Simple and rapid carotene bleaching tests for the detection of lipoxygenase isozymes in soybean seeds.

Abstract

New techniques using the carotene bleaching activity of. Iipoxygenase were developed for the detection of soybean seed lipoxygenases, L-1, L-2, and L-3 isozymes, respectively. The carotene bleaching tests for the three isozymes were performed as follows : (Test I for L-3) the seed extract was incubated with linoleic acid and carotene at pH 6.8. When the seed extract contained L-3, carotene was bleached in a few minutes at 20°C. In the absence of L-3 carotene bleaching was not observed ; (Test II far L-2) the seed extract was incubated with arachidonic acid and carotene at pH 6.8. After incubation for 3 minutes at 20°C, methanol was added into the mixture to stop the bleaching reaction. Since L-2 is much more active than L-1 and L-3 towards arachidonic acid at pH 6. 8, the lack of L-2 significantly decreased the level of the carotene bleaching activity. The presence or absence of L-2 in the F2 seeds from Suzuyutaka (Lx2/Lx2) x P186023 (lx2/lx2) soybeans was accurately determined by the application of Test II ; (Test III for L-1) the seed extract was preincubated with linoleic acid and carotene at pH 9. 0. After preincubation for 60 seconds at 20°C, the mixture. was adjusted to pH 6.8. When the seed extract did not contain L-1, carotene bleaching was observed within thirty minutes at 20°C. The levels of the carotene bleaching activity in the three tests were determined visually or by measurements in a spectrophotometer at 452 nm within 60 minutes. Since the L-3 isozyme is indispensable for the carotene bleaching activity, seed extracts from soybeans la.cking L-3 do not cooxidize carotene. Therefore, in order to determine the presence or absence of L-2 or L-1 in the seeds lacking L-3, seed extracts from the soybeans lacking L-2 and L-1 in addition to the seed extracts to be examined were added to 60mpensate the lack of L-3 isozyme into the reaction mixture, respectively. The other procedures were the same as those applied in Tests II and III, respectively. These tests which are simple, rapid, and accurate, require only a few mg of seed sample.