Abstract
It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.
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Abbreviations
- NBD-F:
-
4-fluoro-7-nitro-2,1,3-benzoxadiazole
- HPLC:
-
High performance liquid chromatography
- IPTG:
-
Isopropyl-β-d-thiogalactopyranoside
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Acknowledgements
We thank Prof. Hidenori Watanabe, Department of Applied Biological Chemistry, The University of Tokyo, and Prof. Keiichiro Ogawa, Graduate School of Arts and Sciences, The University of Tokyo, for the helpful discussions.
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Miyamoto, T., Sekine, M., Ogawa, T. et al. Detection of d-amino acids in purified proteins synthesized in Escherichia coli . Amino Acids 38, 1377–1385 (2010). https://doi.org/10.1007/s00726-009-0348-2
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DOI: https://doi.org/10.1007/s00726-009-0348-2