Study of the Dansylation Reaction of Amino Acids, Peptides and Proteins

Abstract

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pK of the group to be dansylated. A complete chromatographic separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidly. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N‐terminal amino acids, and 5–10 nmoles for their quantitative estimation. Some examples (α‐chymotrypsin, trypsinogen, ribonuclease, lysozyme, aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method.

Simultaneous Determination of Free Amino Acids, L-Carnosine, Purine, Pyrimidine, and Nucleosides in Meat by Liquid Chromatography/Single Quadrupole Mass Spectrometry

“…to the literature, optimal pH for the reaction is 9; [22,24,39] our results are similar. Dansylation reaction medium must be also carefully selected from the point of view of solubility of all compounds of interest or else product yield would suffer.…”

“…In principle all primary=secondary aliphatic, heterocyclic, and even aromatic amines undergo the dansylation process, as do also other functional groups such as -SH or -OH (present in cystine and tyrosine, respectively). [24] Versatility of the process helps to quickly determine various classes of chemicals: amino acids=peptides=proteins, [24,22] low-molecularmass amines, [20] aliphatic amines, [25] biogenic amines, [15,19,21,22] xenoestrogens (4-nonylphenol, bisphenol), [18] N-nitrosamines, [26] or even robenidine. [27] Free nitrogen bases (purine=pyrimidine) and their nucleosides are commonly determined using reversed phase liquid chromatography [28,29] or ion chromatography.…”

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: An example of application for protein chemistry

“…This result would strongly indicate the high purity of the synthesized IADC. It also had k max of 250 nm and 330 nm which is characteristic of compounds containing dansyl group [13,14]. Molar absorption coefficient of IADC at 330 nm was 4.6Â10 3 cm À1 .To conclude, these data indicate that the synthesized compound is Scheme 1.…”

Dansyl‐peptides matrix‐assisted laser desorption/ionization mass spectrometric (MALDI‐MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI‐MS/MS analysis of the proteome

“…Moreover, the modification of the lysine side chain was not always quantitative, because the pH of the reaction was kept at 9.0 while the pKa value of the ε‐amine lysine is 10.5. However, increasing the pH labeling conditions would increase the rate of dansyl chloride hydrolysis 29. Thus, pH 9.0 was maintained for all experiments and in some cases the same peptide was detected twice, both with modified and unmodified lysine ε‐amine.…”