H. Aoshima, J. Sekiya, T. Kajiwara
Sep 1, 1977
Agricultural and biological chemistry
A spin trapping method involves the use of nitrosoalkanes (spin trap) which are very reactive with free radicals whose lifetimes are too short to allow detection by ESR, and the reaction yields a relatively longlived free radical product (spin adduct) which can be studied by ESR. The involvement of linoleic acid free radical in soybean lipoxygenase reaction1) or in potato lipoxygenase reaction2) has been demonstrated by using 2-methyl-2-nitrosopropanol or 2-methyl-2nitrosopropane as a spin trap. The light induced generation of a superoxide by chloroplasts in the presence of oxygen has been shown by the production of the 02adduct of the spin trap, 5, 5-dimethyl-lpyrroline-l-oxide.3) Recently, a new enzyme system which cleaves linoleic acid into n-hexenal in the presence of oxygen was found in the chloroplasts of Thea sinensis leaves.4) Similar enzyme and systems have been found in cucumber,5,6) tomato,') banana) and many other plant leaves.') In this study, we applied a spin trapping technique to a mixture of linoleic acid and chloroplasts of tea leaves, and obtained a spin adduct which has one a-proton hyperfine coupling constant of 2.0 G. Chloroplasts of Thea sinensis (var. Yabukita) leaves were prepared according to the procedure described in a previous paper.') A spin trap, 2-methyl-2-nitrosopropane, was purchased from Aldrich Co., Milwaukee. Linoleic acid, oleic acid and stearic acid were of a high-purity grade purchased from Wako Chemical Co., Osaka. ESR spectra were obtained with an X-band spectrometer of JEOL PE-3X. All experiments were carried out at room temperature in a quartz tube designed for measurement in aqueous solution. The spectrum of Mn2+ in MnO was used as a reference; the splitting between the two central peaks is 86.9+0.1 G. For the spin trapping reaction, 0.2 ml of chloroplasts (0.1 g/ml) dissolved in 0.1 M borate buffer at pH 6.8 was added to the mixture of 0.8 ml of linoleic acid (18 mg/ ml) in 0.1 M borate buffer at pH 6.8 and 0.05 ml of 2methyl-2-nitrosopropane (40 mg/ml) in ethanol.