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

Analysis of Terminal Groups and Stepwise Degradation of Proteins and Peptides
Determination of N-Terminal Groups by the 2,4-Dinitrofluorobenzene Method
Dinitrophenylation of Proteins

The protein is treated with DNFB in a slightly alkaline medium at room Temperature.

Procedure

1 g of the protein is dissolved or suspended in 10 mL of a 10% NaHCO3 solution, and 2 volumes of a 5% solution of DNFB in ethanol are added. The reaction mixture is shaken in the dark at room temperature for 4–72 h. The incubation time depends on the solubility and reactivity of the protein under investigation. If the reaction proceeds in a heterogeneous phase, i.e., a sparingly soluble substance is dinitrophenylated, it is better to increase the incubation time and compare the results obtained after dinitrophenylation for 24, 48, and 72 h. However, if the reaction proceeds in a homogeneous phase, i.e., a bicarbonate-soluble protein is dinitrophenylated or the protein does not precipitate upon The addition of the alcoholic DNFB solution (or only a small precipitate forms), the reaction is complete within 2–4 h. Upon completion of the reaction, the solution or suspension is acidified with a few drops of 0.6 N HCl. The precipitate is separated by centrifugation and washed first with an ethanol–ether (1 : 1) mixture and then with ether. The DNP-protein is stored in a dark place in a vacuum desiccator.



Last update: 06/08/2026

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