Measurement of Protoporphyrinogen Oxidase Activity

Judith M. Jacobs1, Nicholas J. Jacobs1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.5
DOI:  10.1002/0471140856.tx0805s00
Online Posting Date:  May, 2001
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Abstract

Protoporphyrinogen oxidase catalyzes the oxidation of protoporphyrinogen to protophyrin. It is a membrane‐bound mitochondrial enzyme and it is the target of photobleaching herbicides. The basic assay presented in this unit for measuring oxidase activity is based on oxidation of the colorless, nonfluorescent substrate, protoporphyrinogen, to the colored, fluorescent protophyrin. Alternate protocols are provided for the measuring the accumulation of protoporphyrinogen resulting from a decrease in oxidase activity due to treatment with diphenylether herbicides or oxidase inhibitor.

     
 
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Table of Contents

  • Basic Protocol 1: Indirect Assay to Measure Protoporphyrinogen Oxidase Activity
  • Alternate Protocol 1: Direct Assay to Measure Protoporphyrinogen Oxidase Activity
  • Alternate Protocol 2: Detection of Accumulated Protoporphyrinogen
  • Support Protocol 1: Preparing Sodium Amalgam
  • Support Protocol 2: Preparation of E. Coli Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Indirect Assay to Measure Protoporphyrinogen Oxidase Activity

  Materials
  • 0.5 M Tris⋅Cl, pH 7.5 and pH 8.7 ( appendix 2A)
  • 0.05 M Tris⋅Cl, pH 8.7 ( appendix 2A)
  • 0.01 M EDTA in 0.05 M Tris⋅Cl, pH 8.7
  • 0.05 M dithiothreitol (DTT) or glutathione in 0.5 M Tris⋅Cl, pH 7.5
  • Enzyme sample to be assayed, e.g., rat liver mitochondria, fibroblasts, or etiolated chloroplasts
  • Protein assay kit (Bio‐Rad) or equivalent
  • Heat‐inactivated control: enzyme sample that has been heated 15 min at 75°C
  • 10% (v/v) Tween 20 or Tween 80 (optional)
  • ∼500 µM protoporphyrin stock solution (see recipe)
  • Ultra‐high‐purity nitrogen gas
  • 0.01 N KOH
  • 40% phosphoric acid, degassed with nitrogen
  • 3.8% sodium amalgam (see protocol 4)
  • 2.7 N HCl
  • 3‐ml disposable, plastic, square spectrofluorometric cuvettes (12 × 45 mm; Fisher)
  • Fluorometer
  • Blak‐Ray UV lamp (UVP 4‐Watt, Fisher)
  • Filter funnel with fritted disc, fine porosity of 4 to 5.5 µm (Fisher)
  • Applicator sticks
  • pH paper (Hydrion pH test papers, range 6.0 to 8.0 and 8.0 to 9.5, Fisher)
  • Quartz spectrophotometer cuvettes

Alternate Protocol 1: Direct Assay to Measure Protoporphyrinogen Oxidase Activity

  Materials
  • 0.5 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 0.1 M EDTA in 0.05 M Tris⋅Cl, pH 8.7
  • 10% (v/v) Tween 20
  • 0.05 M and 0.1 M DTT in 0.5 M Tris⋅Cl, pH 7.5
  • Sonicated mitochondria or other enzyme sample to be assayed (see protocol 1)
  • Heat‐inactivated control: enzyme sample that has been heated 15 min at 75°C
  • ∼500 µM protoporphyrin stock solution (see recipe)
  • Sodium amalgam (see protocol 4)
  • 3‐ml, disposable spectrofluorometric cuvettes or 1.5‐ml disposable semi‐micro spectrophotometric cuvettes (Fisher 14‐385‐938; see Background Information)
  • Fluorometer
  • 1‐ml screw‐cap microcentrifuge tubes, with securely fitted O rings
  • Mortar and pestle
  • Vortex mixer
  • Blak‐Ray UV lamp (UVP 4‐Watt, Fisher)
  • Applicator sticks
  • pH paper (Hydrion pH test papers with narrow range, Fisher)

Alternate Protocol 2: Detection of Accumulated Protoporphyrinogen

  Materials
  • Sample (reaction mixture or cell suspension) containing protoporphyrinogen (see protocol 1; protocol 2)
  • E. coli membranes (see protocol 5)
  • 10% (v/v) PCA/methanol
  • ∼500 µM protoporphyrin stock solution (see recipe)
  • 0.5 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 3‐ml, disposable spectrofluorometric cuvettes
  • Fluorometer

Support Protocol 1: Preparing Sodium Amalgam

  Materials
  • Mercury metal
  • Nitrogen gas
  • Sodium metal
  • Light mineral oil
  • Phosphorus pentoxide
  • Chemical safety hood
  • Face shield
  • Chemical protective gloves (Fisher)
  • Mercury respirator (Fisher)
  • 50‐ml side‐arm flask
  • Single‐hole rubber stopper with glass tube vent

Support Protocol 2: Preparation of E. Coli Membranes

  Materials
  • E. coli (ATCC #25922)
  • Bacterial Nutrient Broth (Difco)
  • 0.5 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Protein assay kit (Bio‐Rad) or equivalent
  • Centrifuge
  • Sonicator (e.g., Branson W185D or equivalent)
  • Ultracentrifuge
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Figures

Videos

Literature Cited

   Birchfield, N.B. and Casida, J.E. 1996. Protoporphyrinogen oxidase: High affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide‐binding site in mouse liver mitochondria. Chem. Res. Toxicol. 9:1135‐1139.
   Bishop, D.F. and Desnick, R.J. 1982. Assays of the heme biosynthetic enzymes. Enzymes 28:91‐93.
   Brenner, D.A. and Bloomer, J.R. 1980. A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue. Clin. Chim. Acta 100:259‐266.
   Camadro, J.‐M., Abraham, N.G., and Levere, R.D. 1985. Kinetic properties of the membrane‐bound human Liver mitochondrial protoporphyrinogen oxidase. Arch. Biochem. Biophys. 242:206‐212.
   Camadro, J.‐M., Matringe, M., Scalla, R., and Labbe, P. 1993. Fluorometric assay of protoporphyrinogen oxidase in chloroplasts, and in plant, yeast and mammalian mitochondria. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds (P. Boger and G. Sandmann, eds.) pp. 29‐34. Lewis Publishers, Ann Arbor, Mich.
   Dailey, T.A. and Dailey, H.A. 1997. Expression, purification, and characteristics of mammalian protoporphyrinogen oxidase. Methods Enzymol. 281:340‐349.
   Dailey, H.A. and Karr, S.W. 1987. Purification and characterization of murine protoporphyrinogen oxidase. Biochemistry 26:2697‐2701.
   Fieser, L.F. and Fieser, M. 1967. Reagents for Organic Synthesis. John Wiley & Sons, New York.
   Jacobs, J.M. and Jacobs, N.J. 1993. Porphyrin accumulation and export by isolated barley (Hordeum vulgare) plastids. Plant Physiol. 101:1181‐1187.
   Jacobs, J.M., Jacobs, N.J., Sherman, T.D., and Duke, S.O. 1991. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. Plant Physiol. 97:197‐203.
   Jacobs, N.J. and Jacobs, J.M. 1979. Microbial oxidation of protoporphyrinogen: An intermediate in heme and chlorophyll biosynthesis. Arch. Biochem. Biophys. 197:396‐403.
   Jacobs, N.J. and Jacobs, J.M. 1982. Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme 28:206‐219.
   Klemm, D.J. and Barton, L.L. 1987. Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium Desulfovibriogigas. J. Bacteriol. 169:5209‐5215.
   Labbe, P., Camadro, J.‐M., and Chambon, H. 1985. Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase. Anal. Biochem. 149:248‐260.
   Lee, H.J., Duke, M.V., and Duke, S.O. 1993. Cellular localization of protoporphyrinogen‐oxidizing activities of etiolated barley (Hordeum vulgare L.) leaves. Plant Physiol. 102:881‐889.
   Li, F., Lim, C.K., and Peters, T.J. 1987. An hplc assay for protoporphyrinogen oxidase activity in rat liver. Biochem. J. 243:863‐866.
   Mauzerall, D. and Granick, S. 1958. Porphyrin biosynthesis in erythrocytes III. Uroporphyrinogen and its decarboxylase. J. Biol. Chem. 232:1141‐1162.
   Nicolaus, B., Sandmann, G., and Boger, P. 1993. Inhibition of protoporphyrinogen oxidase from maize. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds. (P. Boger and G. Sandmann, eds.) pp. 29‐34. Lewis Publishers, Ann Arbor, Mich.
   Poulson, R. 1976. The enzymatic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria. J. Biol. Chem. 251:3730‐3733.
   Yamato, S., Katagiri, M., and Ohkana, H. 1994. Purification and characterization of protoporphyrinogen oxidase from tobacco cultured cells. Pestic. Biochem. Physiol. 50:72‐82.
Key References
   Camadro et al., 1993. See above.
   Presents slightly different methodologies than those given here, from a laboratory with extensive experience with this difficult assay (e.g., use of ascorbic acid instead of DTT as an antioxidant, and useful hints about preparing and clarifying protoporphyrinogen solutions).
   Dailey and Dailey, 1997. See above.
   Further descriptions of protoporphyrinogen oxidase assays.
   Fieser and Fieser, 1967. See above.
   Alternate procedure for sodium amalgam preparation—the classical method, using enclosed glassware, and recommended over the method described here, although biological laboratories may find it prohibitively inconvenient.
   Labbe et al., 1985. See above.
   First version of the direct fluorometric assay.
   Nicolaus et al., 1993. See above.
   Another version of the direct fluorometric assay with an alternate method for sodium amalgam preparation.
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