Purification and Characterization of Heme Oxygenase

Angela Wilks1

1 University of Maryland, Baltimore, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.9
DOI:  10.1002/0471140856.tx0909s15
Online Posting Date:  May, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

High‐yield expression and purification of human heme oxygenase isozyme 1 provided the breakthrough in characterizing the protein from mechanistic and structural standpoints. This unit provides a protocol for high‐level expression and subsequent purification of HO‐1. The commentary includes a discussion of subsequent biochemical and biophysical characterizations.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: High‐Yield Expression of Human HO‐1 in E. Coli
  • Basic Protocol 2: Purification of HO‐1 Protein
  • Support Protocol 1: Preparation of NADPH–Cytochrome P450 Reductase
  • Spectrophotometric Analysis of the Heme‐Heme Oxygenase Complex
  • Support Protocol 2: UV/Visible Spectra of the Heme‐HO‐1 Complex
  • Support Protocol 3: UV/Visible Spectra of Heme Oxygenase Reaction Intermediates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: High‐Yield Expression of Human HO‐1 in E. Coli

  Materials
  • pBHO‐1 vector (P.R. Ortiz de Montellano; )
  • E. coli DH5αF′ (Life Technologies)
  • LB medium and plates containing 100 µg/ml ampicillin (see recipe)
  • Low‐phosphate induction medium (see recipe) containing 75 µg/ml ampicillin
  • Temperature‐controlled shaking incubator (New Brunswick Model G20 or equivalent)
  • UV/visible spectrophotometer (Varian Cary Bio‐100 or equivalent)
  • Low‐speed centrifuge (Beckman Avanti J25‐I with JA‐10 rotor or equivalent model)

Basic Protocol 2: Purification of HO‐1 Protein

  Materials
  • Cell pellets (see protocol 1)
  • Lysis buffer I (see recipe)
  • Ammonium sulfate
  • 10, 20, and 100 mM potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • 250 mM potassium chloride (KCl)
  • 10 mM diaminopropane
  • Gradient from 0 to 250 mM KCl in 10 mM diaminopropane
  • 1.5 mM hemin solution (see recipe)
  • NADPH–cytochrome P450 reductase (see protocol 3)
  • 2.5 to 3.0 µmol/min/mg biliverdin reductase (Mahin D. Maines; )
  • Hemin/BSA solution (see recipe)
  • 1 mM NADPH: dilute 1 M NADPH stock (see recipe) in 100 mM potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • Fisher Scientific 60 Sonicator Dismembrator (or equivalent)
  • Low‐speed centrifuge (Beckman Avanti J25‐I with JA‐10 and JA‐20 rotors or equivalent) and corresponding 50‐ml centrifuge tubes
  • Dialysis membrane, MWCO 10 kDa (also see appendix 3H)
  • FPLC system (Amersham Pharmacia Biotech) with Mono‐Q (HR 10/10) column or conventional chromatography system (peristaltic pump, fraction collector, and gradient maker) with 3 × 10–cm Q‐Sepharose column (also from Amersham Pharmacia Biotech)
  • 3 × 10–cm chromatography column packed with QAE anion‐exchange resin (Amersham Pharmacia Biotech) and equilibrated with 10 mM diaminopropane, pH 9.0
  • 1.5 × 6.0–cm chromatography column packed with Bio‐Gel HTP hydroxyapatite resin (Bio‐Rad) and equilibrated with 10 mM potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • UV/visible spectrophotometer (Varian Cary Bio‐100 or equivalent)
  • Amicon filtration unit with YM10 membrane (10 kDa molecular weight cut‐off)
  • Additional reagents and equipment for dialysis ( appendix 3H) and SDS‐PAGE ( appendix 3F)
NOTE: All procedures should be carried out at 4°C.

Support Protocol 1: Preparation of NADPH–Cytochrome P450 Reductase

  Materials
  • E. coli BL21(DE3)
  • pETOR (pET vector containing gene for human NADPH cytochrome P450 reductase; )
  • LB medium containing 100 µg/ml ampicillin (see recipe)
  • Terrific broth containing 100 µg/ml ampicillin (see recipe)
  • 1 M IPTG stock (see recipe)
  • Lysis buffer II (see recipe)
  • Resuspension buffer (see recipe)
  • Bio‐Rad protein assay kit
  • Lubrol PX detergent (polyoxyelthylene 9 lauryl ether; Sigma‐Aldrich)
  • Column wash buffers I and II (see reciperecipes)
  • Elution buffer: column wash buffer II (see recipe) containing 2.5 mM 2′‐AMP
  • 20 mM potassium phosphate buffer, pH 7.4 ( appendix 2A) containing 20% (v/v) glycerol
  • 300 mM potassium phosphate buffer, pH 7.7 ( appendix 2A)
  • 1 mM bovine cytochrome c
  • 1 mM NADPH: dilute 1 M NADPH (see recipe) with 300 mM potassium phosphate buffer, pH 7.7 (see appendix 2A for buffer)
  • UV/visible spectrophotometer (Varian Cary Bio‐100 or equivalent model)
  • Temperature‐controlled shaking incubator (New Brunswick Model G25 or equivalent model)
  • Low‐speed centrifuge(Beckman Avanti J25‐I with JA‐10 rotor or equivalent model)
  • Fisher Scientific 60 Sonicator Dismembrator (or equivalent)
  • High‐speed centrifuge (Beckman L8‐70 with 45 Ti rotor, or equivalent)
  • 1 × 10–cm chromatography column packed with 2′,5′‐ADP‐Sepharose and equilibrated with equilibration buffer (see recipe for buffer)

Support Protocol 2: UV/Visible Spectra of the Heme‐HO‐1 Complex

  Materials
  • 10 to 20 µM heme‐HO‐1 complex (see protocol 2)
  • 20 mM potassium phosphate buffer, pH 7.0 ( appendix 2A)
  • Carbon monoxide (CO) gas source
  • 1 mM (17.4 mg/ml) sodium hydrosulfite (sodium dithionite) in 10 mM potassium phosphate buffer, pH 7.0 (see appendix 2A for buffer), prepared immediately before use
  • 1‐cm quartz cuvettes
  • UV/visible spectrophotometer (Varian Cary Bio‐100 or equivalent model)
  • 1 × 10–cm Bio‐Gel P‐10 desalting column (Bio‐Rad), equilibrated with 20 mM potassium phosphate buffer, pH 7.0 (see appendix 2A for buffer)

Support Protocol 3: UV/Visible Spectra of Heme Oxygenase Reaction Intermediates

  Materials
  • NADPH–cytochrome P450 reductase (see protocol 3)
  • Heme‐HO‐1 (see protocol 2)
  • 100 mM potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • Carbon monoxide (CO) gas source
  • 1 M NADPH (see recipe)
  • 1‐cm cuvette
  • UV/visible spectrophotometer (Varian Cary Bio‐100 or equivalent model)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Balla, J., Jacob, H.S., Balla, G., Nath, K., Eaton, J.W., and Vercellotti, G.M. 1993. Endothelial‐cell heme uptake from heme proteins: Induction of sensitization and desensitization to oxidant damage. Proc. Natl. Acad. Sci. U.S.A. 90:9285‐9259.
   Barañano, D.E., Rao, M., Ferris, C.D., and Snyder, S.H. 2002. Biliverdin reductase: A major physiologic cytoprotectant. Proc. Natl. Acad. Sci. U.S.A. 99:16093‐16098.
   Bothwell, T.H., Charlton, R.W., and Motulsky, A.G. 1995. Hemochromatosis. In The Metabolic and Molecular Basis of Inherited Diseases (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 2237‐2269. McGraw‐Hill, New York.
   Cheek, J. and Dawson, J. 2000. Magnetic circular dichroism spectroscopy of heme proteins and model systems. In The Porphyrin Handbook, vol. 7. (K.M. Kadish, K.M. Smith, and R. Guilard, eds.) pp. 339‐369. Academic Press, London.
   Craig, S.P., 3rd, Yuan, L., Kuntz, D.A., McKerrow, J.H., Wang, C.C. 1991. High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 88:2500‐2504.
   Dore, S., Takahashi, M., Ferris, C.D., Hester, L.D., Guastella, D. and Snyder, S.H. 1999. Bilirubin, formed by activation of heme oxygenase‐2, protects neurons against oxidative stress injury. Proc. Natl. Acad. Sci. U.S.A. 96:2445‐2450.
   Hawkins, R.D., Zhuo, M., Arancio, O. 1994. Nitric oxide and carbon monoxide as possible retrograde messengers in hippocampal long‐term potentiation. J. Neurobiol. 25:652‐665.
   Hawkins, B.K., Wilks, A., Powers, L.S., Ortiz de Montellano, P.R., Dawson, J.H. 1996. Ligation of the iron in the heme‐heme oxygenase complex: X‐ray absorption, electronic absorption and magnetic circular dichroism studies. Biochim. Biophys. Acta. 1295:165‐173.
   Hernandez, G., Wilks, A., Paolesse, R., Smith, K.M., Ortiz de Montellano, P.R., La Mar, G.N. 1994. Proton NMR investigation of substrate‐bound heme oxygenase: Evidence for electronic and steric contributions to stereoselective heme cleavage. Biochemistry 33:6631‐6641.
   Immenschuh, S. and Ramadori, G. 2000. Gene regulation of heme oxygenase‐1 as a therapeutic target. Biochem. Pharmacol. 60:1121‐1128.
   Kitagawa, T., Nagai, K., Tsubaki, M. 1979. Assignment of the Fe‐Nepsilon (His F8) stretching band in the resonance Raman spectra of deoxy myoglobin. FEBS Lett. 104:376‐378.
   Liu, Y. and Ortiz de Montellano, P.R. 2000. Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase‐1. J. Biol. Chem. 275:5297‐5307.
   Maines, M.D. 1992. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, Fla.
   Maines, M.D., Trakshel, G.M., 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., 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.
   Ortiz de Montellano, P.R. 2000. The mechanism of heme oxygenase. Curr. Opin. Chem. Biol. 4:221‐227.
   Ortiz de Montellano, P.R. and Wilks, A. 2000. Heme oxygenase structure and mechanism. Adv. Inorg. Chem. 51:359‐402.
   Otterbein, L.E. and Choi, A.M. 2000. Heme oxygenase: Colors of defense against cellular stress. Am. J. Physiol. Lung Cell Mol. Physiol. 279:L1029‐L1037.
   Otterbein, L., Sylvester, S.L., Choi, A.M. 1995. Hemoglobin provides protection against lethal endotoxemia in rats: The role of heme oxygenase‐1. Am. J. Respir. Cell. Mol. Biol. 13:595‐601.
   Otterbein, L.E., Bach, F.H., Alam, J., Soares, M., Tao Lu, H., Wysk, M., Davis, R.J., Flavell, R.A., Choi, A.M. 2000. Carbon monoxide has anti‐inflammatory effects involving the mitogen‐activated protein kinase pathway. Nat. Med. 6:422‐428.
   Poss, K.D. and Tonegawa, S. 1997a. Heme oxygenase 1 is required for mammalian iron reutilization. Proc. Natl. Acad. Sci. U.S.A. 94:10919‐10924.
   Poss, K.D. and Tonegawa, S. 1997b. Reduced stress defense in heme oxygenase 1‐deficient cells. Proc. Natl. Acad. Sci. U.S.A. 94:10925‐10930.
   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.
   Schuller, D.J., Wilks, A., Ortiz de Montellano, P.R., Poulos, T.L. 1999. Crystal structure of human heme oxygenase‐1. Nat. Struct. Biol. 6:860‐867.
   Shibahara, S., Muller, R., Taguchi, H., Yoshida, T. 1985. Cloning and expression of cDNA for rat heme oxygenase. Proc. Natl. Acad. Sci. U.S.A. 82:7865‐7869.
   Shibahara, S., Yoshizawa, M., Suzuki, H., Takeda, K., Meguro, K., Endo, K. 1993. Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J. Biochem. (Tokyo) 113:214‐218.
   Smulevich, G., Mauro, J.M., Fishel, L.A., English, A.M., Kraut, J., Spiro, T.G. 1988. Heme pocket interactions in cytochrome c peroxidase studied by site‐ directed mutagenesis and resonance Raman spectroscopy. Biochemistry 27:5477‐5485.
   Snyder, S.H., Jaffrey, S.R., Zakhary, R. 1998. Nitric oxide and carbon monoxide: Parallel roles as neural messengers. Brain Res. Brain Res. Rev. 26:167‐175.
   Spiro, T.G. and Czernuszewicz, R.S. 1995. Resonance Raman spectroscopy of metalloproteins. Methods Enzymol. 246:416‐460.
   Stevens, C.F. and Wang, Y. 1993. Reversal of long‐term potentiation by inhibitors of haem oxygenase [see comments]. Nature 364:147‐149.
   Stocker, R. 1990. Induction of haem oxygenase as a defence against oxidative stress. Free Radic. Res. Commun. 9:101‐112.
   Stocker, R., Yamamoto, Y., McDonagh, A.F., Glazer, A.N., Ames, B.N. 1987. Bilirubin is an antioxidant of possible physiological importance. Science 235:1043‐1046.
   Sun, J., Wilks, A., Ortiz de Montellano, P.R., Loehr, T.M. 1993. Resonance Raman and EPR spectroscopic studies on heme‐heme oxygenase complexes. Biochemistry 32:14151‐14157.
   Verma, A., Hirsch, D.J., Glatt, C.E., Ronnett, G.V., Snyder, S.H. 1993. Carbon monoxide: A putative neural messenger [see comments] [published erratum appears in Science 1994 263:15]. Science 259:381‐384.
   Weber, C.M., Eke, B.C., Maines, M.D. 1994. Corticosterone regulates heme oxygenase‐2 and NO synthase transcription and protein expression in rat brain. J. Neurochem. 63:953‐962.
   Wilks, A. and Ortiz de Montellano, P.R. 1993. Rat liver heme oxygenase: High level expression of a truncated soluble form and nature of the meso‐hydroxylating species. J. Biol. Chem. 268:22357‐22362.
   Wilks, A., Black, S.M., Miller, W.L., Ortiz de Montellano, P.R. 1995. Expression and characterization of truncated human heme oxygenase (hHO‐1) and a fusion protein of hHO‐1 with human cytochrome P450 reductase. Biochemistry 34:4421‐4427.
   Wilks, A., Torpey, J., Ortiz de Montellano, P.R. 1994. Heme oxygenase (HO‐1): Evidence for electrophilic oxygen addition to the porphyrin ring in the formation of alpha‐meso‐hydroxyheme. J. Biol. Chem. 269:29553‐29556.
   Yasukochi, Y. and Masters, B.S. 1976. Some properties of a detergent‐solubilized NADPH‐cytochrome c (cytochrome P‐450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251:5337‐5344.
   Yoshida, T., Noguchi, M., Kikuchi, G. 1980. A new intermediate of heme degradation catalyzed by the heme oxygenase system. J. Biochem. (Tokyo) 88:557‐563.
   Yoshida, T., Noguchi, M., Kikuchi, G. 1982. The step of carbon monoxide liberation in the sequence of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system. J. Biol. Chem. 257:9345‐9348.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library