Abstract
The expression and purification procedure [Richards, K.M. and Marnett, L.J. (1997) Biochemistry 36, 6692–6699] for leukocyte 12‐lipoxygenase was updated for the production of the large quantities of protein necessary for the characterization of its physical properties. A two‐step FPLC ion exchange chromatography procedure was adequate for purification as evaluated by SDS PAGE, specific activity measurement, isoelectric focusing, and electrospray ionization mass spectrometry. The solution properties of the enzyme were examined by variable temperature circular dichroism spectroscopy, differential scanning microcalorimetry, and dynamic light scattering measurements. The redox characteristics of the active site iron atom were determined by EPR spectroscopy following treatment with a variety of fatty acid hydroperoxides. Leukocyte 12‐lipoxygenase was found to be more similar to reticulocyte 15‐lipoxygenase than to soybean lipoxygenase‐1 or human 5‐lipoxygenase.(Supported by the National Institutes of Health GM 62140)
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