts) = distance traveled by solute /
distance traveled by solvent
 Other types are thin layer Chromatography, ion exchange
Chromatography, Gas Chromatography, affinity
Chromatography, high performance liquid
Chromatography ,gel filtration Chromatography, etc.

103. Electrophorsis
 It is a technique for the separation of the
charged particles by means of electric
current.
 In an electric field, a charged particle
migrates towards cathode or anode based
on its net charges.
 Paper, agar, polyacrylmide gel
electrophoresis are the different types.

104. Biological significance of A.A
 They are involved in protein synthesis.
 Energy yield is 4 kcal / gm.
 Glycine is required for purine, haem,
creatine, glutathione synthesis.
 Cysteine is needed for glutathione synthesis.
Methionine is involved is one carbon
transfer reactions.

105. Tyrosine is the precursor of thyroid
hormones, catecholamines ,dopamine,
melanin pigment.
Tryptophan is the precursor for niacin,
serotonin and melatonin.
Glutamic acid is the precursor of
gamma amino butyic acid (GABA).
Aspartate , glutamine are needed for
purines and pyrimidines synthesis.

106. Denaturation
 Change in native conformation of proteins
due to altered non covalent forces is called
denaturation.
 There will be loss of activity of the proteins.
 Primary structure is not altered.
 Denaturation involves the secondary,
tertiary, quaternary structure of the
molecules.

107. Causes of denaturation
 Chemical agents such as Acid, alkali,
mercury, lead, alcohol
 Physical agents such as heat, UV rays,
ionizing radiation
 Mechanical means like vigorous shaking,
grinding etc.
 It can be reversible some times.

108. uses
 Denaturation causes unfolding of proteins.
 It helps in the digestion of dietary proteins
by proteolytic enzymes.
 Denatured proteins form aggregates and
precipitation occurs.
 Helps in the isolation of proteins of interest.
 Ex: Precipitation of proteins by ammonium
sulfate in the initial stages of isolation of
proteins.

109.  Floccculation
 Denatured protein at isoelectric pH gets
more precipitated forming floccules which
still can be reversible.
 Coagulation
 If the proteins are permanently and
irreversibly disorganized, it results heavy
precipitates called as coagulum and
coagulation is irreversible. Eg: Heat
coagulation.

110. Ultracentrifugation
 Proteins can be separated based on size, shape and
molecular weight based on their sedimentation
velocities.
 Using ultracentrifuge-lipoproteins can be separated
by high speed centrifugation.
 The ultracentrifuge - high as 2,00,000 g.
 Preparative ultracentrifuge is used to separate cellular
organelles.
 Analytical centrifuge is used to obtain information
about the shape, conformation and size distribution
of the molecules.

111. Thank you