Measurement of Uroporphyrinogen Decarboxylase Activity

John D. Phillips1, James P. Kushner1

1 University of Utah Medical School, Salt Lake City, Utah
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.4
DOI:  10.1002/0471140856.tx0804s00
Online Posting Date:  May, 2001
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Abstract

Uroporphyrinogen decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of UROD Activity Using Enzymatically Reduced Porphyrins as Substrates
  • Alternate Protocol 1: Measuement UROD Activity Using Chemically Reduced Porphyrins as Sustrates
  • Support Protocol 1: Purification of PBGD from Rhodobacter spheroides
  • Support Protocol 2: Preparation of Erythrocyte Lysates for UROD Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of UROD Activity Using Enzymatically Reduced Porphyrins as Substrates

  Materials
  • 0.1 M dithiothreitol (DTT) in 0.1 M Tris⋅Cl, pH 7.65 (see appendix 2A for Tris⋅Cl buffer)
  • 2.4 mM porphobilinogen (Porphyrin Products) in 0.1 M Tris⋅Cl, pH 7.65 (see appendix 2A for Tris⋅Cl)
  • Porphobilinogen deaminase (PBGD; see protocol 3), diluted in 10 mM KH 2PO 4, pH 7.6 so that 150 µl will produce 30 to 60 µM uroporphyrinogen in 30 min in the reaction mix in step
  • N 2 source
  • 0.15 M KH 2PO 4, pH 4.45 (mix 1 M KH 2PO 4 and 1 M K 2HPO 4 to a pH of 4.5 and dilute to 0.15 M with H 2O)
  • UROD source to be assayed (see, e.g., protocol 4)
  • 0.1 M DTT in 0.1 M KH 2PO 4, pH 6.8 (see appendix 2A for potassium phosphate buffer)
  • 3 M HCl
  • 3.5 µM mesoporphyrin IX dihydrochloride (Porphyrin Products)
  • Methanol (HPLC grade)
  • Methanol/phosphate (see recipe)
  • Porphyrin standards, dissolved in 3.0 M HCl (Porphyrin Acids Chromatographic Marker Kit, Porphyrin Products)
  • 12 × 75–mm tube to prepare substrate
  • Red lamp
  • Woods lamp (UV, “black light”)
  • HPLC system fitted with a gradient maker and fluorescence detector (excitation 404 nm, emission 618 nm)
  • C18 column for separation of porphyrins (e.g., Waters µBondapak 3.9 × 300 mm)

Alternate Protocol 1: Measuement UROD Activity Using Chemically Reduced Porphyrins as Sustrates

  • Porphyrin dihydrochloride (generally uroporphyrin I or III or pentacarboxyl porphyrin I or III; Porphyrin Products)
  • 2 M NH 4OH
  • 1.5 M HCl
  • 1 M and 50 mM KH 2PO 4, pH 6.8 ( appendix 2A)
  • 5% sodium amalgam (Anachemia, or as prepared in Straka et al., )
  • 1 M DTT
  • 2 M H 3PO 4
  • pH paper, 5 to 10 range
  • Desiccator jar large enough for a tube rack and ice, with a 3‐way stopcock
  • Spectrophotometer (able to scan from 350 nm to 550 nm)

Support Protocol 1: Purification of PBGD from Rhodobacter spheroides

  Materials
  • R. spheroides (Porphyrin Products)
  • 10 mM KH 2PO 4, pH 7.6 ( appendix 2A)
  • 100% ethanol, ice cold
  • (NH 4) 2SO 4
  • 3 mM KH 2PO 4, pH 6.8
  • 0 to 0.50 M KCl gradient in 3 mM KH 2PO 4, pH 6.8
  • 0.01 M DTT
  • 0.1 M Tris⋅Cl, pH 7.6 ( appendix 2A)
  • 0.54 mg/ml porphobilinogen, (PBG; Porphyrin Products) in 0.1 M Tris⋅Cl, pH 7.6
  • 3 M HCl
  • 0.15 M KH 2PO 4, pH 4.45
  • 50 mM KH 2PO 4, pH 6.8
  • Probe sonicator
  • Refrigerated centrifuge accommodating 1‐liter buckets and 50‐ml centrifuge tubes
  • Dialysis tubing, 12,000 to 14,000 MWCO
  • 70°C water bath
  • DE‐52 column, 50 ml bed volume, equilibrated with 3 mM KH 2PO 4, pH 6.8 (see recipe)
  • Concentrator (e.g., Amicon Centriprep 10)
  • Fraction collector
  • UV monitor to measure absorbance at 280 nm, or UV spectrophotometer
  • Woods lamp
  • Additional reagents and equipment for dialysis ( appendix 3A)

Support Protocol 2: Preparation of Erythrocyte Lysates for UROD Assays

  Materials
  • Isoton II (Beckman Coulter)
  • Drabkin's reagent (see recipe)
  • 5 mM and 0.1 M KH 2PO 4, pH 6.8
  • 0.5 M KCl in 5 mM KH 2PO 4, pH 6.8
  • Heparinized Vacutainers
  • Tabletop centrifuge
  • DE‐52 column, equilibrated with 5 mM KH 2PO 4, pH 6.8 (see recipe)
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Figures

Videos

Literature Cited

   Adjarov, D.G. and Elder, G.H. 1988. A simplified method for determination of uroporphyrinogen decarboxylase activity in human blood. Clin. Chim. Acta 177:123‐130.
   De Matteis, F. and Marks, G.S. 1996. Cytochrome P450 and interactions with the heme biosynthetic pathway. Can. J. Pharmacol. 74:1‐8.
   de Verneuil, H., Grandchamp, B., and Nordmann, Y. 1980. Some kinetic properties of human red cell uroporphyrinogen decarboxylase. Biochim. Biophys. Acta. 611:174‐186.
   de Verneuil, H., Sassa, S., and Kappas, A. 1983. Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes: A single enzyme catalyzing the four sequential decarboxylations of uroporphyrinogens I and III. J. Biol. Chem. 258:2454‐2460.
   Elder, G.H. 1975. The differentiation of porphyria cutanea tarda symptomatica from other types of porphyria by the measurement of isocoproporphyrin in faeces. J. Clin. Pathol. 28:601‐607.
   Elder, G.H. and Wyvill, P.C. 1982. Measurement of uroporphyrinogen decarboxylase using porphyrinogens prepared by chemical reduction. Enzyme 28:186‐195.
   Elder, G.H., Tovey, J.A., and Sheppard, D.M. 1983. Purification of uroporphyrinogen decarboxylase from human erythrocytes: Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver. Biochem. J. 215:45‐55.
   Ford, R.E., Ou, C.N., and Ellefson, R.D. 1981. Liquid‐chromatographic analysis for urinary porphyrins. Clin. Chem. 27:397‐401.
   Francis, J.E. and Smith, A.G. 1984. Assay of mouse liver uroporphyrinogen decarboxylase by reverse‐phase high‐performance liquid chromatography. Anal. Biochem. 138:404‐410.
   Franklin, M.R., Phillips, J.D., and Kushner, J.P. 1997. Cytochrome P450 induction, uroporphyrinogen decarboxylase depression, porphyrin accumulation and excretion, and gender influence in a 3‐week rat model of porphyria cutanea tarda. Toxicol. Appl. Pharmacol. 147:289‐299.
   Jordan, P.M. and Shemin, D. 1973 Purification and properties of uroporphyrinogen I synthetase from Rhodopseudomonas spheroides. J. Biol. Chem. 248:1019‐1024.
   Kappas, A., Sassa, S., Galbraith, R.A., and Nordmann, Y. 1995. The porphyrias. In The Metabolic and Molecular Bases of Inherited Disease, Vol. II (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 2103‐2160. McGraw‐Hill, New York.
   Lambrecht, R.W., Jacobs, J.M., Sinclair, P.R., and Sinclair, J.F. 1990. Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P‐450‐mediated uroporphyrinogen oxidation. Biochem. J. 269:437‐441.
   Lowry, O.H., Rosebrough, N.J., Foss, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   McManus, J. Blake and D. Ratnalke, S. 1988. An assay of uroporphyrinogen decarboxylase in erythrocytes. Clin. Chem. 34:2355‐2357.
   Mukerji, S.K., Pimstone, N.R., and Burns, M. 1984. Dual mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron: Its potential role in the genesis of porphyria cutanea tarda. Gastroenterology. 87:1248‐1254.
   Sassa, S., Sugita, O., Ohnuma, N., Imajo, S., Okumura, T., Noguchi, T., and Kappas, A. 1986. Studies of the influence of chloro‐substituent sites and conformational energy in polychlorinated biphenyls on uroporphyrin formation in chick‐embryo liver cell cultures. Biochem. J. 235:291‐296.
   Schmid, R. 1960. Cutaneous porphyria in Turkey. New Engl. J. Med. 263:397‐398.
   Sinclair, P.R., Gorman, N., Dalton, T., Walton, H.S., Bement, W.J., Sinclair, J.F., Smith, A.G., and Nebert, D.W. 1998. Uroporphyria produced in mice by iron and 5‐aminolaevulinic acid does not occur in Cyp1a2(−/−) null mutant mice. Biochem. J. 330:149‐153.
   Smith, A.G., Francis, J.E., Kay, S.J., and Greig, J.B. 1986. Mechanistic studies of the inhibition of hepatic uroporphyrinogen decarboxylase in C57BL/10 mice by iron‐hexachlorobenzene synergism. Biochem. J. 238:871‐878.
   Straka, J.G. and Kushner, J.P. 1983. Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase. Biochemistry 22:4664‐4672.
   Straka, J.G., Kushner, J.P., and Pryor, M.A. 1982. Uroporphyrinogen decarboxylase: A method for measuring enzyme activity. Enzyme 28:170‐185.
   Wyckoff, E.E., Phillips, J.D., Sowa, A.M., Franklin, M.R., and Kushner, J.P. 1996. Mutational analysis of human uroporphyrinogen decarboxylase. Biochim. Biophys. Acta. 1298:294‐304.
Key References
   Jordan and Shemin, 1973. See above.
   Describes the purification of PBGD for use in the enzymatic synthesis of uroporphyrinogen I.
   Straka et al., 1982. See above.
   Methods described in this unit are a modification of those described in this reference. This is a very detailed manuscript that provides additional information not presented here and is a good reference for measuring activity of UROD from various sources.
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