Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Terminal group analysis and stepwise degradation of proteins and peptides
Determination of the N-terminal amino acid sequence by reaction with phenyl isothiocyanate

PITC degradation was proposed by Edman [4, 51. The MAIN STAGES OF the reaction with PITC are shown in the scheme (p. 282); by interacting with free amino groups, PITC forms a phenylthiocarbamyl derivative (PTC derivative). In an acidic medium, this compound undergoes cyclization, followed by the Cleavage of the phenylthiohydantoin derivative (PTH derivative) of the N-terminal amino acid. The remaining part of the protein (or peptide) represents the starting material, but without the terminal residue.

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Since the residue possesses a new N-terminus, it will obviously react with PITC again. Consequently, in the next step of the reaction, the second residue of the peptide chain likewise forms a PTC derivative and can be identified.

Repeated cycles of the Edman reaction, therefore, make it possible to determine the N-terminal Amino Acid Sequence of the peptide under study. This method is of paramount importance for sequencing Peptides isolated from enzymatic hydrolysates, and consequently, for elucidating the Introduction/19.html">Primary Structure of high-molecular-weight Proteins.

PITC degradation is carried out in several steps: 1) addition of PITC, 2) cyclization of the PTC derivative, 3) extraction of the free PTH derivative, 4) Identification and determination of the PTH derivative or Analysis of the shortened peptide, 5) repeated degradation, identification, etc. Due to the varying Solubility of proteins and peptides, the Edman reaction with them must be carried out under different experimental conditions.

PTH derivatives can be identified directly or indirectly. Direct identification is achieved by Chromatography of PTH fractions. In indirect identification, PTH derivatives are first degraded by adding Ba(OH)₂ or 6 N HCl, and then the resulting free Amino Acids are determined. The indirect method is unsuitable for quantitative analysis, since such Treatment causes extensive destruction of the substances.

In The Study of small peptides, a suitable method of Amino acid analysis, for example, using an automatic amino acid analyzer, can quite definitely establish the COMPOSITION OF THE peptide deprived of its terminal group. By monitoring The change in the Amino Acid Composition at successive stages of degradation, The amino acid sequence of the peptide under study can be reconstructed.

Edman Degradation can be combined with the dansyl method. In this case, after the cleavage of the terminal residue, the N-terminus of the remaining peptide is determined, and the sequence is reconstructed based on the N-terminal analysis data.