16 research outputs found
The metabolic pathway of BYZX in HLMs and human liver cytosol.
<p>The metabolic pathway of BYZX in HLMs and human liver cytosol.</p
Pair of Stereodynamic Chiral Benzylicaldehyde Probes for Determination of Absolute Configuration of Amino Acid Residues in Peptides by Mass Spectrometry
This
paper describes a simple method to determine the absolute
configuration of amino acids residues in peptides by mass spectrometry
using a newly developed pair of mass-tagged chiral probes without
the requirement of reference standards. A pair of benzylicaldehyde
probes, 1-(S)-<sup>1</sup>H in S configuration and 2-(R)-<sup>2</sup>D in deuterium-labeled R configuration with the ratio of 1:1, were
synthesized for in situ condensation with amino acid residues and
transformed into a pair of stereodynamic imine products. The characteristic
intensity difference observed in mass spectrometry can be used to
determine the absolute configuration and to quantify the enantiomeric
composition of chiral amino acid residues. Significant chiral recognition
ability was achieved for 18 natural chiral amino acids and for one
β-amino acid by comparing the ion intensity ratio of imine products
I<sub>[1‑(S)‑</sub><sup>1</sup><sub>H‑AA]</sub><sup>–</sup> to I<sub>[2‑(R)‑</sub><sup>2</sup><sub>D‑AA]</sub><sup>–</sup>. For 16 kinds of amino
acids, the <i>L</i> form of the amino acids was more reactive
with 1-(S)-<sup>1</sup>H, while <i>D</i> configuration amino
acids preferred to react with 2-(R)-<sup>2</sup>D. However, for three
kinds of amino acid, the opposite result was obtained. The configurations
of the residues in the peptides, Phe-Tyr-Ala, <i>D</i>-Phe-Tyr-Ala,
Val-Pro-Phe-<i>D</i>-Leu-Met, Val-Pro-Phe-Leu-<i>D</i>-Met, as well as in a natural peptide with unknown chirality were
determined by acid hydrolysis followed by the present method. In addition,
molecular modeling results illustrate that the recognition process
is mainly controlled by kinetic factors. Using the new probes coupled
with a mass spectrometry approach avoids time-consuming workup and
separation steps. We expect that the probes could be applied as tools
to determine the absolute configuration of amino acid residues in
proteins in future research
Km and Vmax values for CYP3A4 and CYP2B6 enzymes with different mutants of POR were determined by their specific substrates testosterone and bupropion.
<p>The ratio of Vmax to Km was used as an index of catalytic efficiency; the activity of each POR mutant co-expressed with CYPs was calculated and expressed as a percentage of the activity of wild-type POR, arbitrarily set at 100%.</p><p>The data were represented as mean ± S.D. of three independent experiments.</p><p>CL<i>int = V</i>max/<i>K</i>m.</p>*<p>: <i>p</i><0.01 in comparison with the cells expressing wild-type (WT) POR.</p>#<p>: <i>p</i><0.05 in comparison with the cells expressing wild-type (WT) POR.</p><p>Dash (–) indicates not detectable.</p
LC-MS chromatograms and mass spectrums of BYZX and its metabolites.
<p>A, total ion chromatograms of BYXZ (20 µM) metabolism in pooled HLMs for 20 min without and with NADPH. The peaks at 5.1 and 5.6 min also existed in the incubation matrix. B, MS spectrums of BYZX and its metabolites in the total ion chromatogram. C, MS/MS spectrums and ion fragments analysis of BYZX and its metabolites in the total ion chromatogram.</p
Kinetics of the formation of M3 from BYZX (A) and M1 from M2 (B) in HLMs, CYP3A4 and CYP2C8, and M2 from BYZX (C) in HLMs and human liver cytosol.
<p>Each inset shows the Eadie-Hofstee plot of the experimental data.</p
NMR assignments for BYZX, M1 and M2.
<p>The numbers in the first column correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059882#pone-0059882-g007" target="_blank">Fig. 7</a>. All of the signals were assigned with the help of 2D-NMR. The values of <b>δ</b><sub>H</sub> at position 8∼10 for BYZX are distinct from those for M1 and M2, due to the splitting signals resulting from C = C reduction. The carbonyl signal still remained (Position 19).</p