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

• We have established a new method to detect innate d-amino acid residues in proteins.
• Our new method was applied to β-galactosidase which is produced in E. coli cells.
• The 0 h-extrapolating values by an orthodox method reflected artificial d-amino acids.
• l-Amino acid residues are presumably isomerized in a very early stage of hydrolysis.
• Our new method is effective to detect innate d-amino acid residues in proteins.

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.

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