Polypeptides dinitrophenyl derivatives

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

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. 

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