Biochemistry & Natural Products

Determination of N-Terminal by Edman

Method
Edman Degradation
• Developed by Pehr Edman.
• Method to sequence amino acids in a peptide
• Amino terminal residue is labelled and cleaved from the
peptide without disturbing the peptide bonds between other
amino acid residues.

N-Terminal Edman Sequencing

•A cyclic peocedure where we cleave the amino acid residue one
at a time.
•Then identified by chromatography
Reaction Mechanism
Importance of Edman Method
• The Edman degradation is by far the most important method in
sequence analysis.
• Guaranteed N-terminal sequence of proteins
• No sample pre-treatment required
• Differentiation of isobaric amino acids (leucine and
isoleucine)
• Identification possible even from unknown proteins not
registered in databases
 Limitations of Edman Degradation
• As theEdman degradation proceeds from the N-terminus of the
protein, it will not work if the N-terminus has been chemically
modified.
(e.g. by acetylation or formation of pyroglutamic acid).
• Sequencing will stop if a non-α-amino acid is encountered
(e.g. isoaspartic acid), since the favored five-membered ring
intermediate is unable to be formed.
• Edman degradation is generally not useful to determine the
positions of disulfide bridges. 
 Coupled analysis
• Edman degradations can be performed directly from a
 polyvinylidene difluoride (PVDF) membrane.
• N-terminal residue sequencing resulting in five to ten
amino acid may be sufficient to identify a Protein of
Interest (POI).
 Determination of C-terminal by enzymes
• The C-terminal residue is determined by the use of either a
chemical reagent or the enzyme carboxypeptidase.
• The chemical reagent hydrazine forms
aminoacyl hydrazides with every residue except the C
terminus.
• The C terminus is readily identified by chromatographic
procedures.
 Importance of C-Terminal sequencing
•C-terminal sequencing would greatly help in verifying the
primary structures of proteins predicted from DNA
sequences

•This method will be very useful in the case of polypeptides

and protein-blocked N termini.

Limitations of C-Terminal sequencing

• The disadvantage of hydrazinolysis is that the entire sample is
used to determine just one residue
• Determination of C-terminal amino acid residues by use of
hydrazine.
 References:
• Protein
Isolation and Determination of Amino Acid Sequence
N.V. BHAGAVAN, in Medical Biochemistry (Fourth Edition),
2002

• https://chem.libretexts.org/

•https://medium.com/@primebio/principles-and-influencing-factors-
of-edman-sequencing-41acb0a023cb