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

End-group analysis and stepwise degradation of proteins and peptides
Determination of amino acid sequences in peptides by Edman degradation and quantitative amino acid analysis
Identification and determination of PTH derivatives

In stepwise peptide degradation, the sequence can be determined simply by analyzing the Amino Acid Composition of the residual peptide remaining after each successive Cleavage step. Suppose a peptide has the composition A, B, C, D, E. If we determine the COMPOSITION OF THE shortened peptide after each step of PTC degradation, we obtain the following results:

after degradation

I: A, C, D, E;

residue B lost;

after degradation

II: A, D, E;

residue C lost;

after degradation

III: D, E;

residue A lost;

after degradation

IV: E;

residue D lost

From the obtained data, it is obvious that the peptide has the sequence B-C-A-D-E.

However, this method is only practical when Amino acid analysis can be carried out rapidly and with sufficient accuracy.

PTH derivatives are identified and determined by paper Chromatography either directly or after their degradation.

DIRECT IDENTIFICATION

Whatman 1 filter paper is moistened with a starch solution and air-dried. Three solvent mixtures are used for chromatography: heptane–pyridine (7 : 3); heptane–butanol–formic acid (4 : 4 : 2); heptane–butanol–formic acid (4 : 2 : 4), upper phase.

Ascending chromatography is run for 5–8 hours. Spots are visualized by spraying the paper with a freshly prepared iodine-azide reagent; PTH derivatives appear as white spots on a brown Background.

Notes. The chromatographic spots lack sharp boundaries and exhibit significant tailing due to adsorption. Spot visualization is not entirely satisfactory: its Specificity is insufficient, and furthermore, the spots fade rapidly, leaving a very short time window to evaluate the chromatogram. Faint spots are poorly defined.

INDIRECT IDENTIFICATION [6]

PTH derivatives are hydrolyzed in 6 N HCl at 150°C (16 h). Neutralized samples are chromatographed in a butanol system in the same manner as free Amino Acids.

Note. PTH derivatives of Ser, Thr, Cys, and Pro are completely destroyed; PTH-Trp yields Gly.



Last update: 19/08/2026

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