Amino Acids, Peptides and Proteins - Devenyi T., Gergely J. 1976

End-group analysis and stepwise degradation of proteins and peptides
Determination of N-terminal groups by the 2,4-dinitrofluorobenzene method

Following the classic work by Sanger [17], the 2,4-dinitrofluorobenzene method became one of the most important techniques in Protein Chemistry. The method is based on the following reactions:

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The reactions proceed in a slightly alkaline medium: in the presence of NaHCО₃, 2,4-dinitrofluorobenzene (DNFB) binds to the free NH₂ groups of the peptide chain. Upon Hydrolysis of the dinitrophenylated protein (DNP-protein) with 20% НСl, yellow-colored DNP-Amino Acids are formed alongside free amino acids. The DNP-amino acids corresponding to the N-terminal residue of the protein (or peptide) carry a Substitution at the a-carbon atom. Other DNP-Amino Acid Derivatives (ε-NH ₂-DNP-Lys, O-DNP-Tyr) typically originate not from terminal amino acids, but from residues located within the chain! Thus, by quantitatively determining and identifying a-DNP-amino acids, we obtain data on the N-terminal amino acids, which enables us to calculate the number of peptide chains in the protein under study.

DNFB is a highly reactive compound that reacts quantitatively with Proteins under mild conditions without side Cleavage of peptide bonds. Therefore, one need not worry that “new” terminal groups might appear during the analysis As a result of peptide bond cleavage.

DNP derivatives, with the exception of certain ones formed by reaction with internal chain residues, are yellow in color, which facilitates their identification and assay. These compounds are light-sensitive; therefore, experiments must be conducted in the dark or, at least, direct sunlight should be avoided in favor of artificial lighting.

The analysis consists of the following stages: 1) dinitrophenylation, 2) removal of excess reagent, 3) hydrolysis of the DNP derivative, 4) dissolution of the DNP-amino acids.