Complete amino acid analysis of proteins by dabsyl derivatization and reversed-phase liquid chromatogrphy

Abstract

A method is described for the quantification of the primary amino acids in protein hydrolysates by dabsylation and high-performance liquid chromatography. Improvements in the established conditions for the formation and storage of amino acid dabsyl derivatives and the use of new reversed-phase columns allow the chromatographic analysis in 25 min of all the proteinogenic amino acids in well resolved peaks of homogenous and highly reproducible size. The method permits the simultaneous quantification of tryptophan residues by previous hydrolysis of the protein with sulphonic acids. The other acid-labile residues, asparagine and glutamine, can also be analysed by previous conversion into diaminopropionic and diaminobutyric acids, respecitvely, by treatment of the protein with [bis(trifluoroacetoxyiodo]-benzene. An extended chromatographic gradient programme allows the separation of many modified amino acids, naturally occurring or produced after chemical modification of proteins. The above characteristics together with a demonstrated high reproducibility (relative standard deviation 2.1%), flexibility, sensitivity (below 100 pmol), and inertness to extraneous chromatographic contamination make this improved method a good alternative to other currently used chromatographic methods for amino acid analysis.