A.V. Ogorodnikova et al. / Phytochemistry 69 (2008) 2793–2798
2797
4.2. Substrate preparations
esters of products were preliminarily separated by RP-HPLC and
NP-HPLC (when specified). Products were separated as methyl es-
ters by RP-HPLC on Macherey-Nagel Nucleosil 5 ODS column
(250 ꢁ 4.6 mm) using the solvent mixture methanol–water (linear
gradient from 76:24 to 96:4, by volume) at a flow rate of 0.4 ml/
min. Products were collected and re-chromatographed by NP-HPLC
on two serially connected Separon SIX columns (150 ꢁ 3.2 mm;
5 mm) eluted with hexane–diethyl ether 99.4:0.6 (by volume),
flow rate 0.6 ml/min.
(9Z,11E,13S)-13-Hydroperoxy-9,11-octadecadienoic acid (13-
HPOD) was prepared by incubation of linoleic acid with soybean
lipoxygenase at 0 °C, pH 9.0, under continuous oxygen bubbling
followed by purification by NP-HPLC. (9S,10E,12Z)-9-Hydroper-
oxy-10,12-octadecadienoic acid (9-HPOD) was prepared by incu-
bation of linoleic acid with tomato fruit lipoxygenase at 0 °C, pH
6.0, under the continuous oxygen bubbling followed by purifica-
tion by NP-HPLC.
4.7. Spectral analyses
4.3. Cell-free preparations from Lily-of-the-Valley roots
The UV spectra of isolated products were recorded with Varian
Cary 50 spectrophotometer. Alternatively, the UV spectra of com-
pounds that were purified by HPLC were recorded on-line by using
an SPD-M20A diode array detector (Shimadzu). GC–MC analyses
were performed by using a Shimadzu QP5050A mass spectrometer
connected to Shimadzu. GC-17A gas chromatograph equipped with
an MDN-5S (5% phenyl 95% methylpolysiloxane) fused capillary
Cut roots (10 g) of Lily-of-the-Valley were suspended in cold (0–
4 °C) 0.1 M Tris/HCl buffer (20 ml, pH 7.5) and homogenized with
Ultra-Turrax. Homogenate was filtered through cheesecloth and
centrifuged at 15,000 g for 15 min. The supernatant was decanted
and used for incubations with linoleic acid or its hydroperoxides.
Alternatively, the 15,000 g supernatant was adjusted to 70% of
(NH4)2SO4 saturation, stirred for 15 min and centrifuged at
15,000 g for 15 min. The supernatant was decanted and the pellet
obtained after the addition of (NH4)2SO4 was frozen with liquid
nitrogen and stored at ꢀ85 °C prior use for incubations. Before
use the preparation was unfrozen and suspended in 0.1 M Tris/
HCl buffer, pH 7.5 at 0–4 °C. Aliquots of this suspension were used
for incubations.
column (length, 30 m; i.d. 0.25 mm; film thickness, 0.25
lm). He-
lium at a flow rate of 30 cm/s was used as the carrier gas. Injections
were made in the split mode by using an initial column tempera-
ture of 120 °C. The temperature was raised at 10 °C/min until
240 °C. Full scan or selected-ion monitoring (SIM) analyses were
both performed by using ionization energy of 70 eV. The 1H NMR
and 2D-COSY spectra were recorded with Bruker Avance 400
instrument, 400 MHz, [2H3]acetonitrile, 296 K.
4.4. Incubation of enzyme preparations with linoleic acid and its
hydroperoxides
Acknowledgements
Standard incubations were initiated by the addition of unla-
belled linoleic acid (0.5 mg) to aliquot of 15,000 g supernatant
preparation, corresponding to 5 g of the roots. The reaction mix-
ture was stirred for 30 min under the continuous oxygen bubbling.
The incubations aliquot of 15,000 g supernatant preparation, corre-
sponding to 2 g of the roots with fatty acid hydroperoxides (0.5 mg
of 13-HPOD, or 9-HPOD) were performed in the same way for
5 min, but without oxygen bubbling. For micro-preparative isola-
tion of compounds 1 and 2 the 15,000 g supernatant obtained from
10 g of roots was incubated with 1 mg 13-HPOD or 9-HPOD.
Incubations with labelled substrates were performed as follows.
Incubations were initiated by the addition of either [1-14C]linoleic
This work was supported by Grant 06-04-48430 from Russian
Foundation for Basic Research and a Grant from Russian Academy
of Sciences (program ‘‘Molecular and Cell Biology”). The authors
appreciate the 1H NMR and COSY records by Dr. Oleg I. Gnezdilov.
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