Dansyl Amino Acids

Derivatization of free amino group of amino acids with 5-methylaminonapthalene-1-sulfonyl (dansyl) chloride has become increasingly popular for N-ter-minal determinations in proteins and for manual Edman degradation. In addition, dansylation has also been used as one of the most sensitive methods for quantitative amino acid analysis.

Two-dimensional TLC on polyamide sheets using water-formic acid (200 : 3, v/v) for the first-direction run and benzene-acetic acid (9 : 1, v/v) for development at right angles to the first run has mostly been employed in conjunction with the Edman dansyl technique for sequencing peptides. These solvents cannot resolve Dns-Glu/Asp, Dns-Thr/Ser, and a-Dns-Lys/ s-Dns-Lys/Arg/His. However, a third run in ethyl

Table 8 Characteristic colours of PTH amino acids following ninhydrin application

PTH derivative

Colour properties

NH4OH colour change

Proline

UV, colourless

Light blue after heating

Alanine

Purple

Deeper colour

Glycine

Orange

Serine

UV, purple

Serine breakdown

Faint orange

Weak red

Asparagine

Yellow

More intense

Carboxymethylcysteine

UV, purple

Methioninesulfone

Light tan

Methionine

Faint tan

Lysine

Very faint pink

Weak blue after heating

Tyrosine

UV, yellow before spray

Intense yellow

Threonine

Colourless

Light tan

Glutamine

Dark green

Dark blue

Phenylalanine

UV, colourless

Faint yellow

Tryptophan

UV, yellow before spray

Deep yellow

Aspartic acid

UV, pink

Darker

Glutamic acid

Grey

Dark blue

Silica gel plates, without fluorescent indicator, developed in heptane-CH2Cl2-propionic acid (45:25:30) and xylene-MeOH (80:10), sprayed with iodine-azide and 1.7% ninhydrin in MeOH-collidine-HOAc(15 : 2 : 5), heated at 90°C for 20 min; colour changes by blowing a saturated ammonia atmosphere over ninhydrin plate.

Silica gel plates, without fluorescent indicator, developed in heptane-CH2Cl2-propionic acid (45:25:30) and xylene-MeOH (80:10), sprayed with iodine-azide and 1.7% ninhydrin in MeOH-collidine-HOAc(15 : 2 : 5), heated at 90°C for 20 min; colour changes by blowing a saturated ammonia atmosphere over ninhydrin plate.

acetate-acetic acid-methanol (20 : 1 : 1) in the direction of solvent 2 resolves Dns-Glu/Asp, and Dns-Thr/Ser. A further run in the direction of solvent 2 and 3 using 0.05 mol L_1 trisodium phosphate-ethanol (3 : 1, v/v) resolves the monosubstituted basic Dns amino acids. Use of molarity ammonia-ethanol (1:1, v/v) as a third solvent for two-dimensional chromatograms, for the separation of basic dansyl amino acids in particular, has been effective. Most of the TLC systems reported up to 1978 required more than two runs for complete resolution of all Dns amino acids. A few solvent systems to yield separations of basic, acidic and hydroxyl derivatives in the presence of other amino acids without resorting to the third solvent system and RF values are given in Table 9. Additionally, a large number of solvent systems for one- or two-dimensional resolution of dansyl amino acids on silica gel or polyamide have been summarized in Table 10. Bhushan and Reddy reviewed the TLC of dansyl, and DNP amino acids and evolved several successful and effective solvent systems for the resolution of almost all the dansyl amino acids on silica gel plates (Tables 11 and 12).

Detection of dansyl amino acids In all cases, dansyl amino acids, being fluorescent, have been detected under a UV lamp (254 nm).

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