Separation of dansyl-amino acids by polyamide layer chromatography

Publisher Summary The methods of amino-terminal analysis and sequence determination based on dansyl chloride have application because of the ease with which one can study minute amounts of peptides and proteins. Dansyl chloride is reactive toward a variety of bases. In sequence analysis, the most important of these are the primary and secondary amines present as the terminal amino groups of peptides and proteins. Compared with most other end-group reagents, dansyl chloride is rather susceptible to hydrolysis by water and hydroxyl ions. Many substances greatly quench the fluorescence of dansyl amino acids, including water, acids, and pyridine. Any dansyl chloride that remains is subsequently broken down during the acid hydrolysis; no labeling of newly exposed amino groups occurs under these conditions. The very simple procedure recommended for peptides is usually unsuitable for proteins, mainly because of a need to use much more dansyl chloride, and larger volumes.

[8] End-group analysis using dansyl chloride.

Publisher Summary This chapter discusses the formation and stability of dansyl chloride procedure Dansyl chloride is used for providing fluorescent “handles” for the study of proteins The reaction of the dye with α-chymotrypsin led them to propose its use as a means of studying the α-amino and other reactive groups of proteins and peptides It is useful in determining the amino acid sequences of small amounts of peptides Dansyl chloride is a typical aromatic sulfonyl chloride, and reacts with a wide variety of bases, forming derivatives of differing stabilities Some recommended procedure for peptides discussed are (1) reagents, buffers, and marker mixtures, (2) labeling of peptide, (3) hydrolysis, (4) visualization of compounds on paper, and (5) interpretation of results The increased requirement for dansyl chloride brings with it the necessity for removing the large amounts of DNS-OH formed by its hydrolysis The removal procedures used are labeling, removal of salt, urea, DNS-OH, hydrolysis, and identification of end groups Some of the advantages and limitations of the method are mentioned The main advantage is the ease and rapidity with which end groups may be determined on very small amounts of peptides

[12] Dansyl chloride procedure

A decrease of the absorbance at 578 nm of a cell wall suspension of mid log phase E. coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55% of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = 1: 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.



This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is -lysine. After trypsin digestion lysine is the only additional amino acid remaining at the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine–N-acetylmuramic acid–l-alanine-d-glutamic acid–meso-diaminopimelic acid–d-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5:4 in the native molecule. The lipoprotein has an unusual amino acid comosition since it contains about 65% polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40% of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About 105 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein.



The lipoprotein has an important function in stabilizing the total structure of the cell wall. It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic exmination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00707.x

Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall. The specific effect of trypsin on the membrane structure.

Use of the Dansyl Reaction in Biochemical Analysis

Some solvent systems are described for the separation of 1-dimethyl-amino-naphthalene-5-sulphonylamino acids on thin layer chromatograms. 10−10 mole per amino acid can be detected on a two-dimensional chromatogram, when the yellow fluorescence of these compounds is excited by a UV-lamp.

Zum Nachweis von Aminosäuren im 10−10-Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen

For the sensitive determination of amines their 1-dimethyl-amino-naphthalene-5-sulphonyl derivatives were separated in two-dimensional thin-layer chromatograms. 10−10–10−11 mole per amine can be detected, when the fluorescence of the DANSyl-compounds is excited by the 365 mµ Hg line.

N. Seiler

Papers

Zum Nachweis von Aminosäuren im 10−10-Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen

Zum Nachweis von Aminen im 10 −10 -Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonsäure-amiden auf Dünnschichtchromatogrammen