Co‐Oxidation by Cyclooxygenases

Lawrence M. Szewczuk1, Trevor M. Penning2

1 GlaxoSmithKline, Collegeville, Pennsylvania, 2 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.30
DOI:  10.1002/0471140856.tx0430s42
Online Posting Date:  November, 2009
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Abstract

Cyclooxygenases (COXs; prostaglandin H2 synthases) catalyze the bis‐dioxygenation of arachidonic acid (AA) to generate prostaglandin (PG) G2 followed by the peroxidative cleavage of PGG2 to yield PGH2, the precursor to all of the vasoactive PGs. These enzymes utilize a Fe‐protoporhyrin IX (heme) co‐factor to catalyze peroxide bond cleavage, which puts the Fe at a higher oxidation state (Fe3+ → Fe5+). The heme Fe requires two electrons (e) to return to its resting state (Fe3+) for the next round of catalysis. Peroxide bond cleavage thus occurs via compound I and compound II, observed for horseradish peroxidase. To return to Fe3+, electrons come from “co‐reductants” and their subsequent oxidation by the enzyme is known as “co‐oxidation”. The protocols in this unit are aimed at characterizing this side reaction of COXs. Curr. Protoc. Toxicol. 42:4.30.1‐4.30.14. © 2009 by John Wiley & Sons, Inc.

Keywords: peroxidase; co‐reductant; co‐substrate; self‐inactivation; resveratrol; phenol

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining if a Compound is Co‐Oxidized by Cyclooxygenase
  • Basic Protocol 2: Determining the Effect of Test Compound Co‐Oxidation on Cyclooxygenase
  • Basic Protocol 3: Performing Product Profiling for the Co‐Oxidation Reaction
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determining if a Compound is Co‐Oxidized by Cyclooxygenase

  Materials
  • 1 M Tris⋅Cl, pH 8.0 (Sigma)
  • Tween‐20 (Sigma)
  • Glycerol (Fisher Scientific)
  • COX‐1 (Biomol)
  • 1 mM hematin (Sigma) prepared in 0.1 M NaOH
  • Micro‐BCA assay kit (Pierce)
  • 10 mM test compound(s) prepared in DMSO
  • 30% H 2O 2 (Sigma), prepare dilutions fresh in water and keep on ice
  • 1 M sodium citrate, pH 4.0 (Fisher Scientific)
  • 10% methanol
  • 10,000 MWCO Slide‐A‐Lyzer cassette (Pierce)
  • Centriprep‐30s (Millipore)
  • 1.5‐ml plastic tubes
  • Waters Xterra RP 18 column (3.5 µm; 4.6 × 150‐mm)
  • Waters Alliance 2690 HPLC system with a Waters 996 photodiode array detector and a beta‐RAM in‐line radiometric detector (IN/US Systems)
NOTE: If preferred, COX‐1 can be purified in house from ram seminal vesicles as previously described (Marnett et al., ). A 250‐g seminal vesicle preparation typically yields 20 to 30 mg of pure protein.

Basic Protocol 2: Determining the Effect of Test Compound Co‐Oxidation on Cyclooxygenase

  Materials
  • 1 M Tris⋅Cl, pH 8.0 (Sigma)
  • 40 mM N,N,N′,N′‐tetramethyl‐1,4‐phenylene‐diamine (TMPD; Sigma), prepared fresh in ethanol and kept ice cold and protected from light
  • Dialyzed holo‐COX‐1 (see protocol 1)
  • 30% H 2O 2 (8.82 M; Sigma), prepare dilutions fresh in water and keep on ice
  • 2 µM hematin
  • 10 mM test compound(s) prepared in DMSO
  • DMSO (Fisher Scientific)
  • 20 mM phenol (Fisher Scientific) prepared in DMSO, optional
  • 10 mM resveratrol (Sigma) prepared in DMSO, optional
  • 1‐ml quartz cuvette
  • UV/Vis spectrophotometer
  • 0.5‐ml plastic tubes

Basic Protocol 3: Performing Product Profiling for the Co‐Oxidation Reaction

  Materials
  • 1 M Tris⋅Cl, pH 8.0 (Sigma)
  • Dialyzed holo‐COX‐1 (see protocol 1)
  • 10 mM radiolabeled test compound, prepared in DMSO (25,000 cpm/nmol)
  • 30% H 2O 2 (Sigma), 1 mM prepared fresh in water and kept on ice
  • Solvents for HPLC (water, acetonitrile, methanol)
  • C 18 column (e.g., Phenomenex Spherisorb C 18 column, 5 µm, 4.6 × 250–mm; Waters)
  • HPLC with in‐line diode array and radiometric detectors
  • HPLC with MSn detection
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Figures

Videos

Literature Cited

Literature Cited
   Dietz, R., Nastainczyk, W., and Ruf, H. 1988. Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2. Eur. J. Biochem. 171:321‐328.
   Divi, R. and Doerge, D. 1994. Mechanism‐based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33:9668‐9674.
   Egan, R., Paxton, J., and Kuehl, F. 1976. Mechanism for irreversible self‐deactivation of prostaglandin synthetase. J. Biol. Chem. 251:7329‐7335.
   Flower, R. 1974. Drugs which inhibit prostaglandin biosynthesis. Pharmacol. Rev. 26:33‐67.
   Hamberg, M. and Samuelsson, B. 1967a. On the mechanism of the biosynthesis of prostaglandins E1 and F1α. J. Biol. Chem. 242:5336‐5343.
   Hamberg, M. and Samuelsson, B. 1967b. Oxygenation of unsaturated fatty acids by the vesicular gland of sheep. J. Biol. Chem. 242:5344‐5354.
   Hamberg, M. and Samuelsson, B. 1973. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc. Natl. Acad. Sci. U.S.A. 70:899‐903.
   Hamberg, M., Svensson, J., Wakabayashi, T., and Samuelsson, B. 1974. Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. Natl. Acad. Sci. U.S.A. 71:345‐349.
   Hla, T. and Nielson, K. 1992. Human cyclooxygenase‐2 cDNA. Proc. Natl. Acad. Sci. U.S.A. 89:7384‐7388.
   Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. 1988. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction. Eur. J. Biochem. 171:313‐320.
   Markey, C., Alward, A., Weller, P., and Marnett, L. 1987. Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities. J. Biol. Chem. 262:6266‐6279.
   Marnett, L., Siedlik, P., Ochs, R., Pagels, W., Das, M., Honn, K., Warnock, R., Tainer, B., and Eling, T. 1984. Mechanism of the stimulation of prostaglandin H synthase and prostacyclin synthase by the antithrombotic and antimetastatic agent, nafazatrom. Mol. Pharmacol. 26:328‐335.
   Patrono, C., Coller, B., Dalen, J., FitzGerald, G., Fuster, V., Gent, M., Hirsh, J., and Roth, G. 2001. Platelet‐active drugs: The relationships among dose, effectiveness, and side effects. Chest 119:39S‐63S.
   Rouzer, C. and Marnett, L. 2003. Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases. Chem. Rev. 103:2239‐2304.
   Ruf, H., Raab‐Brill, U., and Blau, C. 1993. A model for the catalytic mechanism of prostaglandin endoperoxide synthase. Biochem. Soc. Trans. 21:739‐744.
   Samuelsson, B. 1965. On the incorporation of oxygen in the conversion of 8,11,14‐eicosatrienoic acid to prostaglandin E1. J. Amer. Chem. Soc. 87:3011‐3013.
   Smith, W. and Lands, W. 1972. Oxygenation of polyunsaturated fatty acids during prostaglandin biosynthesis by sheep vesicular gland. Biochemistry 11:3276‐3285.
   Szewczuk, L., Forti, L., Stivala, L., and Penning, T. 2004. Resveratrol is a peroxidase‐mediated inactivator of COX‐1 but not COX‐2: A mechanistic approach to the design of COX‐1 selective agents. J. Biol. Chem. 279:22727‐22737.
   Szewczuk, L., Lee, S.H., Blair, I., and Penning, T. 2005. Viniferin formation by COX‐1: Evidence for radical intermediates during co‐oxidation of resveratrol. J. Nat. Prod. 2005:36‐42.
   Tsai, A. and Kulmacz, R. 2000. Tyrosyl radicals in prostaglandin H synthase‐1 and ‐2. Prostaglandins Other Lipid Mediat. 62:231‐254.
   Vane, J. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin‐like drugs. Nature 231:232‐235.
   Wu, G., Vuletich, J., Kulmacz, R., Osawa, Y., and Tsai, A. 2001. Peroxidase self‐inactivation in prostaglandin H synthase‐1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX. J. Biol. Chem. 276:19879‐19888.
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