An HPLC Method to Detect Heme Oxygenase Activity

Stefan W. Ryter1, Rex M. Tyrrell2

1 Southern Illinois University School of Medicine, Springfield, Illinois, 2 University of Bath, Bath, United Kingdom
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.6
DOI:  10.1002/0471140856.tx0906s05
Online Posting Date:  May, 2001
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This unit presents a method to calculate heme oxygenase enzymatic activity from the formation of bilirubin equivalents [biliverdin‐Ixalpha (BV) and bilirubin‐IXalpha (BR)]. The BV and BR generated in the reaction are separated by reversed‐phase HPLC and detected using visible absorbance spectroscopy. Since both metabolites of heme degradation are directly quantifiable, the assay eliminates the requirement for biliverdin reductase supplementation.

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Table of Contents

  • Basic Protocol 1: Determining Heme Oxygenase Activity Using HPLC
  • Support Protocol 1: Preparation of Microsomal Protein Extracts from Cultured Cells
  • Alternate Protocol 1: Detection of BVR Activity
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Determining Heme Oxygenase Activity Using HPLC

  • Microsomal protein extract from cultured cells (see protocol 2)
  • Solution A (see recipe)
  • Partially purified biliverdin reductase (BVR; optional)
  • 20 mM β‐NADPH (see recipe)
  • 40 mM glucose 6‐phosphate (see recipe)
  • 1 U/µl glucose‐6‐phosphate dehydrogenase (see recipe)
  • 2.5 mM protoporphyrin IX (Sn PPIX; see recipe; optional)
  • 2.5 mM hemin stock solution (see recipe)
  • Stop solution with mesoporphyrin internal standard (see recipe)
  • HPLC buffer A (see recipe)
  • HPLC buffer B: methanol (HPLC grade; Fisher; degassed 2 min with compressed He)
  • 20 µM biliverdin and bilirubin standards (see reciperecipes)
  • 1.5‐ and 0.5‐ml microcentrifuge tubes
  • High performance liquid chromatography (HPLC) system with two solvent reservoirs and pumps (e.g., Kontron or equivalent)
  • Data acquisition system (e.g., MT 450 MS‐DOS or higher; Kontron or equivalent)
  • Autosampler (e.g., HPLC 360; Kontron, or equivalent)
  • Visible detector (e.g., UVIKON 720 LC microdetector; Kontron or equivalent)
  • Reversed‐phase C18 steel cartridge HPLC column (e.g., Novapak 3.9 mm × 15 cm) or phenyl reversed‐phase column (both available from Waters Chromatography)
  • Compressed helium tank

Support Protocol 1: Preparation of Microsomal Protein Extracts from Cultured Cells

  • Cultures of cells
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A), ice‐cold
  • PBS/EDTA solution (see recipe), ice‐cold
  • Solution A (see recipe)
  • 1000× protease inhibitor stock solutions (see recipe)
  • Rubber cell scrapers
  • 15‐ml centrifuge tubes (e.g., 16 × 125–mm tissue culture tubes with screw caps; Corning)
  • Refrigerated tabletop centrifuge (e.g., GPK centrifuge; Beckman, or equivalent)
  • Sonicator (e.g., Branson sonifier B‐12 sonicator; Branson Ultrasonics, or equivalent)
  • Ultracentrifuge (e.g., Centrikon T20‐60 ultracentrifuge; Kontron, or equivalent)
  • Fixed‐angle rotor capable of 105,000 × g (e.g., TFT65.13 rotor; Kontron, or equivalent)
  • Polyallomar ultracentrifuge tubes, (e.g., 13.5‐ml; Kontron)
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)
NOTE: Perform all steps at 4°C.

Alternate Protocol 1: Detection of BVR Activity

  • Protein sample to be analyzed for BVR activity
  • 2.5 µM biliverdin (BV) solution in DMSO (see recipe for 20 µM stock)
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Literature Cited

Literature Cited
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   Durante, W. and Schafer, A. 1998. Carbon monoxide and vascular cell function (review). Int. J. Mol. Med. 2:255‐262.
   Keyse, S.M. and Tyrrell, R.M. 1989. Heme oxygenase is the major 32‐kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. U.S.A. 86:99‐103.
   Kutty, R.K. and Maines, M.D. 1982. Oxidation of heme C derivatives by purified heme oxygenase. Evidence for the presence of one molecular species of heme oxygenase in the rat liver. J. Biol. Chem. 257:9944‐9952.
   Lee, T.C. and Ho, I.C. 1994. Expression of heme oxygenase in arsenic‐resistant human lung adenocarcinoma cells. Cancer Res. 54:1660‐1664.
   Lincoln, B.C., Mayer, A., and Bonkovsky, H. 1988. Microassay of heme oxygenase by high performance liquid chromatography. Application to assay of needle biopsies of human liver. Anal. Biochem. 170:485‐490.
   Lincoln, B., Aw, T.Y., and Bonkovsky, H. 1989. Heme catabolism in cultured hepatocyte: Evidence that heme oxygenase is the predominant pathway and that a proportion of the synthesized heme is converted rapidly to biliverdin. Biochim. Biophys. Acta. 992:49‐58.
   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 second‐messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517‐554.
   Maines, M.D., Trakshel, G.M., and Kutty, R.K. 1986. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J. Biol. Chem. 261:411‐419.
   McCoubrey, W.K., Huang, T.J., and Maines, M.D. 1997a. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase‐3. Eur. J. Biochem. 247:725‐732.
   McCoubrey, W.K., Huang, T.J., and Maines, M.D. 1997b. Heme oxygenase‐2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis. J. Biol. Chem. 272:12568‐12575.
   Noguchi, M., Yoshida, T., and Kikuchi, G. 1982. Identification of the product of heme degradation catalyzed by the heme oxygenase system as biliverdin‐IXα by reverse phase high performance liquid chromatography. J. Biochem. (Tokyo). 91:1479‐1483.
   Ryter, S. and Tyrrell, R.M. 1997. The role of heme oxygenase‐1 in the mammalian stress response: Molecular aspects of regulation and function. In Oxidative Stress and Signal Transduction (H.J. Forman and E. Cadenas, eds.) pp.343‐386. Chapman and Hall, New York.
   Ryter, S. and Tyrrell, R.M. 2000. The heme synthesis and degradation pathways: Role in oxidant sensitivity. Heme oxygenase has both pro‐ and antioxidant properties. Free Radic. Biol. Med. 28:289‐309.
   Ryter, S., Kvam, E., Richman, L., Hartmann, F., and Tyrrell, R. M. 1998. A chromatographic assay for heme oxygenase activity in cultured human cells: Application to artificial heme oxygenase over‐expression. Free Radic. Biol. Med. 24:959‐971.
   Ryter, S., Kvam, E., and Tyrrell, R.M. 2000. The heme oxygenases: Current methods and applications. Methods Mol. Biol. 99:369‐391.
   Schacter, B. 1978. Assay for microsomal heme oxygenase in liver and spleen. Methods Enzymol. 52:367‐372.
   Sierra, E. and Nutter, L.M. 1992. A microassay for heme oxygenase activity using thin layer chromatography. Anal. Biochem. 200:27‐30.
   Stocker, R., Yamamoto, Y., McDonagh, A., Glazer, A., and Ames, B.N. 1987. Bilirubin is an antioxidant of possible physiological importance. Science. 235:1043‐1046.
   Tenhunen, R. 1972. Method for microassay of microsomal heme oxygenase activity. Anal. Biochem. 45:600‐607.
   Tenhunen, R., Marver, H.S., and Schmid, R. 1969. Microsomal heme oxygenase, characterization of the enzyme. J. Biol. Chem. 244:6388‐6394.
   Tenhunen, R., Ross, M.E., Marver, H.S., and Schmid, R. 1970. Reduced nicotinamide‐adenine dinucleotide phosphate dependent biliverdin reductase: Partial purification and characterization. Biochemistry. 9:298‐303.
   Vile, G.F. and Tyrrell, R.M. 1993. Oxidative stress resulting from ultraviolet‐A irradiation of human skin fibroblasts leads to a heme oxygenase‐dependent increase in ferritin. J. Biol. Chem. 268:14678‐14681.
   Weber, C.M., Eke, B.C., and Maines, M.D. 1994. Corticosterone regulates heme oxygenase‐2 and NO synthase transcription and protein expression in the rat brain. J. Neurochem. 63:953‐962.
Key References
   Ryter, S., Kvam, E., and Tyrrell, R.M. 1999. Determination of heme oxygenase activity by high performance liquid chromatography. Methods Enzymol. 300:322‐336.
  This paper also provides detailed methodology for the analysis of HO activity using high performance liquid chromatography.
   Ryter et al., 1998. See above.
  This paper shows graphic examples of the application of the HPLC technique to the detection of heme oxygenase activity.
   Maines, 1992. See above.
  This book provides a comprehensive account of the basic background information related to the heme oxygenase system.
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