Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

Abstract

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and δ-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70°C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-μm Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methanesulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the expewrimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 μg) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-μm particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions. However, in this case it is necessary to elute the excess reagent from the column at the end of the day, and this can only be achieved using a high concentration of solvent B (85% acetonitrile). The present method (isocratic elution) is particularly useful for checking, firstly, the purity of the dimethyaminoazobenzene thiocarbamoyl-peptides, separated by reversed-phase HPLC (J.-Y. Chang (1981) Biochem. J. 199, 537–545) by their N-terminal amino acid analysis (J.-Y. Chang (1988) Anal. Biochem. 170, 542–556) and, secondly, for microsequencing studies of proteins and peptides, to the picomole level, using the dimethylaminoazobenzene isothiocyanate-phenylisothiocyanate double-coupling method. We believe that the two different HPLC methods described in this paper could be very useful for amino acid analysis and microsequencing studies at very high sensitivity.