D. Gani et al. / Bioorg. Med. Chem. 7 (1999) 977±990
989
ammonia in sulphuric acid was then oxidised to dini-
(2S)-Aspartic acid
trogen using excess sodium hypobromite solution. The
evolved dinitrogen was introduced directly into one
port of the dual-inlet isotope ratio mass spectrometer
and the 15N/14N ratio (R0) was determined against a
reference standard of dinitrogen connected to the other
port. Three complete duplicate determinations were
carried out for the sample. The entire procedure was
repeated for the substrate (2S,3S)-[3-2H]-3-methylas-
partic acid.
Similar experiments to those described above were per-
formed using (2S)-aspartic acid and (2S,3R)-[3-2H1]-
aspartic acid at pL 9.0 and 9.4 in water and in deuter-
ium oxide in the presence of 1 mM KCl and 20 mM
MgCl2.
(2S,3R)-3-Methylaspartic acid
Similar experiments to those described above were per-
formed using (2S,3R)-3-methylaspartic acid and
(2S,3R)-[3-2H]-3-methylaspartic acid at pH 9.0 in water
in the presence of 1 mM KCl and 20 mM MgCl2.
R was determined using ammonia produced from a
partial deamination reaction performed under the spe-
ci®c conditions described below. Incubations (total
volume 10 ml) were performed at 300.1 ꢁC, in buf-
fered solution with 20 mM MgCl2, and KCl as speci-
®ed below. The substrate concentration was in the
range 0.15 to 0.2 M and the reaction was initiated by
addition of enzyme solution (50 ml, ca. 6 units). The
extent of the deamination reaction (f) was measured
spectrophotometrically by withdrawing small aliquots
of the solution at various time intervals and deter-
mining the concentration of mesaconic acid from the
UV absorbance at 240 nm.9,18 After 10±20% of the
methylaspartic acid had been deaminated, the reaction
was quenched by the addition of concentrated sul-
phuric acid (1.0 ml) and the product ammonia was
distilled into dilute sulfuric acid in a stream of air, see
above. The dinitrogen obtained upon hypobromite
oxidation was analysed as described above to give the
15N/14N ratio in the product (R). Three duplicate
incubations were carried out for each deamination
experiment.
Acknowledgements
The authors thank Professor A. Jackson (University of
Southampton) for performing isotope ratio mass spec-
trometry and the Biomolecular Sciences committee of
the BBSRC and EPSRC for grant B05857.
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Similar experiments to those described above were
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