Heme Oxygenase Activity Current Methods and Applications

  • Stefan W. Ryter
  • Egil Kvam
  • Rex M. Tyrrell
Part of the Methods in Molecular Biology™ book series (MIMB, volume 99)


The heme oxygenase enzymes (HO-1 and HO-2) oxidize heme to biliver-din-IXα (BVIXα), releasing carbon monoxide (CO) and iron (Fig. 1). HO enzymes control the rate of heme degradation and, consequently, also control the redistribution of the heme iron (1). The CO generated from the HO reaction affects signal transduction pathways in neuronal and vascular systems (2).
Fig. 1.

The pathway of heme metabolism. Heme oxygenase (HO-1), first described as a microsomal mixed-function oxygenase (E.C. 1:14:99:3, heme-hydrogen donor:oxygen oxidoreductase), catalyzes the rate-determining step in heme metabolism. Both heme oxygenase isozymes (HO-1 and HO-2) oxidize heme (ferriprotoporphyrin IX) to the bile pigment biliverdin-IXa, in a reaction requiring 3 mol of molecular oxygen. The accessory enzyme, NADPH:cytochrome p-450 reductase, reduces the ferric heme iron as a prerequisite for each cycle of oxygen binding and (continued) oxygen activation. The cleavage of the heme ring frees the coordinated iron, as well as the α-methene bridge carbon as carbon monoxide. The principle HO reaction product, biliverdin-IXα, is further metabolized by divalent reduction to form bilirubin-IXα, by NAD(P)H:biliverdin reductase. Heme side chains are designated as M, methyl; V, vinyl; and P, propionate.


  1. 1.
    Tenhunen R., Marver H. S., and Schmid R. (1969) Microsomal heme oxygenase, characterization of the enzyme. J. Biol. Chem. 244, 6388–6394.PubMedGoogle Scholar
  2. 2.
    Maines M. D. (1997) The heme oxygenase system: a regulator of second messenger gasses. Annu Rev. Pharmacol. Toxicol. 37, 517–554.PubMedCrossRefGoogle Scholar
  3. 3.
    Keyse S. M. and Tyrrell R. M. (1987) Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts. J. Biol. Chem. 262, 14,821–14,825.PubMedGoogle Scholar
  4. 4.
    Applegate L. A., Luscher P., and Tyrrell R. M. (1991) Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 51, 974–978.PubMedGoogle Scholar
  5. 5.
    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. USA 86, 99–103.PubMedCrossRefGoogle Scholar
  6. 6.
    Keyse S. M. and Tyrrell R. M. (1989) Induction of the heme oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365nm) radiation: evidence for the involvement of hydroxyl radical. Carcinogenesis 11, 787–791.CrossRefGoogle Scholar
  7. 7.
    Ryter S. W. and Tyrrell R. M. (1998) Singlet molecular oxygen: a possible effector of eukaryotic gene expression. Free Radic. Biol. Med. 24, 1520–1534.PubMedCrossRefGoogle Scholar
  8. 8.
    Murphy B. J., Laderoute K. R., Short S. M., and Sutherland R. M. (1991) The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells. Br. J. Cancer 64, 69–73.PubMedCrossRefGoogle Scholar
  9. 9.
    Motterlini R., Foresti R., Intaglietta M., and Winslow R. M. (1996) NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol. 270(1 Pt 2), H107–H114.PubMedGoogle Scholar
  10. 10.
    Kappas A. and Drummond G. S. (1984) Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals, and synthetic metalloporphyrins. Env. Health Perspect. 57, 301–306.CrossRefGoogle Scholar
  11. 11.
    Caltabiano M. M., Koestler T. P., Poste G., and Grieg R. G. (1986) Induction of 32-and 34-kDa stress proteins by sodium arsenite, heavy metals and thiol-reactive agents. J. Biol. Chem. 261, 13,381–13,386.PubMedGoogle Scholar
  12. 12.
    Taketani S., Sato H., Yoshinaga T., Tokunaga R., Ishii T., and Bannai S. (1990) Induction in mouse peritoneal macrophages of a 34 kDa stress protein and heme oxygenase by sulfhydryl reactive agents. J. Biochem. (Tokyo) 108, 28–32.Google Scholar
  13. 13.
    Maines M. D. (1992) Heme Oxygenase: Clinical Applications and Functions. CRC, Boca Raton, FL.Google Scholar
  14. 14.
    Ryter S. W. 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, (Forman H. J. and Cadenas E., eds.), Chapman and Hall, New York, 343–386.Google Scholar
  15. 15.
    Tyrrell R. M. (1997) Approaches to define pathways of redox regulation of a eukaryotic gene: the heme oxygenase 1 example. Methods 11, 313–318.PubMedCrossRefGoogle Scholar
  16. 16.
    Lautier D., Luscher P., and Tyrrell R. M. (1992) Endogenous glutathione levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygenase gene. Carcinogenesis 13, 227–232.PubMedCrossRefGoogle Scholar
  17. 17.
    Basu-Modak S. and Tyrrell R. M. (1993) Singlet oxygen: a primary effector in the ultraviolet A/near-visible light induction of the human heme oxygenase gene. Cancer Res. 53, 4505–4510.PubMedGoogle Scholar
  18. 18.
    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.PubMedGoogle Scholar
  19. 19.
    Applegate L. A., Noel A., Vile G., Frenk E., and Tyrrell R. M. (1995) Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress. Photochem. Photobiol. 61, 285–291.PubMedCrossRefGoogle Scholar
  20. 20.
    Trakshel G. M., Kutty R. K., and Maines M. D. (1986) Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform. J. Biol. Chem. 261, 11,131–11,137.PubMedGoogle Scholar
  21. 21.
    Cruse I. and Maines M. D. (1988) Evidence suggesting that the two forms of heme oxygenase are products of different genes. J. Biol. Chem. 263, 3348–3353.PubMedGoogle Scholar
  22. 22.
    Trakshel G. M., Kutty R. K., and Maines M. D. (1988) Resolution of rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1). Arch. Biochem. Biophys. 260, 732–739.PubMedCrossRefGoogle Scholar
  23. 23.
    Zakhary R., Gaine S., Dinerman J., Ruat M., Flavahan N., and Snyder S. (1996) Heme oxygenase 2: Endothelial and neuronal localization and role in endothelium dependent relaxation. Proc. Natl. Acad. Sci. USA 93, 795–798.PubMedCrossRefGoogle Scholar
  24. 24.
    Vercellotti G. M., Balla G., Balla J., Nath K., Eaton J. W., and Jacob H. S. (1994) Artif. Cells Blood Substit. Immobil. Biotechnol. 22, 207–213.PubMedCrossRefGoogle Scholar
  25. 25.
    Stocker R. (1990) Induction of haem oxygenase as a defense against oxidative stress. Free Rad. Res. Comm. 9, 101–112.CrossRefGoogle Scholar
  26. 26.
    Stocker R., Yamamoto Y., McDonagh A. F., Glazer A. N., and Ames B. N. (1987) Bilirubin is an antioxidant of possible physiological importance. Science 235, 1043–1045.PubMedCrossRefGoogle Scholar
  27. 27.
    Vile G. F., Basu-Modak S., Waltner C., and Tyrrell R. M. (1994) Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts. Proc. Natl. Acad. Sci. USA 91, 2607–2610.PubMedCrossRefGoogle Scholar
  28. 28.
    Eisenstein R. S. and Munro H., (1990) Translationalregulation of ferritin synthesis by iron. Enzyme 44, 42–58.PubMedGoogle Scholar
  29. 29.
    Eisenstein R. S., Garcia-Mayol D., Pettingel W., and Munro H. (1991) Regulation of ferritin and heme oxygenase synthesis in rat fibroblasts by different forms of iron. Proc. Natl. Acad. Sci. USA 88, 688–692.PubMedCrossRefGoogle Scholar
  30. 30.
    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 in-crease in ferritin. J. Biol. Chem. 268, 14,678–14,681.PubMedGoogle Scholar
  31. 31.
    Lin F. and Girotti A. W. (1998) Hemin-enhanced resistance of human leukemia cells to oxidative killing: antisense determination of ferritin involvement. Arch. Biochem. Biophys. 352, 51–58.PubMedCrossRefGoogle Scholar
  32. 31a.
    Ryter S. and Tyrrell R. M. (2000) Free Radic. Biol. Med. 28, in press.Google Scholar
  33. 32.
    Poss K. D. and Tonegawa S. (1997) (I) Heme oxygenase 1 is required for mammalian iron reutilization. (II) Reduced stress defense in heme oxygenase 1-defi-cient cells. Proc. Natl. Acad. Sci. USA 94, 10,919–10,930.PubMedCrossRefGoogle Scholar
  34. 33.
    Dennery P. A., Spitz D. R., Yang G., Tatarov A., Lee C. S., Shegog M. L., and Poss K. D. (1998) Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2. J. Clin. Invest. 101, 1001–1011.PubMedCrossRefGoogle Scholar
  35. 34.
    Verma A., Hirsch D. J., Glatt C. E., Ronnett G. V., and Snyder S. H. (1993) Carbon monoxide: a putative neural messenger. Science 259, 381–384.PubMedCrossRefGoogle Scholar
  36. 35.
    Dawson T. and Snyder S. H. (1994) Gasses as biological messengers: nitric oxide and carbon monoxide in the brain. J. Neurosci. 14, 5147–5159.PubMedGoogle Scholar
  37. 36.
    Zakhary R., Poss K. D., Jaffery S. R., Ferris C. D., Tonegawa S., and Snyder S. H. (1997) Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide. Proc. Natl. Acad. Sci. USA 94, 14,848–14,853.PubMedCrossRefGoogle Scholar
  38. 37.
    Stevens C. F. and Wang Y. (1993) Reversal of long-term potentiation by inhibitors of haem oxygenase. Nature 364, 147–149.PubMedCrossRefGoogle Scholar
  39. 38.
    Johnson R. A., Lavesa M., Askari B., Abraham N., and Nasjletti A. (1995) A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor function in rats. Hypertension 25, 166–169.PubMedGoogle Scholar
  40. 39.
    Furchgott R. F. and Jothianandan D. (1991) Endothelium dependent and independent vasodilation involving cyclic GMP: Relaxation induced by nitric oxide, carbon monoxide, and light. Blood vessels 28, 52–61.PubMedGoogle Scholar
  41. 40.
    Stone J. and Marletta M. (1994) Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33, 5636–5640.PubMedCrossRefGoogle Scholar
  42. 41.
    Burnett A. L., Johns D. G., Kriegsfeld L. J., Klein S. L., Calvin D. C., Demas G. E., Schramm L. P., Tonegawa S., Nelson R. J., Snyder S. H., and Poss K. D. (1998) Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2. Nat. Med. 4, 84–87.PubMedCrossRefGoogle Scholar
  43. 42.
    Poss K. D., Thomas M. J., Ebralidze A. K., Odell T. J., and Tonegawa S. (1995) Hippocampal long term potentiation is normal in heme oxygenase-2 mutant mice. Neuron 15, 867–873.PubMedCrossRefGoogle Scholar
  44. 43.
    Tyrrell R. M. and Basu-Modak S. (1994) Transient enhancement of heme oxygenase-1 mRNA accumulation: a marker of oxidative stress to eukaryotic cells. Methods. Enzymol. 234, 224–235.PubMedCrossRefGoogle Scholar
  45. 44.
    Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.PubMedCrossRefGoogle Scholar
  46. 45.
    Yoshida T., Biro P., Cohen T., Müller R. M., and Shibahara S. (1988) Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur. J. Biochem. 171, 457–461.PubMedCrossRefGoogle Scholar
  47. 46.
    Shibahara S., Müller R. M., Taguchi H., and Yoshida T. (1985) Cloning and expression of cDNA for rat heme oxygenase. Proc. Natl. Acad. Sci. USA 82, 7865–7869.PubMedCrossRefGoogle Scholar
  48. 47.
    Kageyama H., Hiwasa T., Tokunaga K., and Sakiyama S. (1988) Isolation and characterization of a complementary DNA clone for a MR 32,000 protein which is induced with tumor promoters in BALB/c 3T3 cells. Cancer Res. 48, 4795–4798.PubMedGoogle Scholar
  49. 48.
    Suzuki T., Sato M., Ishikawa K., and Yoshida T. (1992) Nucleotide sequence of cDNA for porcine heme oxygenase and its expression in Escherichia coli. Biochem. Int. 28, 887–893.Google Scholar
  50. 49.
    Evans C. O., Healey J. F., Greene Y., and Bonkovsky H. L. (1991) Cloning, sequencing and expression of cDNA for chick liver haem oxygenase. Biochem. J. 273, 659–666.PubMedGoogle Scholar
  51. 50.
    Alam J., Shibahara S., and Smith A. (1989) Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. J. Biol. Chem. 264, 6371–6375.PubMedGoogle Scholar
  52. 51.
    Keyse S. M., Applegate L. A., Tromvoukis Y., and Tyrrell R. M. (1990) Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts. Mol. Cell. Biol. 10, 4967–4969.PubMedGoogle Scholar
  53. 52.
    Kutty R. K., Kutty G, Nagineni C. N., Hooks J. J., Chader G. J., and Wiggert B. (1994) RT-PCR assay for heme oxygenase-1 and heme oxygenase-2: a sensitive method to estimate cellular oxidative damage. Ann. NY Acad. Sci. 738, 427–430.PubMedCrossRefGoogle Scholar
  54. 53.
    Saunders E. L., Maines M. D., Meredith M. J., and Freeman M. J. (1991) Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells. Arch. Biochem. Biophys. 288, 368–373.PubMedCrossRefGoogle Scholar
  55. 54.
    Hiwasa T., and Sakiyama S. (1986) Increase in the synthesis of a MR 32,000 protein in BALB/c 3T3 cells after treatment with tumor promoters, chemical carcinogens, metal salts and heat shock. Cancer Res. 46, 2474–2481.PubMedGoogle Scholar
  56. 55.
    Ishizawa S., Yoshida T., and Kikuchi G. (1983) Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues as well as the induced enzymes. J. Biol. Chem. 258, 4220–4225.PubMedGoogle Scholar
  57. 56.
    Greene Y. J., Healey J. F., and Bonkovsky H. L. (1991) Immunochemical studies of haem oxygenase. Preparation and characterization of antibodies to chick liver haem oxygenase and their use in detecting and quantifying amounts of haem oxygenase protein. Biochem. J. 279(Pt 3), 849–854.PubMedGoogle Scholar
  58. 57.
    Schacter B., Cripps V., Troxler R. F., and Offner G. D. (1990) Structural studies on bovine spleen heme oxygenase. Arch. Biochem. Biophys. 282, 404–412.PubMedCrossRefGoogle Scholar
  59. 58.
    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.PubMedCrossRefGoogle Scholar
  60. 59.
    Schacter B. (1978) Assay for microsomal heme oxygenase in liver and spleen. Methods Enzymol. 52, 367–372.PubMedCrossRefGoogle Scholar
  61. 60.
    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.PubMedGoogle Scholar
  62. 61.
    Tenhunen R. (1972) Method for microassay of microsomal heme oxygenase activity. Anal. Biochem. 45, 600–607.PubMedCrossRefGoogle Scholar
  63. 62.
    Sierra E. and Nutter L. (1992) A microassay for heme oxygenase activity using thin layer chromatography. Anal. Biochem. 200, 27–30.PubMedCrossRefGoogle Scholar
  64. 63.
    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.PubMedGoogle Scholar
  65. 64.
    Lee T. C. and Ho I. C. (1994) Expression of heme oxygenase in arsenic-resistant human lung adenocarcinoma cells. Cancer Res. 54, 1660–1664.PubMedGoogle Scholar
  66. 65.
    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.PubMedCrossRefGoogle Scholar
  67. 66.
    Bonkovsky H. L., Wood S. G., Howell S. K., Sinclair P. R., Lincoln B., Healy J. F., and Sinclair J. F., (1986) High performance liquid chromatography separation and quantitation of tetrapyrroles from biological materials. Anal. Biochem. 155, 56–64.PubMedCrossRefGoogle Scholar
  68. 67.
    Ryter S. W., Kvam E., and Tyrrell R. M. (1999) Determination of heme oxygenase activity by high performance liquid chromatography. Methods Enzymol. 300, 322–336.PubMedCrossRefGoogle Scholar
  69. 68.
    Ryter S. W., 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 overexpression. Free Radic. Biol. Med. 24, 959–971.PubMedCrossRefGoogle Scholar
  70. 69.
    Vremen H. and Stevenson D. (1988) Heme oxygenase activity as measured by carbon monoxide production. Anal. Biochem. 168, 31–38.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Stefan W. Ryter
    • 1
  • Egil Kvam
    • 2
  • Rex M. Tyrrell
    • 2
  1. 1.Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Internal MedicineSouthern Illinois University School of MedicineSpringfield
  2. 2.Department of Pharmacy and PharmacologyUniversity of BathBath

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