Heat in Activation Studies of 15-Lipoxygenase Isoenzyme-1
Skoures, Evan Tony
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Since research on lipoxygenase is still nascent, one of our research interests is to characterize the enzyme as much as possible. The X-ray structure, as already mentioned, has already been solved and will provide a good basis for solving other lipoxygenase structures such as iosenzymes 2, and 3 of soybean 15-lipoxygenase and perhaps mammalian lipoxygenases as well. Site directed mutagenesis studies will also be done to gain further insight into some of the structural aspects and how it relates to the function. However, the issue that will be addressed in this paper is to elucidate what happens to the enzyme when it is heat inactivated at some temperature for some time interval. Scanning calorimetry data show that the enzyme unfolds irreversibly at 65°C and hence the enzyme has been denatured.9 However, experiments were performed in which the enzyme was heated at 65°C for certain time intervals, 0 to 20 minutes, and the activity was measured via the lipoxygenase kinetics spectrophotometric assay. The results, as expected, showed a significant decrease in enzyme activity but the remaining question was why and how? In attempt to answer these questions, Electron Paramagnetic Resonance (EPR) was employed to monitor the iron of the active site via changes in the shapes and line widths of the spectrum.