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Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non-heme iron

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Lipids

Abstract

An efficient three-step purification technique has been developed for the reticulocyte 15-lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated byN-terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g-atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid resulted in the appearance of an EPR signal around g6.

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Abbreviations

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

EPR:

electron paramagnetic spectroscopy

MCD:

magnetic circular dichroism

NMR:

nuclear magnetic resonance

PVDF:

polyvinylidenedifluoride

SDS:

sodium dodecylsulfate

UV:

ultraviolet

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Authors and Affiliations

  1. Department of Chemistry, University of Toledo, 2801 W. Bancroft Street, 43606, Toledo, Ohio

    Richard T. Carroll, Jessica Muller, Jennifer Grimm & Max O. Funk Jr.

  2. Department of Medicinal Chemistry, University of Toledo, 43606, Toledo, Ohio

    Richard T. Carroll, Jessica Muller, Jennifer Grimm & Max O. Funk Jr.

  3. Biophysics Research Division, University of Michigan, 48109, Ann Arbor, Michigan

    William R. Dunham & Richard H. Sands

Authors
  1. Richard T. Carroll
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  2. Jessica Muller
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  3. Jennifer Grimm
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  4. William R. Dunham
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  5. Richard H. Sands
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  6. Max O. Funk Jr.
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Carroll, R.T., Muller, J., Grimm, J. et al. Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non-heme iron. Lipids 28, 241–244 (1993). https://doi.org/10.1007/BF02536646

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  • DOI: https://doi.org/10.1007/BF02536646

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