Measurement of Heme Concentration

Peter R. Sinclair1, Nadia Gorman1, Judith M. Jacobs1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.3
DOI:  10.1002/0471140856.tx0803s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Heme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P‐450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed‐phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.

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

Table of Contents

  • Basic Protocol 1: Spectrophotometric Heme Assay of Pyridine Hemochrome
  • Alternate Protocol 1: Fluorescence Heme Assay
  • Alternate Protocol 2: Assay of Heme and Biosynthetic Intermediates by Reversed‐Phase HPLC
  • Alternate Protocol 3: Assay of Radioactive Heme by Solvent Extraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Spectrophotometric Heme Assay of Pyridine Hemochrome

  Materials
  • 5% (w/v) tissue sample suspension: tissue homogenate, or a homogenate or sonicate of tissue culture cells (minimally, 2 mg protein)
  • Reagent‐grade pyridine (discard if yellow)
  • 1.0 M NaOH
  • Sodium dithionite crystals
  • 3 M potassium ferricyanide
  • 12 × 75–mm disposable glass tubes (Fisher)
  • Sensitive scanning spectrophotometer
  • Appropriate cuvettes with caps

Alternate Protocol 1: Fluorescence Heme Assay

  Materials
  • Tissue culture cells or tissue (sonicate or homogenate; see protocol 1 materials)
  • recipe2 M oxalic acid solution (see recipe)
  • recipe1 mg/ml heme standard solution (see recipe) or protoporphyrin standard solution (Porphyrin Products)
  • Heating block at 100°C
  • 6 × 50–mm disposable glass tubes (Fisher)
  • Spectrofluorometer equipped with cell holder and preferably with red‐sensitive phototube (Hamamatsu R928, R446, or R3896, or equivalent)

Alternate Protocol 2: Assay of Heme and Biosynthetic Intermediates by Reversed‐Phase HPLC

  Materials
  • Tissue culture cells or tissue (sonicate or homogenate; see protocol 1 materials)
  • Aqueous cell homogenate or tissue culture cells
  • Acetone/HCl/water (see recipe)
  • Acetone/HCl (see recipe)
  • Buffer A (see recipe)
  • HPLC‐grade methanol (buffer B)
  • Heme/porphyrin standard injection mixture (see recipe)
  • HPLC system with multisolvent delivery system, absorbance detection for heme (400 nm), and fluorescence detection for porphyrins (excitation 400 nm, emission 600 nm)
  • C18 HPLC column (e.g., Waters µBondapak; 39 × 300 mm) and C18 precolumn

Alternate Protocol 3: Assay of Radioactive Heme by Solvent Extraction

  Materials
  • Radiolabeled tissue culture cells
  • 2 M HCl
  • Ethyl ether
  • Aqueous scintillation fluid
  • ∼45°C water bath
  • Additional reagents and equipment for preparing acetone extracts (see protocol 3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Berry, E.A. and Trumpower, B.L. 1987. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161:1‐15
   Bonkovsky, H., Wood, S., Howell, S., Sinclair, P., Lincoln, B., Healey, J., and Sinclair, J. 1986. High performance liquid chromatographic separation and quantitation of tetrapyrolles from biological materials. Anal. Biochem. 155:56‐64.
   Fuhrhop, J.H. and Smith, K.M. 1975. Laboratory methods in porphyrin and metalloporphyrin research, pp. 48‐51. Elsevier Scientific Publishing, Amsterdam.
   Healey, J.F., Bonkowsky, H.L., Sinclair, P.R., and Sinclair, J.F. 1981. Conversion of 5‐aminolaevulinate into haem by liver homogenates. Biochem. J. 198:595‐604.
   Jacobs, J.M., Sinclair, P.R., Gorman, N., Jacobs, N.J., Sinclair, J.F., Bement, W.J., and Walton, H.S. 1992. Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes. J.Biochem. Toxicol. 7:87‐95.
   Jacobs, J.M., Sinclair, P.R., Sinclair, J.F., Gorman, N., Walton, H.S., Wood, S.G., and Nichols, C. 1998. Formation of zinc protoporphyrin in cultured hepatocytes: Effects of ferrochelatase inhibition, iron chelation or lead. Toxicology 125:95‐105.
   Paul, K.G., Theorell, H., and Akeson, A. 1953. The molar light absorption of pyridine ferroprotoporphyrin (pyridine hemochromogen). Acta Chem. Scand. 7:1284‐1287.
   Sassa, S. 1976. Sequential induction of heme enzymes during erythroid differentiaion of mouse Friend leukemia virus–infected cells. J. Exp. Med. 143:305‐315.
   Sassa, S. and Kappas, A. 1977. Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. J. Biol. Chem. 252:2428‐2436.
   Taketani, S., Immenschuh, S., Go, S., Sinclair, P.R., Stockert, R.J., Liem, H.H., and Muller Eberhard, U. 1998. Hemopexin from four species inhibits the association of heme with cultured hepatoma cells or primary rat hepatocytes exhibiting a small number of species specific hemopexin receptors. Hepatology 27:808‐814.
   Tzagoloff, A., Nobrega, M., Gorman, N., and Sinclair, P. 1993. On the functions of the yeast COX10 and COX11 gene products. Biochem. Mol. Biol. Int. 31:593‐598.
   Waterman, M.R. 1978. Spectral characteristics of human hemoglobin and its derivatives. Methods Enzymol. 52:456‐463.
Key References
   Bonkovsky et al., 1981 see above.
   Describes basic HPLC method.
   Healey et al., see above.
   Describes the method of extracting radioactive heme from liver homogenates.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library