Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Protein Structure
Protein Structure
Determination of the Structure of N-Terminal Residues in Peptide Chains

The Structure of the N-terminal residue of peptide chains can be determined using the following Methods.

> Using Sanger's reagent (proposed in 1945), which is 1,5-difluoro-2,4-dinitrobenzene (DNFB). The free unprotonated amino group of the polypeptide's N-terminal amino acid reacts with DNP in an alkaline medium to form yellow-colored 2,4-dinitrophenyl derivatives (DNP-Amino Acid Derivatives). This derivative remains stable during the acid Hydrolysis of the peptide and is easily isolated from The amino acid mixture upon completion of Cleavage. The STRUCTURE OF THE N-terminal amino acid is identified using a reference standard ("witness") via Chromatography of the reference amino acid DNP derivatives.

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The First stage of the reaction is carried out at T = 25 - 37⁰С, рН=7 - 8. The Second Stage involves standard acid hydrolysis of the protein (110 ∘С, in 6N HCl medium).

This method has limited Specificity. DNP reacts not only with the α-amino group of the terminal amino acid, but also with the ε-amino groups of Lysine, SH groups of cystine, OH groups of Tyrosine, and imidazole groups of Histidine. Nevertheless, ε-DNP-lysine is easily distinguished using a specific reference standard, while all other side DNP derivatives are colorless and do not interfere with the analysis for the N-terminal amino acid.

Instead of DNP, pipsyl chloride (p-iodobenzenesulfonyl chloride) can be used to determine the N-terminal amino acid, which forms the following addition product with the N-terminal amino acid:

A key feature of this method is that radioactive iodine is introduced into pipsyl chloride as a label, and the pipsyl derivative is quantified radiometrically.

> A more convenient METHOD FOR DETERMINING the N-terminal amino acid is the Edman Degradation. It employs phenylisothiocyanate (PTIC), known as Edman's reagent. PITC reacts with the N-terminal amino acid of the peptide to form a phenylthiocarbamoyl derivative. Subsequent acid Treatment induces its cyclization into a phenylthiohydantoin derivative of the N-terminal amino acid. During this cyclization process, the amino acid is cleaved from the peptide chain while the remainder of the peptide chain stays intact. Thus, the Edman method allows the modified N-terminal residue to be cleaved without destroying the polypeptide chain.

Reaction scheme with phenylisothiocyanate:

> Occasionally, aminopeptidases are used to determine the N-terminal residue. These Enzymes sequentially cleave Amino Acids starting from the N-terminus of the peptide chain. The best-characterized enzyme of this type is leucine aminopeptidase, which is isolated from porcine Kidneys. This enzyme was notably used to determine the N-terminal amino acids of Insulin and Ribonuclease. However, the enzyme is inefficient at cleaving terminal amino acids if they are linked to lysine, Arginine, or aromatic amino acids.

Once the structure of the N- and C-terminal amino acids has been established, the Determination of the Amino Acid Sequence of the polypeptide chain can proceed.