Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Chemistry
Structural organization of proteins
Methods for determining the N-terminal amino acid

Several Methods have been proposed to determine The Nature of the N-terminal amino acid, most notably the Sanger method (developed by F. Sanger). It is based on the arylation of a polypeptide using 2,4-dinitrofluorobenzene (DNFB), which yields a yellow-colored 2,4-dinitrophenyl derivative of the N-terminal amino acid*. The polypeptide solution is treated with DNFB, which reacts specifically with the free NH-group of the peptide's N-terminal amino acid.

* For The Development of this method, F. Sanger was awarded the Nobel Prize in 1958. In 1980, he received a second Nobel Prize, shared with W. Gilbert and P. Berg, for developing methods to determine the primary Structure of Nucleic Acids.

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Following acid Hydrolysis of the reaction product—the dinitrophenylpeptide—only the N-terminal amino acid remains linked to the reagent as a 2,4-dinitrophenylamino acid (which is stable during hydrolysis). Unlike the other free Amino Acids released during polypeptide hydrolysis, this derivative is yellow and is identified using Chromatography.

Edman's phenylthiohydantoin method is much more widely used for determining the N-terminal amino acid due to its high sensitivity and The ability to repeatedly analyze the same sample. Phenylisothiocyanate reacts with the free $\alpha$-NH₂ group of the polypeptide's N-terminal amino acid to form a phenylthiocarbamoyl peptide.

Treatment of the reaction product with an acid induces cyclization and the release of the N-terminal amino acid phenylthiohydantoin, the identity of which is established chromatographically. The shortened polypeptide, lacking one amino acid, is then subjected to further analysis.

This Procedure of stepwise peptide Cleavage from the N-terminus can be repeated numerous times, allowing one amino acid after another to be identified sequentially. Edman's degradation serves as the chemical basis for determining the Introduction/19.html">Primary Structure of Proteins and Peptides. It has been automated in a specialized device called a Sequencer (derived from the English word "sequence"), which makes it possible to determine the Amino Acid Sequence from the peptide's N-terminus for up to 50–60 amino acid residues.

In addition to these Reagents, potassium cyanate, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), and dabsyl chloride are used to determine The amino acid sequence. Exopeptidases, such as Alanine and leucine aminopeptidases, are also occasionally employed for this purpose. These Enzymes cleave peptide bonds at the end of the polypeptide bearing a free NH₂ group, releasing the N-terminal amino acid (for The Mechanism of exopeptidase action, see Chapter 12).