Origin of D-amino acids detected in the acid hydrolysates of purified Escherichia coli β-galactosidase.

In previous report, we detected D-amino acids in the acid hydrolysates of purified recombinant β-galactosidase. Here, we employed a deuterium-hydrogen exchange method to discriminate innate D-amino acids from those generated during hydrolytic incubation. After hydrolysis of β-galactosidase in DCl/D2O, amino acids were derivatized with NBD-F and separated on a reverse-phase column, followed by liquid chromatography-tandem mass spectrometry equipped with a chiral column. Our results show an absence of innate D-amino acid residues in the protein and suggest that the protein undergoes isomerization during a very early stage of hydrolytic incubation.