Detection of Heme Oxygenase Activity by Measurement of CO

Hendrik J. Vreman1, David K. Stevenson1

1 Stanford University Medical Center, Stanford, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.2
DOI:  10.1002/0471140856.tx0902s00
Online Posting Date:  May, 2001
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Heme oxygenase (HO) is the first and rate‐limiting step in degradation of heme, and in the presence of NADPH‐cytochrome P‐450 reductase it produces equimolar amounts of biliverdin and CO. CO produced in a closed system can be quantified as described in this unit by gas chromatography as a measure of HO activity in tissue slices, tissue homogenates, and tissue fractions.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1:

  • Tissue or cell samples
  • 0.1 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 0.9% (w/v) NaCl (optional, appendix 2A)
  • HO substrate (see recipe)
  • 4.5 mM NADPH (see recipe)
  • NADPH–cytochrome P‐450 reductase (optional)
  • Anhydrous magnesium perchlorate (anhydrone; Fisher)
  • GC calibration gas: 10.8 µl CO/liter air (Scott Specialty Gases)
  • CO gas, 99.9% pure (e.g., Matheson Gas Products), optional
  • 12 × 32–mm amber vials with polypropylene screw caps fitted with septa (e.g., Alltech Associates)
  • Vial racks (e.g., Fisher or equivalent)
  • Hamilton gas‐tight syringe with repeating dispenser
  • 2.5‐mm‐thick blue silicone sheets (Alltech Associates), for use in making septa
  • Hopcalite (CuO/MnO) catalytic converter (Trace Analytical)
  • Vial‐purging assembly (see Fig. A)
  • Headspace‐sampling assembly (see Fig. B)
  • 0.0625‐in. (1.59‐mm) o.d. sleeve connector
  • 18‐G side‐port needles, 5 and 7 cm
  • 68 × 0.53–cm (i.d.) stainless steel column
  • 40‐ to 60‐mesh molecular sieve, 13X (Alltech Associates)
  • Gas‐flow meter (J & W Scientific)
  • Gas‐chromatograph system with reduction gas detector (Trace Analytical)
  • Sample injection valve with Model 451 Recycling Intervalometer (Gralab Instruments Division)
  • Recorder: 10‐mV recorder (Linear Instrument) or integrating recorder (e.g., CR‐3A, Shimadzu Scientific Instruments)
  • 20‐G side‐port needle.
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)
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Literature Cited

Literature Cited
   Cavallin‐Stahl, E., Jonsson, F.‐I., and Lundh, B. 1978. A new method for determination of microsomal haem oxygenase (E.C. based on quantitation of carbon monoxide formation. Scand. J. Clin. Lab. Invest. 38:69‐76.
   Dennery, P.A., McDonagh, A.F., Spitz, D.R., Rodgers, P.A., and Stevenson, D.K. 1995. Hyperbilirubinemia results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia. Free Radic. Biol. Med. 19:395‐404.
   Levitt, M.D., Ellis, C., Springfield, J., and Engel, R.R. 1995. Carbon monoxide generation from hydrocarbons at ambient and physiological temperature: A sensitive indicator of oxidant damage? J. Chromatogr. 695:324‐328.
   Lodola, A., Hendry, A.F., and Jones, O.T.G. 1979. Haem oxygenase: A reappraisal of the stoichiometry. FEBS Lett. 104:45‐50.
   Lowry, O.H., Rosebrough, H.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   Maines, M.D. 1992. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, Fla.
   Maines, M.D. 1997. The heme oxygenase system: A regulator of 2nd‐messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517‐554.
   Maines, M.D., Ibrahim, N.G., and Kappas, A. 1977. Solubilization and partial purification of heme oxygenase from rat liver. J. Biol. Chem. 252:5900‐5903.
   McCoubrey, W.K., Jr., Huang, T.J., and Maines, M.D. 1997. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase‐3. Eur. J. Biochem. 247:725‐732.
   Meffert, M.K., Haley, J.E., Schuman, E.M., Schulman, H., and Madison, D.V. 1994. Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long term potentiation by metalloporphyrins. Neuron 13:1225‐1233.
   Ostrander, C.R., Stevenson, D.K., Neu, J., Kerner, J.A., and Moses, S.W. 1982. A sensitive analytical apparatus for measuring hydrogen production rates. I. Application to studies in small animals. Evidence of the effects of an α‐glucoside‐hydrolase inhibitor in the rat. Anal. Biochem. 119:378‐386.
   Sunderman, F.W., Jr., Downs, J.R., Reid, M.C., and Bibeau, L.M. 1982. Gas chromatographic assay for heme oxygenase activity. Clin. Chem. 28:2026‐2032.
   Tenhunen, R. 1972. Method for microassay of microsomal heme oxygenase activity. Anal. Biochem. 45:600‐607.
   Tenhunen, R., Marver, H.S., and Schmid, R. 1968. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Nat. Acad. Sci. U.S.A. 61:748‐755.
   Vallier, H.A., Rodgers, P.A., and Stevenson, D.K. 1993. Inhibition of heme oxygenase after oral vs intraperitoneal adminstration of chromium porphyrins. Life Sci. 52:79‐84.
   Vreman, H.J. and Stevenson, D.K. 1988. Heme oxygenase activity as measured by CO production. Anal. Biochem. 168:31‐38.
   Vreman, H.J., Kwong, L.K., and Stevenson, D.K. 1984. Carbon monoxide in blood: An improved micro‐blood sample collection system, with rapid analysis by gas chromatography. Clin. Chem. 30:1382‐1386.
   Vreman, H.J., Stevenson, D.K., Henton, D., and Rosenthal, P. 1988. Correlation of carbon monoxide and bilirubin production by tissue homogenates. J. Chromatog. Biomed. Appl. 427:315‐319.
   Vreman, H.J., Gillman, M.J., Downum, K.R., and Stevenson, D.K. 1990a. In vitro generation of carbon monoxide from organic molecules and synthetic metalloporphyrins mediated by light. Dev. Pharmacol. Ther. 15:112‐124.
   Vreman, H.J., Rodgers, P.A., and Stevenson, D.K. 1990b. Zinc protoporphyrin administration for suppression of increased bilirubin production by iatrogenic hemiolysis in rhesus neonates. J. Pediatr. 117:292‐297.
   Vreman, H.J., Lee, O.K., and Stevenson, D.K. 1992. In vitro and in vivo characteristics of the heme oxygenase inhibitor: ZnBG. Am. J. Med. Sci. 302:335‐341.
   Vreman, H.J., Ekstrand, B.C., and Stevenson, D.K. 1993. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr. Res. 33:195‐200.
   Vreman, H.J., Wong, R.J., Sanesi, C.A., Dennery, P.A., and Stevenson, D.K. 1998. Simultaneous production of carbon monoxide and thiobarbituric acid reactive substances in rat tissue preparations by an iron/ascorbate system. Can. J. Physiol. Pharmacol. 76:1057‐1065.
Key References
   Maines, 1992. See above.
  This book provides a comprehensive review of heme oxygenase.
   Maines, 1997. See above.
  This review presents the most up‐to‐date information on the heme oxygenase system.
   Vreman and Stevenson, 1988. See above.
  This paper provides technical details on measuring HO activity by assaying CO production.
   Vreman et al., 1984. See above.
  This article presents technical details on determining CO concentrations by GC.
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