Sanger's reagent

Sanger's reagent, or 1-fluoro-2,4-dinitrobenzene (commonly called dinitrofluorobenzene or DNFB), is a chemical used for polypeptide sequencing. In 1945, Frederick Sanger described its use for determining the N-terminal amino acid in polypeptide chains, in particular insulin. Sanger's initial results suggested that insulin was a smaller molecule than previously estimated (molecular weight 12,000), and that it consisted of four chains (two ending in glycine and two ending in phenylalanine), with the chains cross-linked by disulfide bonds. Sanger's continued work on insulin, using dinitrofluorobenzene in combination with other techniques, eventually resulted in the complete sequence of insulin (consisting of only two chains, with a molecular weight of 6,000).Joseph Fruton, "Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology". New Haven: Yale University Press, 1999. p. 216]

Following Sanger's initial report of the reagent, the dinitrofluorobenzene method was widely adopted for studying proteins, until it was superseded by other reagents for terminal analysis (e.g., diaminonaphthylsulfonyl chloride and later aminopeptidases and carboxypeptidases) and other general methods for sequence determination (e.g., Edman degradation).

Dinitrofluorobenzene reacts with the amine group in amino acids to produce dinitrophenyl-amino acids. These DNP-amino acids are moderately stable under acid hydrolysis conditions that break peptide bonds. The DNP-amino acids can then be recovered, and the identity of those amino acids can be discovered through chromatography.

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