Identification of N-terminal amino acids by high-performance liquid chromatography

1-Dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride, Dns-Cl) has been widely used in protein chemistry for determination of the N-terminal amino acid of peptides. The dansyl chloride reacts with both α- and Σ-amino groups (Fig. 1). After derivatization the peptide is acid hydrolyzed releasing free amino acids, amino acids such as lysine and histidine with the dansyl group attached to their side chains, together with the derivatized N-terminal amino acid. Traditionally the Dns-amino acids have been separated by thin layer chromatography and viewed by fluorescence in UV light ( Chapter 23 in vol. 1 of this series). This procedure is time-consuming, however, and is not easily quantified. More recently, methods have been developed for separation of the Dns-amino acids by reverse-phase HPLC (1,2), thus allowing for automation of the separation and quantitation of the derivatives. In order to minimize the interference by reagent byproducts, the reaction conditions have been modified from those of the traditional method.