Characterization of crude lipoxygenase extract from green pea using a modified spectrophotometric method

In the spectrophotometric assay of lipoxygenase (LOX), the buffered linoleic acid solution used as the reaction medium is not optically clear enough at neutral or lower pH values, due to its limited solubility, to provide a precise, accurate and reproducible estimation of activity as the increase in absorbance at 234 nm. Therefore, an optically clear solution was obtained by formation of the Na-salt of unreacted linoleic acid before absorbance measurement. The modified method was then used to characterize crude LOX from green peas in terms of pH and temperature optima, thermal stability, and kinetic parameters. The optimum pH and temperature for the activity of LOX from green peas were determined to be 6.0 and 30 degreesC, respectively. LOX was found to be very stable at 60 degreesC, but much less stable at 65 degreesC or higher temperatures. The Michaelis constant (Km) and maximum rate (V-max) for linoleic acid were calculated to be 1666 units per mg protein per min and 2.33 mM, respectively.