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    Heat in Activation Studies of 15-Lipoxygenase Isoenzyme-1

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    Date
    1994
    Author
    Skoures, Evan Tony
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    Abstract
    Since the quaternary structure of soybean 15-lipoxygenase isoenzyme-l has been elucidated by X-ray crystallography, attempts were made to characterize of the enzyme further. Scanning calorimetry studies have revealed that there is a thermal destabilization of the enzyme at 650C. One of our long term goals is to determine which amino acid residues or secondary structures contribute to the stabilization at the transition temperature via site directed mutagenesis. To establish a basis for this long term goal, the present study investigates the effects are observed as the enzyme is exposed to its melting temperature of 65 0C and 70 0C for varying time intervals. It was found that at 65 0C the enzyme activity reached 50% inhibition after heating at approximately 20 minutes. Heating the enzyme at 70 0C for approximately 3 minutes resulted in 50% inhibition. Low temperature Electron Paramagnetic Resonance (EPR) was also employed to determine whether the ferric center was stabilizing the enzyme at the transition temperature. We noted that there was an overall decrease in the peak intensity with increasing duration of heat application which implies that the enzyme was not activated to its catalytically active Fe3+ form upon addition of 1 equivalent of product. Changes in the line shapes were investigated in order to determine if the strain, caused by the heat application, produced a distortion around the octahedral ferric center. This would imply that the ferric center was stabilizing the enzyme. These changes were not evident, however. We conclude that heating the enzyme for various durations at 65 0C results in destabilizing of the enzyme in terms of disruption of the weak intermolecular forces, for short heat applications, and disruption of strong intermolecular forces to a point of irreversible unfolding for longer heat applications. The EPR data support the destabilization but evidence of stabilization by the iron is not conclusive.
    URI
    http://hdl.handle.net/10920/23095
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