Cyclooxygenase Assays

James K. Gierse1, Carol M. Koboldt1

1 Searle Research and Development, Chesterfield, Missouri
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.1
DOI:  10.1002/0471141755.ph0301s00
Online Posting Date:  May, 2001
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Abstract

Cyclooxygenase (COX, also known as prostaglandin H2 synthase, PGH2) is one of the most significant enzymes in pharmacology since COX inhibition is the mechanism of action of most nonsteroidal anti‐inflammatory drugs (NSAIDs). There are two forms: COX‐1 is a constitutive form of the enzyme that has been linked to the production of physiologically important prostaglandins that may play a role in homeostasis (gastric, renal, etc.). COX‐2 is inducible by cytokines and growth factors, and induction of COX‐2 is linked to inflammatory cell types and tissues, so COX‐2 is believed to be the target for the anti‐inflammatory actions of NSAIDs. COX‐2 also exists in a constitutive form in kidney and brain. Thus, there is an ongoing effort to identify compounds that will inhibit COX‐2 in preference to COX‐1 since such an agent may be a safer, and perhaps more efficacious, NSAID. Three assays are provided in this unit for measuring COX activity: one is to measure the final product of the reaction, PGE2, by ELISA; another is to measure directly the oxygen uptake that occurs during the first step of the reaction using an oxygen sensor; and the third is to measure the second reaction (peroxidase function) spectrophotometrically.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Measurement of the Product of the Cyclooxygenase Reaction by PGE2 ELISA
  • Alternate Protocol 1: Cell‐based Measurement of Cyclooxygenase Activity by PGE2 ELISA
  • Alternate Protocol 2: Assay of Prostaglandins Extracted from Frozen Tissue
  • Basic Protocol 2: Direct Measurement of Cyclooxygenase Activity by Measurement of Oxygen Uptake
  • Basic Protocol 3: Peroxidase Assay for Measurement of Cyclooxygenase Activity
  • Alternate Protocol 3: Peroxidase Endpoint Assay for Cyclooxygenase Using a 96‐Well Plate Reader
  • Alternate Protocol 4: Peroxidase Initial Rate Assay for Cyclooxygenase Using a 96‐Well Plate Reader
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Product of the Cyclooxygenase Reaction by PGE2 ELISA

  Materials
  • Affinity‐purified goat anti‐rabbit immunoglobulin G (IgG; Jackson Immunoresearch)
  • 50 mM potassium phosphate, pH 7.4 ( appendix 2A)
  • Saturation buffer (see recipe)
  • Insect cells expressing COX‐1 or COX‐2 (Gierse et al., )
  • Cell harvest buffer (see recipe)
  • 10% (w/v) 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS)
  • Inhibitors, including standard inhibitor (e.g., indomethacin or diclofenac)
  • Dimethylsulfoxide (DMSO)
  • 10 mM arachidonic acid (see recipe), diluted to 100 µM
  • Prostaglandin E 2 (PGE 2) assay buffer (see recipe), with and without 20 µM indomethacin
  • Wash buffer (see recipe)
  • Prostaglandin E 2 (PGE 2) standard (see recipe)
  • ELISA buffer (see recipe)
  • PGE 2‐acetylcholinesterase tracer (Cayman)
  • Anti‐PGE 2 monoclonal antibody (Cayman)
  • Ellman's solution (see recipe)
  • 96‐well NUNC‐Immuno Plate, MaxiSorp type
  • 96‐well round‐bottom plate (Falcon)
  • Multichannel pipet (0 to 50–µl and 50 to 300–µl sizes)
  • 96‐well plate sealer (Falcon)
  • Plate washer
  • 96‐well microplate reader (Molecular Devices)

Alternate Protocol 1: Cell‐based Measurement of Cyclooxygenase Activity by PGE2 ELISA

  • Human fetal dermal fibroblast (HFDF) cells, able to express COX‐2
  • Complete DMEM, with and without 10% FBS (complete DMEM/10% FBS and complete DMEM without serum; see recipe)
  • Interleukin 1 (IL‐1)
  • 96‐well plate for cell culture (Costar)
  • Additional reagents and equipment for cell culture (see Phelan, )

Alternate Protocol 2: Assay of Prostaglandins Extracted from Frozen Tissue

  • Tissue of interest, from frozen storage
  • Tissue extraction buffer (see recipe)

Basic Protocol 2: Direct Measurement of Cyclooxygenase Activity by Measurement of Oxygen Uptake

  Materials
  • Tris/heme/phenol (THP) buffer (see recipe)
  • Sodium dithionite, saturated solution
  • 2 mg/ml cyclooxygenase (COX) enzyme solution (see recipe; 20 to 30 µg per reaction)
  • Inhibitors, dissolved in DMSO
  • Dimethylsulfoxide (DMSO)
  • 10 mM arachidonic acid (see recipe)
  • Methanol
  • Clark‐style polarographic electrode, with 600‐µl water‐jacketed reaction vessel (Instech)
  • Chart recorder (2 V full‐scale input) or IBM‐compatible computer with Intake software (Instech)
  • 37°C recirculating water bath
  • Two peristaltic pumps
  • Two 10‐ and one 50‐µl Hamilton syringes
  • Four‐way valve

Basic Protocol 3: Peroxidase Assay for Measurement of Cyclooxygenase Activity

  Materials
  • Peroxidase assay buffer (see recipe)
  • 2 mg/ml cyclooxygenase (COX) enzyme solution (see recipe)
  • Inhibitors, dissolved in DMSO
  • Dimethylsulfoxide (DMSO)
  • N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine (TMPD; see recipe)
  • 10 mM arachidonic acid (see recipe)
  • Visible spectrophotometer or 96‐well plate reader (Molecular Devices) with 590‐ to 611‐nm filter

Alternate Protocol 3: Peroxidase Endpoint Assay for Cyclooxygenase Using a 96‐Well Plate Reader

  • Endpoint assay mix (see recipe)
  • 96‐well flat‐bottom plates
  • 96‐well microplate reader (Molecular Devices)
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Figures

Videos

Literature Cited

Literature Cited
   Copeland, R.A., Williams, J.M., Giannaras, J., Nurnberg, S., Covington, M., Pinto, D., Pick, S., and Trazaskos, J.M. 1994. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc. Natl. Acad. Sci. U.S.A. 91:11202‐11206.
   Gierse, J.K., Hauser, S.D., Creely, D.P., Koboldt, C., Rangwala, S.H., Isakson, P.C. and Seibert, K. 1995. Expression and selective inhibition of the constitutive and inducible forms of human cyclo‐oxygenase. Biochem. J. 305:479‐484.
   Kargman, S. 1996. Mechanism of selective inhibition of human prostaglandin G/H synthase‐1 and ‐2 in intact cells. Biochem. Pharmacol. 52:1113‐1125.
   Kulmacz, R.J. and Lands, W.E.M. 1987. Cyclo‐oxygenase: Measurement, purification and properties. In Prostaglandins and Related Substances (C. Benedetto, R. McDonald‐Gibson, S. Nigam, and T. Slater, eds.) pp. 209‐227. IRL Press, Oxford.
   Marnett, L.J., Siedik, P.H., Ochs, R.C., Pagels, W.R., Das, M., Honn, K.V., Warnock, R.H., Tainer, B.E., and Eling, T.E. 1984. Mechanism of the stimulation of prostaglandin H synthase and prostacyclin synthase by the antithrombotic and antimetastatic agent Nafaztrom. Mol. Pharm. 26:328‐335.
   Phelan, M.C. 1998. Techniques for mammalian cell tissue culture. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3F.1‐A.3F.14. John Wiley & Sons, New York.
   Raz, A. and Needleman, P. 1990. Differential modification of cyclo‐oxygenase and peroxidase activities of prostaglandin endoperoxidase synthase by proteolytic digestion and hydroperoxides. J. Biol. Chem. 269:603‐607.
   Smith, W.L. and Marnett, L.J. 1991. Review:Prostaglandin endoperoxide synthase: Structure and catalysis. Biochim. Biophys. Acta 1083:1‐17.
Key Reference
   Kulmacz and Lands, 1987. See above.
  Provides general description of cyclooxygenase reaction mechanism and assays.
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