Detection of D-amino acids in purified proteins synthesized in Escherichia coli.
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 beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees 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.
Miyamoto
Tetsuya
T
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Sekine
Masae
M
Ogawa
Tetsuhiro
T
Hidaka
Makoto
M
Homma
Hiroshi
H
Masaki
Haruhiko
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D054832:Urocortins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D054832:Urocortins; D001616:beta-Galactosidase