Bleaching of Methylene Blue as an Index of Lipoxygenase Activity

Abstract

An assay of lipoxygenase activity was developed that depends on measurements of the time needed for the bleaching of methylene blue to begin. This time was inversely related to the level of lipoxygenase activity (y = 114.5132 - 0.0132x; r= -0.99); it was not affected by the concentration of methylene blue, linoleic acid, or hydroperoxide. Lipoxygenase activity can be calculated from the time needed for the bleaching of methylene blue to begin.

Simple and Rapid Method for the Selective Detection of Individual Lipoxygenase Isoenzymes in Soybean Seeds

“…These results indicated that test I could selectively detect the L-l isozyme contained in soybean extracts. Toyosaki (1992) proposed that methylene blue bleaching occurred as a result of the sequence of production of the lipid radical R" and the peroxy radical ROO*. Another simple explanation is that a redox reaction is likely involved in this bleaching reaction, because methylene blue is known to be a redox indicator.…”

“…There are some studies dealing with dye solution bleaching by soybean lipoxygenases. Toyosaki (1992) showed that commercial L-l bleached methylene blue, although it was uncertain whether other isozymes did so. Kikuchi and Kitamura (1987) reported three tests and two versions for the detection of lipoxygenase isozymes by skillful use of the different action among the carotene bleaching activities of three lipoxygenase isozymes; however, the tests involved complex procedures and careful handling for the detection of the L-l or L-2 isozyme in soybean seeds lacking the L-3 isozyme.…”

Colorimetric Method for the Determination of Lipoxygenase Activity

“…Several such methods exist. An assay based on the bleaching (reduction) of methylene blue was developed by Toyosaki (7). This method, although simple and rapid, is not particularly sensitive and is difficult to quantify.…”

High-Throughput Assay for Detection of Soybean Lipoxygenase-1

“…The use of fluorescein for detection of soybean LOX-1 was modeled using the previous methylene blue bleaching method (7). The methylene blue LOX assay measures the bleaching of methylene blue by hydroperoxides formed by the radical-mediated enzymatic oxidation of linoleic acid (15). The new fluorescein assay measures the decrease in fluorescence of fluorescein as it is degraded by hydroperoxide radical attacks.…”

“…There are other methods that quantify LOX activity. For example, the methylene blue assay quantifies the enzyme by measuring time until bleaching begins (15). Measuring the AUC of fluorescence allows for more objective and accurate quantification.…”