The authors of this article describe the determination of D/L-amino acid residues in peptides and proteins at microgram levels. They fluorescently tagged amino acid residues with naphthalene-2,3-dicarboxaldehyde and performed enantioseparation using [Beta]-cyclodextrin-modified micellar electrokinetic chromatography in the presence of methanol as an organic modifier. Their separation was coupled with laser-induced fluorescence detection for sensitive determination. The authors determined configurations of amino acid residues in peptides with 10 [micro]g of hydrolyzed peptide material. They demonstrated the assessment of enantiomeric purity for synthetic peptides by their proposed method. They also applied the method to determining D-aspartic acid, free and bound in proteins in rat brains. Although they detected high levels of free D-aspartic acid, no D-aspartic acid was bound in proteins.
Amino acids are essential components in biological systems; they serve either as individual compounds or as constituents of proteins. D-Amino acids have been considered rare in nature for a long time; however, unexpectedly high levels of D-amino acids recently were found both free and bound in peptides in samples of various origins. L-Amino acid residues in food proteins can be racemized to D-amino acids during food processing. In some cases, the racemization ratios can be greater than 25% (1). Thus, D-amino acids are continuously consumed by animals and humans. In addition, D-amino acids can be synthesized in vivo by certain microorganisms through transformation of L-amino acids. Enzymes such as amino acid oxidase, transaminases, and epimerases catalyze this type of transformation. Several D-amino acid-containing peptides have been discovered in various animals during the past decade (2-4). These peptides exhibit biological functions such as cardio-excitation and muscle activity modulation. Obviously, it is important to assess the factors that influence the formation and biological functions of D-amino acids and their utilization by animals and humans.
Biologically active peptides used as therapeutic drugs should be stereochemically pure because the stereoisomers may produce different effects. In peptide synthesis, the amino acid residues used for synthesis must be optically pure, and analysts must make efforts to reduce racemization in every synthesis step, including residue coupling, deprotection, and cleavage of the resulting peptides from resin (5). Stereochemical purity assessment for synthetic peptide materials always is necessary. However, it has been a challenge to separate peptide stereoisomers, particularly for peptides with more than five amino acid residues. A convenient, reliable way to assess stereochemical purity for synthetic peptides and to determine the configuration of amino acid residues in novel peptides and proteins is to analyze D/L-amino acid residues after hydrolyzing the peptide-protein material (6).
Analytical chemists have achieved enantioseparation of racemic amino acids mainly by chromatographic methods. Recently, chiral capillary electrophoresis (CE) has been drawing considerable research interest (7-10) since its potential in enantiomeric separations was first demonstrated by Gassmann and co-workers (11) in 1985. Cyclodextrin-modified micellar electrokinetic capillary chromatography (MEKC) has been particularly useful for separating a wide range of enantiomeric compounds (12,13). One of the most significant advantages of CE-based chiral separations is that they are well suited for analysis of mass- or volume-limited samples.
In this...