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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2017/06/30 18:33:27」(JST)

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Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide.^[1] 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

Phenyl isothiocyanate is reacted with an uncharged N-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 derivatization not always going to completion. The derivatization 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 modern machines capable of over 99% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 pico-moles of peptide for the sequencing process. The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up the process^[2] and 100 automated devices were in use worldwide by 1973.^[3]

Limitations

Because the Edman 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. It also requires peptide amounts of 1 picomole or above for discernible results.

Coupled analysis

Following 2D SDS PAGE the proteins can be transferred to a polyvinylidene difluoride (PVDF) blotting membrane for further analysis. Edman degradations can be performed directly from a PVDF membrane. N-terminal residue sequencing resulting in five to ten amino acid may be sufficient to identify a Protein of Interest (POI).

References

^ Edman, P.; Högfeldt, Erik; Sillén, Lars Gunnar; Kinell, Per-Olof (1950). "Method for determination of the amino acid sequence in peptides". Acta Chem. Scand. 4: 283–293. doi:10.3891/acta.chem.scand.04-0283 .
^ Edman P, Begg G (March 1967). "A protein sequenator". Eur J Biochem. 1 (1): 80–91. PMID 6059350. doi:10.1111/j.1432-1033.1967.tb00047.x.
^ Niall HD (1973). "Automated Edman degradation: the protein sequenator". Meth. Enzymol. Methods in Enzymology. 27: 942–1010. ISBN 978-0-12-181890-6. PMID 4773306. doi:10.1016/S0076-6879(73)27039-8.

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English Journal

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog, Odorrana livida.

Yang K1,2, Ma C2, Zhou M2, Wang L2, Li R2, Chen T2, Shaw C2, Li W1.
Journal of peptide science : an official publication of the European Peptide Society.J Pept Sci.2016 Mar;22(3):181-5. doi: 10.1002/psc.2856.
A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by
PMID 26856692

Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.

Corrêa EA1, Kayano AM2, Diniz-Sousa R2, Setúbal SS3, Zanchi FB4, Zuliani JP5, Matos NB6, Almeida JR7, Resende LM7, Marangoni S7, da Silva SL8, Soares AM9, Calderon LA4.
Toxicon : official journal of the International Society on Toxinology.Toxicon.2016 Feb 27;115:13-21. doi: 10.1016/j.toxicon.2016.02.021. [Epub ahead of print]
Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The object
PMID 26927324

Japanese Journal

Isolation and Characterization of an Anti-Insect β-Toxin from the Venom of the Scorpion Isometrus maculatus

KAWACHI Tomoyuki,MIYASHITA Masahiro,NAKAGAWA Yoshiaki [他]
Bioscience, Biotechnology, and Biochemistry 77(1), 205-207, 2013-01
NAID 40019562095

Isolation and Characterization of an Anti-Insect β-Toxin from the Venom of the Scorpion Isometrus maculatus

KAWACHI Tomoyuki,MIYASHITA Masahiro,NAKAGAWA Yoshiaki,MIYAGAWA Hisashi
Bioscience, Biotechnology, and Biochemistry 77(1), 205-207, 2013
Im-3 was isolated from the venom of the scorpion Isometrus maculatus through several steps of HPLC fractionation based on the insect paralytic activity. Injecting Im-3 into crickets induced paralysis, …
NAID 130004138004