Separation of dansyl amino acids in a single analysis

Abstract

The reagent, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl) 1 1 Abbreviations: dansyl or DNS- = 1-dimethylaminonaphthalene-5-sulfonyl-DNP = dinitrophenyl. Conventional abbreviations of amino acids are used for derivatives and peptides. was first proposed as an N-terminal reagent for proteins and peptides by Gray and Hartley (1, 2). They reported separation of most of the dansyl amino acids by high-voltage electrophoresis on paper at pH 4.4. Dansyl-serine, -proline, and -alanine, which migrate together in this system, could be resolved electrophoretically at pH 12.7. The advantages of this method are twofold: the derivatives retain the relative charge properties of the parent amino acids, and their ultraviolet fluorescence extends the sensitivity to 1 nanomole, a 100-fold increase over the DNP method. By using this method on 20 nmoles of a peptide from cytochrome C-551 in conjunction with the Edman degradation, the N-terminal sequence for 6 amino acids was determined (2). Since the original publications of Gray and Hartley, others have reported separation of dansyl amino acids by thin-layer chromatography on silica gel (3–5) and aluminum oxide (3) using various organic solvent systems. None of these methods, however, could give identification of all dansyl amino acids in a single analysis. The method presented here utilizes a combination of adsorption chromatography and high-voltage electrophoresis on cellulose thin-layer plates. The lower limit of detection of dansyl amino acids on thin-layer plates is ten picomoles.