6040 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Hoffman et al.
concentration of 5% (w/v), and protein pellets were separated
by centrifugation. The supernatants were extracted twice with
water-saturated diethyl ether prior to derivatization. SDH
reaction mixtures were derivatized with 2,4-dinitrophenylhy-
drazine (DNPH), and pyruvate formation was monitored on
HPLC as previously described.8,44 AR and TA reaction mix-
tures were derivatized with FDAA and separated according to
the protocol for separation of L- and D-serine described above.
AR mixtures were analyzed for the formation of L-alanine, and
TA mixtures were analyzed for the formation of L-glutamic acid.
Pure L-alanine, D-alanine, L-aspartic acid, and L-glutamic acid
were derivatized and used as calibration standards.
NMR Measurements. An approximately 8 mM stock solution
of PLP was prepared in 100 mM phosphate buffer in D2O, pD
8.4. An appropriate amount of hydroxamic acid (chosen to
ensure an approximately 1:1 molar ratio between the acid and
PLP) was weighed out and mixed into 2 mL of PLP stock
solution. The final pD was adjusted to 7.1-8.1 by the addition
of NaOH powder. For 13C NMR measurements (see Supporting
Information), an approximately 10-fold higher PLP concen-
tration was used.
Mass Spectrometry. A 4, 5, or 30 mM solution of PLP (pH
adjusted to 8.5 with NaOH) was mixed with a 10-fold molar
excess of the test compound and incubated at room temperature
for one day prior to measurement. Mixtures were prepared both
with and without a pH control (200 mM ammonium sulfate, pH
8.0). The mixture was diluted in water 40- to 100-fold and
subjected to ESI-MS in a negative ion mode.
Spectrophotometric Assay for the Reaction of PLP with
Hydroxamic Acids. A 0.6 mM solution of pyridoxal-50-phos-
phate and 10 mM solutions of test compounds were prepared in
100 mM phosphate buffer, pH 8.0. The PLP and inhibitor
solutions were mixed in a 1:1 ratio so that the final concentration
of PLP was 300 μM, and the final concentration of inhibitor was
5 mM. The reaction mixtures were incubated at room tempera-
ture for 1 h prior to measurement. Absorbance spectra were
collected in the 310-500 nm range.
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Acknowledgment. This work was financially supported by
the Ministry of Education of the Czech Republic (grant
1M0508) and the Grant Agency of the Academy of Sciences
of the Czech Republic (grant IAA 400720706).We would like
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ꢁ
´
to thank Mr. Roman Vanıcek for providing us with the very
helpful Sample Reader1 program and Mrs. Ludmila
ꢀ
Soukupova for the synthesis of some hydroxamic acids. We
would also like to thank the anonymous reviewers of this
manuscript for their inspiring comments.
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and resolution of electronic spectra of pyridoxal phosphate and
other 3-hydroxypyridine-4-aldehydes. Biochim. Biophys. Acta
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Supporting Information Available: Synthetic procedures for
compounds 11-14 and additional spectroscopic measurements
(NMR and UV/vis).
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