Peptides are formed by the condensation of amino acids, with a specific structure that includes an N-terminal and C-terminal residue. The primary structure refers to the exact sequence of amino acids in a polypeptide chain, which can be determined through methods such as Sanger's or Edman degradation for N-terminal analysis and enzymatic methods for C-terminal analysis. Various chemical reactions and chromatography techniques are employed to identify and analyze amino acids in the peptide sequence.

1. Presented by:-
Smt. Jyotsnarani Panda
Asst. Professor of Chemistry, F.M. (Auto) College ,
Balasore

2. PEPTIDES
Peptides are
amides formed by
condensation of
amino group of
one alpha amino
acid with the
carboxyl group of
the other amino
acid with the
elimination of a
molecule of
water.

3. N-Terminal & C-Terminal Amino Acid
Residues
In a peptide, the amino acid that contains the free amino group is called
N-terminal residue and the amino acid that contains the free carboxyl
group is called C-terminal residue.
Peptides are always written with the N-terminal residue on the left and
the C-terminal residue on the right.

4. X-ray analysis has revealed that the entire peptide linkage is
flat.
Structure Of Peptide Linkage
In a peptide linkage the oxygen atom and the hydrogen atom
bonded to N, are trans to each other.

5. The lone pair of electrons on the N-atom in the peptide
bond is delocalized over C=O group, so C-N bond has 50%
double bond character.
Rotation around C-N bond is hindered and due to
hindered rotation, the peptide bond can show
geometrical isomerism.

6. Free rotation of a peptide chain can occur only around
the bonds joining the nearly planner amide groups to the
α-carbon.
So it is possible to describe the conformation of the
polypeptide chain in terms of the angle φ between
R - CH –NH and the angle ψ between R - CH –CO
bonds.

7. Determination of Primary Structure of Peptides:-
The exact sequence of the amino acids that are present in a
polypeptide chain, is called the primary structure of the
polypeptide.
The polypeptide is treated with a suitable reagent which
reacts either with the N-terminal or the C-terminal amino acid
residue to form the tagged peptide.
The tagged peptide is then subjected to partial hydrolysis
that releases the tagged amino acid which is then identified.
This process is repeated a number of times till the entire
peptide chain is degraded and all the amino acids are
identified.

8. END GROUP ANALYSIS:-
N-terminal residue analysis:-
Sanger’s Method:-
The peptide is treated with
2,4-dinitrofluorobenzene (DNFB) in presence of
mildly basic solution of aqueous sodium
bicarbonate at room temperature.
Acid hydrolysis gives the DNP derivative of the N-
terminal amino acid which is extracted and
identified by chromatography and comparison with
the standards.

9. Reaction:-

10. Dansyl Method:-
Peptide is treated with dansyl chloride, DNS-Cl 5-(N,N-
dimethyl)- amino naphthalene-1-sulphonyl chloride.
Acid hydrolysis gives a highly fluorescent DNS Amino acid
which is identified in minute amounts by fluriometric
methods.
Reaction:-

12. Edman Degradation:-
Nucleophilic addition of the free amino group of the
polypeptide to the C=N of phenyl isothiocyanate in a mild
basic medium.
Ring closure forms a N-phenylthiohydantoin which
detaches itself from the rest of the peptide which remains
intact with all its sequences.
N-phenylthiohydantoin is identified chromatographically
by comparing with standards. This leads to identification of
N-terminal amino acid.
The residual peptide chain is subjected to Edman
degradation repeatedly and all the amino acids in the
polypeptide are identified.

13. Reaction:-

15. C-terminal Residue Analysis:-
Hydrazinolysis Method (Akabori Method):-
The polypeptide is heated with anhydrous hydrazine
at 100oC.
The C-terminal amino acid separates and other
amino acids of the parent polypeptide chain form the
corresponding α-aminoacyl hydrazides.
The products are subjected to chromatography on a
column of a strong cation-exchange resin.
On elution the basic hydrazides are retained while the
free amino acid is eluted and can be identified.

16. Reaction:-

17. Enzymatic Method (Carboxypeptidase Method ):-
The enzyme carboxypeptidase (obtained from pancreas)
hydrolyses the peptide linkage at the C-terminal which
holds the amino acid with a free carboxyl group.
The free amino acid unit of the C-terminal and the
degraded peptide is formed.
The C-terminal amino acid is identified.
The degraded peptide with a new C-terminal residue is
again hydrolyzed with the enzyme to get a second free
amino acid and a further shortened peptide.
The process is repeated till the sequence of all the amino
acid residues in the protein is worked out.

18. Reaction:-