Amino Acids, Peptides and Proteins - Dévényi 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
Quantitative determination of DNP derivatives

The DNP fraction obtained from mixtures with a known amount of protein or peptide is chromatographed alongside a specified amount of reference DNP-Amino Acids, after which the DNP-Amino acids are eluted from the paper and their content is determined photometrically using a calibration curve plotted for the DNP-amino acids. This method allows for the Determination of the Terminal Groups of Proteins AND Peptides, the number of polypeptide chains in proteins, the minimum Molecular Weight of proteins, and the Amino Acid Composition of protein hydrolysates.

Procedure

A. A specified amount of the reference DNP derivative is hydrolyzed in 6 N HCl under the same conditions as the Hydrolysis of the DNP-protein or DNP-peptide. The hydrolysate is extracted with ether, the organic phase is evaporated, and the residue is dissolved in a small, measured volume of methanol. Aliquots of this reference solution are applied to the paper.

B. A known amount of the DNP fraction under study is chromatographed on paper under the conditions required for the given DNP derivative. Samples of the reference preparation containing various amounts of the substance are chromatographed on the same sheet of paper. After Chromatography is complete, the DNP-amino acid spots are cut out along with the reference spots, the paper is cut into small pieces, and placed into test tubes. To each tube, 5 mL of a 1% NaHCO₃ solution is added, and the samples are eluted for 30 min in the dark with periodic shaking. The absorbance of the solutions at 360 nm is measured using a spectrophotometer. The content of DNP-amino acids in the analyzed fraction is determined from a calibration curve plotted for the reference samples.

NOTES

1. The Use of reference samples treated in the same manner as the test samples eliminates errors associated with degradation during hydrolysis. Since the content of DNP derivatives is determined using calibration curves plotted for reference samples subjected to the exact same Procedures as the test samples, errors arising from uncontrolled experimental factors (such as photodegradation, losses during preparation and extraction, etc.) are compensated for.

2. DNP-Pro is measured photometrically at 385 nm, and DNP-peptides at 350 nm.

3. If previously plotted calibration curves are used, they should be adjusted to the conditions of the current experiment using correction factors.