Sanger N-terminal analysis

Another method for sequence analysis is the Sanger N-terminal analysis, based on the use of 2,4-dinitrofluorobenzene (DNFB). When a polypeptide is treated with DNFB in mildly basic solution, a nucleophilic aromatic substitution reaction (SnAt, Section 21.1 lA) takes place involving the free amino group of the N-terminal residue. Subsequent hydrolysis of the polypeptide gives a mixture of amino acids in which the N-terminal amino acid is labeled with a 2,4-dinitrophenyl group. After separating this amino acid from the mixture, it can be identified by comparison with known standards. 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

Dinitrofluorobenzene will react with any free amino group in a polypeptide, including the e-amino group of lysine, and this fact complicates Sanger analyses. Only the N-terminal amino acid residue of a peptide will bear the 2,4-dinitrophenyl group at its a-amino group, however. Nevertheless, the Edman method of N-terminal analysis is much more widely used. ... 

Several chemical methods have been devised for identifying the N terminal ammo acid They all take advantage of the fact that the N terminal ammo group is free and can act as a nucleophile The a ammo groups of all the other ammo acids are part of amide linkages are not free and are much less nucleophilic Sanger s method for N terminal residue analysis involves treating a peptide with 1 fluoro 2 4 dimtrobenzene which is very reactive toward nucleophilic aromatic substitution (Chapter 23)... 

When Sanger s method for N terminal residue analysis was discussed you may have wondered why it was not done sequentially Simply start at the N terminus and work steadily back to the C terminus identifying one ammo acid after another The idea is fine but It just doesn t work well m practice at least with 1 fluoro 2 4 dimtrobenzene... 

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. 

The high reactivity of di- and trinitrophenyl fluorides towards nucleophiles has been used for the arylation of various N-nucleophiles. A method was developed for the determination of N-terminal amino acids in peptides. Thus, nucleophilic attack of the amino acid nitrogen at Sanger s reagent (2,4-dinitrophenyl fluoride, 4), hydrolysis and subsequent analysis of the N-(2,4-dinitrophenyl)amino acid allows determination of the amino acid.162,163 Although this method has been replaced by more efficient procedures, it marked a milestone in the elucidation of peptide structures (Nobel Prize 1958). A variety of N-nuclcophilcs (no amino acids) which have been used in the nucleophilic substitution of 2,4-dinitrophcnyl fluoride is listed. 

Using procedures such as those outlined in this section more than 100 proteins have been sequenced. This is an impressive accomplishment considering the complexity and size of many of these molecules (see, for example, Table 25-3). It has been little more than two decades since the first amino acid sequence of a protein was reported by F. Sanger, who determined the primary structure of insulin (1953). This work remains a landmark in the history of chemistry because it established for the first time that proteins have definite primary structures in the same way that other organic molecules do. Up until that time, the concept of definite primary structures for proteins was openly questioned. Sanger developed the method of analysis for N-terminal amino acids using 2,4-dinitrofluorobenzene and received a Nobel Prize in 1958 for his success in determining the amino-acid sequence of insulin. 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

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