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Lipoxygenase-Catalyzed Phospholipid Peroxidation: Preparation, Purification, and Characterization of Phosphatidylinositol Peroxides

  • E. Susan O’Connor Butler
  • Jessica N. Mazerik
  • Jason P. Cruff
  • Shariq I. Sherwani
  • Barbara K. Weis
  • Clay B. Marsh
  • Achuthan C. Raghavamenon
  • Rao M. Uppu
  • Harald H. O. Schmid
  • Narasimham L. Parinandi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

The importance of understanding the mechanisms of modulation of cellular signaling cascades by the peroxidized membrane phospholipids (PLs) is well recognized. The enzyme-catalyzed peroxidation of PLs, as opposed to their oxidation by air and metal catalysis, is well controlled and rapid and yields well-defined PL peroxides which are highly desirable for biological studies. Therefore, here, we chose bovine liver phosphatidylinositol (PI), a crucial membrane PL which acts as the substrate for phospholipase C in cellular signal transduction, as a model membrane PL. We successfully generated the PI peroxides with soybean type-I lipoxygenase (LOX) in the presence of deoxycholate, which facilitates the LOX-mediated peroxidation of the polyunsaturated fatty acids esterified to the PL. The LOX-peroxidized PI, after enzymatic catalysis, was separated from the unoxidized PI in the reaction mixture by normal-phase, high-performance liquid chromatography (HPLC). The extent of LOX-mediated peroxidation of PI following HPLC purification was established by the analysis of lipid phosphorus, conjugated dienes by UV spectrophotometry, peroxides, and loss of fatty acids by gas chromatography. This study established the optimal conditions yielding ∼46% of peroxidized PI from 300 μg of neat bovine liver PI that was peroxidized by soybean type-I LOX (50 μg) for 30 min in borate buffer (0.2 M, pH 9.0) containing 10 mM deoxycholate.

Key words

Lipoxygenase phospholipid peroxidation phosphatidylinositol peroxides HPLC 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • E. Susan O’Connor Butler
    • 1
  • Jessica N. Mazerik
    • 1
  • Jason P. Cruff
    • 1
  • Shariq I. Sherwani
    • 1
  • Barbara K. Weis
    • 2
  • Clay B. Marsh
    • 1
  • Achuthan C. Raghavamenon
    • 3
  • Rao M. Uppu
    • 3
  • Harald H. O. Schmid
    • 2
  • Narasimham L. Parinandi
    • 4
  1. 1.Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Internal MedicineThe Ohio State University College of MedicineColumbusUSA
  2. 2.The Hormel Institute, University of MinnesotaAustinUSA
  3. 3.Department of Environmental Toxicology and the Health Research CenterSouthern University and A&M CollegeBaton RougeUSA
  4. 4.Department of Internal Medicine and the Davis Heart and Lung Research InstituteThe Ohio State University Medical CenterColumbusUSA

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