Peptides Sanger degradation

Sanger degradation Peptide sequencing by tagging succe.ssive /V-terminal amino acids—residues at the amino end of the chain. 

Sanger degradation (Section 23.4) A method for determining the amino acid at the amino terminus of a chain using 2,4-dinitrofluorobenzene. Unfortunately, the Sanger degradation hydrolyzes, and thus destroys, the entire peptide. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

Know the meaning of amino acid sequence, Sanger s reagent, Edman degradation, selective peptide cleavage. 

Understand how peptides are sequenced, using Sanger s reagent, the Edman degradation, and enzyme hydrolysis. (Problems 26.37 and 26.38)... 

The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HC1. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified. 

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

The Sanger end-group determination is sometimes used as an alternative to the Edman degradation. In the Sanger method, a peptide is allowed to react witi> 2.4-dinitrolluorobenr ae. the peptide is hydrolyzed, and the N-terminal amino add is identified by separation as its iV-2,4-dinitrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

It is always useful to determine the N-terminal amino acid in the peptide even before the sequencing by Edman degradation is attempted. The N-terminal amino acid is determined by reacting the peptide with Sanger s reagent fluorodinitrobenzene (FDNB), and then the amino acids... 

Chemical reagents that induce degradation of a peptide allow various constituent amino acid residues to be identified. Ninhydrin is a common reagent used to indicate the presence of amino acids. Disulfide linkages can be cleaved with peroxyformic acid or with mercaptoethanol. Sanger s reagent forms a compound that allows the N-terminal amino acid to be identified. Dansyl chloride also reacts with the N-terminal amino acid to form a readily identifiable compound. Phenylisothiocyanate reacts with the N-terminal amino acid to form a phenylthiohydantoin derivative, which is readily identified. 

Mixtures of peptides result from partial degradation of proteins. Separation of the individual components and establishment of their amino acid sequence form the basis for elucidating the chemical structure of proteins for information about the methods of degradation which yield larger and smaller peptides, reference may be made to Sanger [130] (partial hydrolysis) Craig et al. (oxidative fission of S—S... 

Scheme 12.68. A representation of the Sanger method of degradation of a peptide at the N-terminus. The example chosen shows the reaction between the N-terminus amino acid alanine (Ala, A) and 2,4-dinitrofluorobenzene (DNFB). A phenylalanine (Phe, F) is shown adjacent to the terminal alanine (Ala, A) as the second from the N-terminus. The remainder of the peptide is not shown.The products of the reaction are shown as the A-(2,4-dinitrophenyl) alanine and the peptide chain where the phenylalanine (Phe, F) is now the new terminal amino acid.

FIGURE 23.36 The Edman degradation procedure for sequencing polyamino acids uses phenylisothiocyanate.The amino terminus is identified by the structure of the phenylthiohydantoin formed. Notice that this method does not destroy the peptide chain as the Sanger procedure does. 

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