Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation

William K. McCoubrey1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.3
DOI:  10.1002/0471140856.tx0903s00
Online Posting Date:  May, 2001
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Abstract

It is possible to detect changes in the level of expression of the two isozymes of heme oxygenase (HO). The sequence differences allow the production of isozyme‐specific antibodies that can be used in immunoblot analysis. Total HO activity in tissue microsomal fractions or extracts of bacteria transformed with an HO gene can be measured as described in this unit using a coupled assay employing biliverdin reductase, which converts the biliverdin product to bilirubin. Bilirubin is then quantified with a scanning spectrophotometer.

     
 
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Table of Contents

  • Basic Protocol 1: Spectrophotometric Heme Oxygenase Assay
  • Basic Protocol 2: Immunoblotting for Heme Oxygenase 1 and 2
  • Support Protocol 1: Preparation of Microsomal Fraction from Tissue
  • Support Protocol 2: Preparation of Bacterial Cell Extracts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Spectrophotometric Heme Oxygenase Assay

  Materials
  • 1 mM heme (see recipe), prepared fresh
  • 30 U/ml biliverdin reductase (BVR; e.g., StressGen Biotechnologies) or kidney cytosolic fraction (see protocol 3)
  • Cytochrome c reductase (CCR; e.g., Sigma or StressGen Biotechnologies), optional
  • Heme oxygenase (HO) assay buffer (see recipe)
  • Microsomal fraction (adjust protein concentration to <8 mg/ml with resuspension buffer)
  • Resuspension buffer (see recipe)
  • Triton X‐100 (optional)
  • 2.75 mM NADPH (see recipe)
  • Chloroform, chilled on ice (optional)
  • Glass tubes, 13 × 100–mm or 12 × 75–mm
  • Scanning spectrophotometer

Basic Protocol 2: Immunoblotting for Heme Oxygenase 1 and 2

  Materials
  • Fresh or flash‐frozen tissue
  • 0.9% (w/v) NaCl in distilled H 2O, ice cold
  • Homogenization buffer (see recipe), ice cold
  • Resuspension buffer (see recipe)
  • Surgical scissors
  • Dounce homogenizer with tight‐fitting Teflon pestle
  • Variable‐speed drill
  • Centrifuge with fixed‐angle rotor and tubes (e.g., Beckman J2 and JA20 rotor)
  • Ultracentrifuge with fixed‐angle rotor (e.g., Beckman L8 and 50Ti rotor)
NOTE: Microsomes can be prepared from either fresh or frozen tissue. All steps must be carried out on ice or at 4°C unless otherwise noted.

Support Protocol 1: Preparation of Microsomal Fraction from Tissue

  Materials
  • Bacteria on culture plate or in overnight culture
  • Liquid culture medium (e.g., 2× TY medium or superbroth; appendix 3A) containing appropriate antibiotic (e.g., 50 to 100 µg/ml ampicillin)
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG), optional
  • 0.1% (w/v) NaCl in distilled H 2O
  • Resuspension buffer (see recipe)
  • Centrifuge and rotor (e.g., Beckman J2 and JA20 rotor)
  • Water bath, room temperature
  • Sonicator
  • Additional reagents and equipment for culturing bacteria ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Ewing, J.F. and Maines, M.D. 1991. Rapid induction of heme oxygenase‐1 mRNA and protein by hyperthermia in the rat brain: Heme oxygenase‐2 is not a heat shock protein. Proc. Natl. Acad. Sci. U.S.A. 88:5364‐5368.
   Keyse, A.M. and Tyrell, A.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.
  Maines, M.D. 1988. Heme oxygenase: Function, multiplicity, regulatory mechanisms and clinical applications. FASEB J. 2:2557‐2568.
   Maines, M.D. 1992. Heme oxygenase and heme degrading enzymes. In Heme Oxygenase: Clinical Applications and Functions (M.D. Maines, ed.) pp. 43‐108. 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., Ibrahim, N.G., and Kappas, A. 1977. Solubilization and partial purification of heme oxygenase from rat liver. J. Biol. Chem. 252:5900‐5903.
  Maines, M.D., Trakshel, G.M., and Kutty, R.K. 1986. Characterization of two constitutive forms of rat liver heme oxygenase: Only one molecular species is inducible. J. Biol. Chem. 261:411‐419.
  McCoubrey, W.K., Jr., and Maines, M.D. 1993. Domains of rat heme oxygenase‐2: The amino terminus and histidine 151 are required for heme oxidation. Arch. Biochem. Biophys. 302:402‐408.
  McCoubrey, W.K., Jr., and Maines, M.D. 1994. The structure, organization and differential expression of the gene encoding rat heme oxygenase‐2. Gene 139:155‐161.
  McCoubrey, W.K., Jr., Huang, T.J., and Maines, M.D. 1996. Heme oxygenase‐2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis. J. Biol. Chem. 272:12568‐12574.
  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.
  Müller, R.M., Taguchi, H., and Shibahara, S. 1987. Nucleotide sequence and organization of the rat heme oxygenase gene. J. Biol. Chem. 262:6795‐6802.
  Rotenberg, M.O. and Maines, M.D. 1990. Isolation, characterization and expression in Escherichia coli of a cDNA encoding rat heme oxygenase‐2. J. Biol. Chem. 265:7501‐7506.
  Rotenberg, M.O. and Maines, M.D. 1991. Characterization of a cDNA‐encoding rabbit brain heme oxygenase‐2 and identification of a conserved domain among mammalian heme oxygenase isozymes: Possible heme‐binding site? Arch. Biochem. Biophys. 290:336‐344.
  Shibahara, S., Müller, R., Taguchi, H., and Yoshida, T. 1985. Cloning and expression of cDNA for rat heme oxygenase. Proc. Natl. Acad. Sci. U.S.A. 82:7865‐7869.
  Tenhunen, R., Marver, H.S., and Schmid, R. 1968. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. U.S.A. 61:748‐755.
  Zhang, L. and Guarente, L. 1995. Heme binds to a short sequence that serves a regulatory function in diverse proteins. EMBO J. 14:313‐320.
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