Sanger’s method

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... 

FIGURE 3-25 Steps in sequencing a polypeptide, (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger s method for identifying the amino-terminal residue is shown here, (b) The Edman degradation procedure reveals... 

This is surely the case with hemoglobins and globins. Sanger s method involves prolonged exposure (2 hours at room temperature) to over 60% alcohol in the presence of the reagent and sodium bicarbonate (Sanger, 1945). This concentration of alcohol in the presence of saturated bicarbonate denatures about two-thirds of the protein in less than 2 minutes. ... 

Several methods are available to determine the N-terminal amino acid. In Sanger s method, the polypeptide chain is reacted with l-fluoro-2,4-dinitrobenzene. The dinitrophenyl (DNP) derivative of the N-terminal amino acid can be isolated and identified by ion-exchange chromatography after the polypeptide is hydrolyzed. A group of enzymes called the carboxypeptidases are used to identify the C-terminal residue. Carboxypeptidases A and B, both secreted by the pancreas, hydrolyze peptides one residue at a time from the C-terminal end. Carboxypeptidase A preferentially cleaves peptide bonds when an aromatic amino acid is the C-terminal residue. Carboxypeptidase B prefers basic residues. Because these enzymes sequentially cleave peptide bonds starting at the C-terminal residue, the first amino acid liberated is the C-terminal residue. 

A modification of Sanger s method has resulted in the commercial availability of automated DNA sequenators based on Sanger s use of dideoxy analogs of nucleotides. Instead, however, of tagging a primer with P, the purine and pyrimidine base portions of the dideoxynucleotides are each modified to contain a side chain that bears a different fluorescent dye, and all the dideoxy analogs are present in the same reaction. After electrophoretic separation of the products in a single lane, the gel is... 

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