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
Hydrolysis of DNP-protein or DNP-peptide and extraction of DNP-derivatives

The DNP derivative is hydrolyzed with acid. Typically, under experimental conditions, most DNP-Amino Acids remain stable and can be extracted with organic Solvents based on their solubility.

Procedure

The DNP derivative is hydrolyzed using an 80-fold excess of 6 N HCl at 100° C for 16 h in a sealed ampoule under vacuum or in a nitrogen atmosphere. The hydrolyzate is diluted 3-fold with distilled Water and extracted three times with peroxide-free ether. The ether fractions are pooled, washed 2–3 times with distilled water to remove traces of acid, dried over anhydrous Na₂SO₄, evaporated, and then redissolved in a few drops of methanol and 5–10 ml of water. The aqueous phase is freed from acid in vacuo and evaporated again. This procedure is repeated until HCl is completely removed. Finally, the residue is dissolved in a small volume of water.

NOTES

1. Under the described conditions, DNP-Pro is completely destroyed, and DNP-Gly is degraded by 60–70%. DNP-Pro can still be determined by performing the Hydrolysis in 12 N HCl for 4 h. For the determination of DNP-Gly, hydrolysis should be carried out for 3–4 h in 6 N HCl or in a mixture of perchloric and acetic acids.

2. The procedure described above is also recommended for the hydrolysis of DNP-Peptides.

3. If the analysis yields a negative result—i.e., terminal groups cannot be detected—the following potential causes should be considered: 1) the test substance is a cyclic molecule lacking a free α-NH₂ group; 2) the molecule has a poorly reactive (blocked) N-terminus; 3) the terminal group is acetylated (as, for example, in cytochrome c or tobacco mosaic virus protein); and 4) finally, the DNP derivative (DNP-Pro, DNP-Gly) may have decomposed during the assay. It should also be kept in mind that the yields of DNP-Val and DNP-Ile are low due to the resistance of Val-X and Ile-X peptide bonds to hydrolysis; in such cases, hydrolysis should be extended to 72 h.

4. If the terminal residue is bis-DNP-His, it is more convenient to extract the hydrolyzate with ethyl acetate, since bis-DNP-His is sparingly soluble in ether.

5. Nearly all terminal DNP derivatives are soluble in ether, whereas ε-NH₂-DNP-Lys, N-DNP-His, terminal DNP-cysteic acid, and terminal α-DNP-Arg are also soluble in water.