2.3. Electron paramagnetic resonance detection of lipid radicals
Leaves were powdered in liquid nitrogen, homogenized in
100mM potassium phosphate buffer, pH 7.4, and incubated for
1 h at 30 ◦C in the presence of the spin trap -(4-pyridyl-1-
oxide)-N-t-butyl nitrone (POBN), 50mM final concentration. Lipid
radicals were detected using EPR. Spectra were recorded at room
temperature using a Bruker ECS 106 spectrometer, operating at
9.81GHz with 50 kHz modulation frequency. EPR instrument settings
were as follows: microwave power, 20mW; modulation
amplitude, 1.232 G; time constant, 81.92 ms. The lipid radical
adducts were quantified using standard solutions of 4-hydroxy-
2,2,6,6-tetramethyl piperidine-N-oxyl (TEMPOL) as previously
described [43].