Practical Protein Chemistry - A. Darbre 1989

Determination of the amino acid sequence of peptides by the Edman degradation method with identification of dansyl amino acids
Dansylation

Introduction of the dansyl label into the unprotonated $\alpha$-amino group of a peptide (Fig. 11.1) is carried out in an alkaline medium within a mixed aqueous-organic solvent (with the organic component serving to dissolve dansyl chloride). Under these conditions, two competing processes take place—Dansylation and the Hydrolysis of dansyl chloride—and the extent of peptide modification is determined by the relative rates of these reactions. The hydrolysis of dansyl chloride is catalyzed by pyridine [4], whereas triethylamine accelerates both the Condensation reaction and hydrolysis. Therefore, it is recommended to completely remove pyridine salts (formed after the Edman Degradation or Peptide elution from paper) from the medium by thorough sample drying in the presence of triethylamine. Traces of ammonium salts (introduced with the corresponding buffers), which react with dansyl chloride to form dansylamide, are eliminated in the same manner. The Use of triethylamine is accompanied by The formation of yet another dansyl derivative (likely due to the presence of Primary and secondary amines in triethylamine), but on polyamide plates, the spots of these compounds migrate in solvent 2 above dansylproline (Fig. 11.2).

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FIG. 11.1. Dansylation of an Amino Acid and competing hydrolysis of dansyl chloride to dansyl acid.

Modified Peptides are hydrolyzed with 6 M HCl at 100°C (5–15 h) or with a mixture of equal volumes of conc. HCl and propionic acid at 165°C (12 min) (G. Winter, unpublished data). Hydrolysis with 6 M HCl for 5 h minimizes the losses of dansylproline and dansylserine, but fails to completely cleave the peptide bonds of dansylvaline and dansylisoleucine residues with Other Amino Acids (especially with valine and isoleucine). The resulting dansyldipeptides on the chromatogram can be mistaken for dansylamino acids. The positions of the spots for some of these derivatives are shown in Fig. 11.2.

On the chromatogram, dansyldipeptides are located approximately along the line connecting the positions of the dansyl derivatives corresponding to the amino acids making up the dipeptide. The correct identification of an anomalous spot as a dansyl dipeptide derivative can be verified by extended hydrolysis (>24 h). Hydrolysis with the HCl–propionic acid mixture increases the degree of Cleavage of dansylproline and dansylserine and the probability of detecting dansyldipeptides on the chromatogram; however, it practically avoids The oxidation of dansylmethionine that occurs during hydrolysis in 6 M HCl (G. Winter, unpublished data).

FIG. 11.2. Approximate locations of spots of certain dansyldipeptides (hatched spots) relative to the spots of DNS-amino acid markers (dark spots). Amino acid designations are given in the single-letter code: a — after Solvents 1 and 2; b — after solvents 1, 2, and 3. DNS∙OH — dansyl acid, DNS∙NH2 — dansylamide.

The destruction of dansyltryptophan under acidic hydrolysis conditions can be minimized by adding 1% tryptamine to 6 M HCl; dansyltryptophan can also be cleaved using Chymotrypsin, though this is applicable only in specific cases and requires up to 5 nmol of the substance for identification. Differences between amide and carboxyl side-chain groups can be established by electrophoretic Analysis of the peptide at pH 6.5 before and after the Edman cleavage of Asx and Glx residues.