Biochemistry

1. DETERMINATION OF PRIMARY
STRUCTURE OF PROTEINS
PRADEEP SINGH, HINA YASEEN
M.Sc. Medical Biochemistry
HIMSR, JAMIA HAMDARD

2. INTRODUCTION
 Proteins are polymers composed of hundreds or even thousands of
amino acids linked by peptide bonds.
 Proteins are most abundant biomolecules which makes about 75%
of total body weight.
 Carbon, hydrogen, oxygen and nitrogen are major components of
proteins.
 Average molecular weight of proteins range between 5kDa- 220kDa.

3. ORGANISATION OF PROTEIN
 Proteins are usually large molecules with a complex three
dimensional structure.
 Four levels of proteins are commonly defined as:
1. Primary structure
2. Secondary structure
3. Tertiary structure
4. Quaternary structure

4. Primary Structure of Proteins
 It is a linear polymer formed by linking the α-carboxyl group of one
amino acid to the α- amino group of another amino acid .
 This type of linkage is called a peptide bond or amide bond forming
polypeptide chain.

5.  Dipeptide is formed by two amino acids.
Similarly, tripeptide is formed by three
amino acids. Eg: Glutathione is a tripeptide.
 Each amino acid unit in a polypeptide is called residue.
 The sequence of amino acids in a polypeptide chain is
written starting with the amino terminal residue called as
N-terminal residue.

6.  A polypeptide chain consist of a regularly repeating part, called main chain or
backbone, and a variable part, comprising the distinct side chain (R1 , R2 , R3 etc).
 The linear polypeptide chain is cross linked and the most common cross-links are
disulphide bonds.

7. PEPTIDE BOND
All amino acids are linked
by peptide bond.
N terminal end- free NH2
group of amino acid.
C terminal end- free COOH
group of amino acid.
Amino acid are sequenced
from N- terminal end to
the C- terminal end.

8. CHARACTERISTICS OF PEPTIDE BOND
1. Partial double bond character.
2. Rigid and planer.
3. C-N bond is trans in nature.
4. Uncharged but polar.
5. This prevents free rotation
around the bond between the
carbonyl carbon and the nitrogen
of the peptide bond.

9. Secondary Structure
 Secondary structure are formed by a regular pattern of H-bonds and
other non covalent interactions between the side chain residues of
amino acids that are near to one another in the linear sequence.
 Example: Myoglobin
 Types of secondary structure:
1. Alpha Helix
2. Beta Pleated Sheet
3. Loops
4. Bends

10.  α- helix  β - pleated sheet
• Right handed spiral structure.
• H- bonding interaction.
• Proline and hydroxyproline
will not allow the formation
of alpha helix
• Almost fully extended
• Stabilized by h- bonding.
• Zig- zag or pleated pattern
• Adjacent strands in a sheet can
run in the same direction or
opposite direction.

11. Tertiary Structure
 The entire three dimension conformation of a
polypeptide is referred to as tertiary structure.
 Tertiary structure is maintained by weak bonds
(H-bonds, electrostatic forces and other non
covalent interactions).
 Tertiary structure represent the lowest energy
and greatest stability state of a polypeptide
chain .
 Example: Enzymes are globular proteins with a
specific tertiary structure.
Folding of primary structure to
form tertiary structure in
enzymes

12. Quaternary Structure
 Two or more peptides combine together
to form the quaternary structure of
protein.
 These peptides chains are linked to one
another by non covalent interactions.
 Example- Haemoglobin

13. Types of protein on the basis of structure
 Proteins fall into three basic classes according to shape and
solubility:
1. Fibrous Protein – Collagen
2. Globular Protein – Myoglobin, Hemoglobin
3. Membrane Proteins

14. • Regular linear structures
• Play structural roles in the
cell (matrix formation)
• Often water Insoluble
1. Fibrous Protein 2. Globular Protein 3. Membrane Protein
• Roughly spherical in shape
• Often water soluble
• Hydrophobic amino acid side chains
present in the interior of the molecule
while hydrophilic side chains are outside
exposed to the solution
• Hydrophobic amino acid side
chains present outside
• Play role in the cellular
transport
• Often water insoluble but
soluble in detergents
Collagen
Myoglobin GPCR

15. DETERMINATION OF PRIMARY STRUCTURE
OF PROTEIN

16. Why we Need to Determine the Primary Structure
of Protein?
 Protein play important role as “the building block of life”
 Enzyme
 Hormones and hormone receptors
 Sensing device: such as rhodopsin
 Role in immune system
 Expression of genetic information (transcription)
 Constituent of important body part: collagen
 Transporters: Albumin, Myoglobin & Hb.
 To locate the gene of interest in the host cell.
 To artificial synthesis the above products by using the applications
of biotechnology, we need the determine the primary structure of
protein.

17. PURIFICATION OF PROTEINS
 Step 1: Solubilization of protein
A. Homogenization
B. Centrifugation
C. Filtration
 Step 2: Stabilization of proteins
 Step 3: Purification of proteins by various techniques including chromatography,
Gel Electrophoresis etc.
 Step 4: Digestion of proteins into smaller peptides
 Step 5: Sequencing of individual peptide.

18. DETERMINATION OF THE AMINO ACID
COMPOSITION OF A POLYPEPTIDE
 The first step in determining the primary structure of a polypeptide is to identify and
quantitate its constituent amino acids.
 Cleavage of disulphide bonds by the 2-Ercaptoethanol.
 A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid
(6M HCl) at 110°C for 24 hours.
 This treatment cleaves the peptide bonds and releases the individual amino acids,
which can be separated by cation-exchange chromatography.

19.  The separated amino acids contained in the eluate from the
column are quantitated by heating them with Ninhydrin—a
reagent that forms a purple compound with most amino acids,
ammonia, and amines.
 The amount of each amino acid is determined by
spectrophotometrically by measuring the amount of light
absorbed by the Ninhydrin derivative.

20. Determination of amino acid composition
Several Methods are used in combination to get the final composition-
ACID HYDROLYSIS: Purified Sample to be analysed is first hydrolysed by 6N
HCL and heated at 110-120 °C for 24hrs. in sealed vessel .
 The peptide bonds are broken and the hydrolysate is analysed by HLPC to
determine the composition.
 It degrades serine, threonine, tyrosine, tryptophan.

21.  ALKALINE HYDROLYSIS: A Purified sample to be analysed is
hydrolysed with a strong base e.g: NaOH and hydrolysate is
examined.
 It does not destroy tyrosine , tryptophan and glutamine but it destroys
Serine, threonine, arginine.
 ENZYMATIC HYDROLYSIS: To cleave the proteins into a small
number of peptide fragments.
 Cyanogen Bromide (CNBr) splits polypeptide chain only on the
carboxylic side of methionine residues.

22. SEQUENCING OF PROTEIN

23. End group analysis
 Identification of N- terminal and C- terminal amino acids in a
polypeptide chain is called End group analysis.

24. Identification of N- terminal
1) Sanger’s Method
2) Edman’s Degradation Technique
3) Dansyl Chloride Method

25. Sanger’s Method
 This was the first technique to determine the sequence of proteins.
 Sanger’s Reagent – 2,4- Dinitroflouro Benzene.
 Sanger’ s Reagent derivatizes the amino terminal
residues.
 The first proteins to be sequenced by the method
is Insulin by Fredrick Sanger. He got Noble Prize
in 1958.
 Only dipeptides or tripeptides can be sequenced.

26. Steps:
 2,4- dinitro fluorobenzene is reacted with amino group of a peptide
or a proteins to form 2,4- dinitrophenyl derivative of N- terminal
amino acid which is yellow in colour.
 The treated peptide is then subjected to acid hydrolysis which
cleaves all the peptide bonds except the bond between 2,4- DNF
and NH2 group which is resistant to acid hydrolysis .
 Separated by Chromatography.

27. Steps:
+

28. Edman’s Degradation Method
 This process was developed by Pehr Edmen.
 Edman ‘s Reagent – Phenyl isothiocyanate
 It is a technique for identifying specific amino
acids at each position in the peptide chain
beginning at the amino terminal end.
 Edman’s technique can sequence many residues
(5-40)of a single polypeptide sample.

29. METHOD
 Phenyl iso thiocyanate (PITC) react with amino group of a polypeptide
under mild alkaline conditions to form a corresponding phenylthiol-
carbamoyl-peptide.
 It involves a controlled stepwise cleavage of the polypeptide.
 ADVANTAGE: This method over sanger’s Method is that the remaining
peptide after the removal of the N-terminal amino acid is not hydrolysed
and can be used again to detect the next amino acid.

31. DANSYL CHLORIDE METHOD
 Dansyl chloride- 5’-dimethyl-1-naphthalene-sulphonyl-chloride
 NH2 –terminal is reacted with dansyl chloride which is a fluorescent
compound to form a fluorescent dansyl amino acid derivative.
 This derivative is removed from polypeptide by hydrolysis. Amino
terminal residue is separated by Chromatography.
 ADVANTAGE: It is a sensitive method and can detect picomole
quantity

33. Identification of C- Terminal amino acid
 Akabori Method
 By Carboxy Peptidase

34. AKABORI METHOD
 Akabori method - Treatment with hydrazine
 In Akabori method, identification of c-terminus amino acids, involves the
heating of a linear peptide in the presence of anhydrous hydrazine in a sealed
tube for several hours
 The amino group of each peptide bond react with hydrazine to form the
corresponding amino acid hydrazide.
 Identified by chromatography.

35.  The application of microwave irradiation to the Akabori reaction
was investigated . It was observed that microwave irradiation
reduced the time required for the completion of the Akabori
reaction from hours to minutes.
This approach provided information not only about the C-terminus
but also the N-terminus.

37. BY CARBOXYPEPTIDASES
 Carboxy peptidases cleave a polypeptide form C-terminal,
removing one amino acid at a time by cleaving the peptide
bond.
 Removal of one amino acid leaves behind a new polypeptide
which is the target of carboxy peptidase .
 Carboxypeptidases that have a stronger preference for those
amino acids containing aromatic or branched hydrocarbon
chains are called carboxypeptidase A

38.  Carboxypeptidases that cleave positively charged amino acids
(arginine, lysine) are called carboxypeptidase B
 Plants contain carboxypeptidase C that liberates the amino acid
proline as well as being able to release many of the other protein
amino acids.

39. CLEAVAGE OF THE POLYPEPTIDE INTO
SMALLER FRAGMENTS

40. ENZYMATIC METHOD
 To cleave the proteins into a small no. of pure fragments.
 Example- Cyanogen Bromide (CNBr) splits polypeptide chain only on the
carboxylic side of methionine residues.
 Highly specific cleavage is also obtained by trypsin, a proteolytic enzyme
secreted by pancreas.
 Trypsin cleaves polypeptide chain on the carboxylic side of arginine residues.
 Peptides obtained specific chemical or enzymatic cleavage are separated by
some type of chromatography.

41. Reagent
Chemical cleavage
Cleavage site
Cyanogen bromide COOH side of methionine residue
hydroxylamine Asparagine - glycine bond
O-iodosobenzoate COOH side of tryptophan residue
2- Nitro-5-
thiocyanobenzoate
NH2 side of cysteine residue
Enzymatic cleavage
Trypsin Lysin and arginine residue
Chymotrypsin Tyrosine, tryptophan, phenylalanine, leucine and
methionine
Thrombin Arginine
Carbopeptidase A C-terminal amino acid
clostripain Arginine residue

43. • When exposed to trypsin
• When expose to chymotrypsin

45. MASS SPECTROMETRY

46.  PRINCIPLE- A mass spectrometer generates multiple ions from the
sample under investigation, it then separates them according to
their specific mass-to-charge ratio (m/z), and then records the
relative abundance of each ion type.
 Detect ions-

47.  For small organic molecules the MW can be determined to within 5 ppm
or 0.0005% which is sufficiently accurate to confirm the molecular
formula from mass alone
 For large biomolecules the MW can be determined within an accuracy of
0.01% (i.e. within 5 Da for a 50 kD protein)
 Recall 1 dalton = 1 atomic mass unit (1 amu)

48.  Mass spectrometry is a technique for analyzing ionized
forms of molecules in the gas phase.
 It is most readily applied to gases or to volatile liquids that
easily release gas-phase ions.
 Mass measurement obtained by determining how readily
ion is accelerated in an applied electric field.

49. Mass spectrometry

50. METHODS OF MS
Methods are widely used:-
 ESI-QTOF
 Electrospray ionization source + quadrupole mass filter + time-of-flight mass
analyzer
 MALDI-QTOF
 Matrix-assisted laser desorption ionization + quadrupole + time-of-flight
mass analyzer
 Tandem LC and Tandem MS
 Separates by HPLC, ID’s by mass and AA sequence.

51. Different Ionization Methods
 Electron Impact
 small molecules, 1-1000 Daltons
 Fast Atom Bombardment
 peptides, sugars, up to 6000 Daltons
 Electrospray Ionization
 peptides, proteins, up to 200,000 Daltons
 Matrix Assisted Laser Desorption
 peptides, proteins, DNA, up to 500 kD

52. MALDI
 Sample is ionized by bombarding sample with laser light
 Sample is mixed with a UV absorbent matrix (sinapinic acid for
proteins, 4-hydroxycinnaminic acid for peptides)
 Light wavelength matches that of absorbance maximum of matrix
so that the matrix transfers some of its energy to the analyte (leads
to ion sputtering)

53. Tandem Mass Spectrometry
 Purpose is to fragment ions from parent ion to provide structural
information about a molecule
 Also allows mass separation and AA identification of compounds
in complex mixtures
 Uses two or more mass analyzers/filters separated by a collision
cell filled with Argon or Xenon
 Collision cell is where selected ions are sent for further
fragmentation

54. Advantages of Tandem Mass
Spectrometry
 FAST
 No Gels
 Determines MW and AA sequence
 Can be used on complex mixtures-including low copy #
 Can detect post-translational modif.-ICAT
 High-thoughput capability

56. Enzyme linked immunosorbent assays
Western blot of SDS- polyacrylamide gel
Immunofluorescence microscopy
Other Techniques To Investigate The
Protein

57. Sickle Cell Disease
 It is an genetic inherited blood disorder.
 It occur due to a single amino acid substitution, in which glutamate
at position 6th has been replaced with Valine.

58.  Replacement of the charged glutamate with the nonpolar
valine forms a protrusion on the β-globin that fits into a
complementary site on the β chain of another
hemoglobin molecule in the cell.
 Sickled cells frequently block the flow of blood in the
narrow capillaries.
 Sickled cells have a decreased ability to deform and an
increased tendency to adhere to vessel walls, and so have
difficulty moving through small vessels.

59. Thank You