Measurement of Ferrochelatase Activity

Shigeru Taketani1

1 Kansai Medical University, Moriguchi, Osaka
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.7
DOI:  10.1002/0471140856.tx0807s02
Online Posting Date:  May, 2001
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Abstract

Ferrochelatase is the terminal enzyme in the heme biosynthesis pathway. Under anaerobic conditions it catalyzes the insertion of ferrous iron into the protoporphyrin IX ring to form protoheme. In the absence of iron and under aerobic conditions, the enzyme will use zinc or mercury as a substitute. The assay described in this unit uses zinc under aerobic conditions and the artificial substrate mesoporphyrin to analyze the formation of zinc‐mesoporphyrin by HPLC with UV or fluorescence detection.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Zinc‐Mesoporphyrin Formation
  • Alternate Protocol 1: Assay of Cobalt‐Chelating Activity
  • Alternate Protocol 2: Assay of Iron‐Chelating Activity
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Zinc‐Mesoporphyrin Formation

  Materials
  • 1 mM mesoporphyrin solution (see recipe)
  • Tris/palmitate/Tween buffer (see recipe)
  • Enzyme source (see recipe): Mitochondrial preparation or cell homogenate (0.5 to 2.5 µg protein/µl)
  • 2 mM zinc acetate
  • Stop solution: 3:7 (v/v) dimethyl sulfoxide/methanol with 0.1 mM EDTA
  • Ammonium acetate/methanol solution (see recipe)
  • Zinc‐mesoporphyrin (Porphyrin Products)
  • Dimethylsulforide (DMSO)
  • Ultrasonicator
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • C18 column: 4‐µm Novapak C18 silica column (4.6 × 150–mm; Waters) or Cosmosil 5C18‐AR column (4.6 × 160–mm Nacalai Tesque)
  • 0.2‐µm precolumn filter (0.4 mm; Millipore)
  • HPLC system with 0.2‐µm precolumn filter (2.5 × 2.5 cm), fluorescence spectrophotometric detector,and spectrophotometric visual detector

Alternate Protocol 1: Assay of Cobalt‐Chelating Activity

  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 1 mM mesoporphyrin solution (see recipe)
  • 100 mM dithiothreitol (DTT)
  • 10% (w/v) Triton X‐100
  • Enzyme source (see recipe): Mitochondrial preparation or cell homogenate (0.5 to 2.5 µg protein/µl)
  • 10 mM iron citrate (see recipe)
  • Nitrogen (N 2) gas
  • 0.1 M iodoacetamide
  • 1 N NaOH
  • Pyridine
  • Sodium dithionite
  • Thunberg tube (Top Labo‐Ware)
  • Spectrophotometer with scanning wavelength (i.e., Hitachi U‐2010) and cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Camadro, J.M. and Labbe, P. 1988. Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae Evidence for a precursor form of the protein. J. Biol. Chem. 263:11675‐11682.
   Goldberg, A., Ashenbrucker, M., Cartwright, G.E., and Wintrobe, M.M. 1956. Studies on the biosynthesis of heme in vitro by avian erythrocytes. Blood 11:821‐833.
   Li, F., Lim, C.K., and Peters, T.J. 1987. An HPLC assay of rat liver ferrochelatase activity. Biomed. Chromatogr. 2:164‐168.
   Rossi, E., Costin, K.A., and Garcia‐Webb, P. 1988. Ferrochelatase activity in human lymphocytes as quantified by a new high‐performance liquid‐chromatographic method. Clin. Chem. 34:2481‐2485.
   Sawada, H., Takeshita, M., Sugita, Y., and Yoneyama, Y. 1969. Effect of lipid on protoheme ferrolyase. Biochim. Biophys. Acta 178:145‐155.
   Taketani, S. 1994. Molecular and genetic characterization of ferrochelatase. In Regulation of Heme Protein Synthesis (H. Fujita, ed.) pp. 41‐54. AlphaMed Press, Dayton, Ohio.
   Taketani, S. and Tokunaga, R. 1981. Rat liver ferrochelatase. Purification, properties and stimulation by fatty acids. J. Biol. Chem. 256:12748‐12753.
Key References
   Furukawa, T., Kohno, H., Tokunaga, R., and Taketani, S. 1995. Nitric oxide‐mediated inactivation of mammalian ferrochelatase in vivo and in vitro: Possible involvement of the iron‐sulphur cluster of the enzyme. Biochem. J. 310:533‐538.
   Description of ferrochelatase activity of mouse macrophage cell line and recombinant human ferrochelatase expressed in E. coli. Some results are shown in Figure .
   Rossi et al., 1988. See above.
   Description of assay conditions of zinc‐mesoporphyrin formation by HPLC.
   Taketani, 1994. See above.
   Description of properties and kinetics of ferrochelatase and genetic demonstration of mammalian ferrochelatase.
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