Edman degradation

Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. [Edman, P. "Acta Chem. Scand." 1950, "4", 283.] In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

Mechanism

Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical "phenylthiocarbamoyl" derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues (and in practice, under 30). The peptide length is limited due to the cyclical derivitization not always going to completion. The derivitization problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino acids with 98% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 picomoles of peptide for the sequencing process. Edman degradation reaction is automated to speed up the process. [cite journal |author=Niall HD |title=Automated Edman degradation: the protein sequenator |journal=Meth. Enzymol. |volume=27 |issue= |pages=942–1010 |year=1973 |pmid=4773306 |doi= |url=]

Limitations of the Edman degradation

Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminal amino acid has been chemically modified or if it is concealed within the body of the protein. It also requires the use of either guesswork or a separate procedure to determine the positions of disulfide bridges.

References

ee also

*Bergmann degradation

*Dansyl Chloride