Detection of Biliverdin Reductase Activity

Tian‐Jun Huang1

1 New York Blood Center, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.4
DOI:  10.1002/0471140856.tx0904s00
Online Posting Date:  November, 2002
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The conversion of biliverdin to the bile pigment bilirubin is catalyzed by biliverdin reductase, a cytosolic enzyme with two pH optima with different cofactors. The enzyme is assayed at pH 8.7 with NADPH as a cofactor and at 6.75 with NADH. The production of bilirubin is detected as described in this unit with a spectrophotometric assay. The enzyme can be qualitatively assayed by immunoblotting and changes in reductase isoform expression can be detected by two‚Äźdimensional electrophoresis.

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Table of Contents

  • Basic Protocol 1: Kinetic Assay of Biliverdin Reductase Activity
  • Basic Protocol 2: Immunoblotting Assay for Biliverdin Reductase
  • Basic Protocol 3: Two‐Dimensional Gel Isoelectric Focusing
  • Support Protocol 1: Preparation of BVR Fusion Protein from Bacterial Cell Extracts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Kinetic Assay of Biliverdin Reductase Activity

  • Fresh or flash‐frozen tissue
  • 0.9% (w/v) NaCl in distilled H 2O, ice cold
  • Homogenization buffer (see recipe)
  • BVR assay buffers, pH 8.7 and 6.75 (see reciperecipes)
  • 300 µM biliverdin (see recipe)
  • 20 mg/ml bovine serum albumin (BSA)
  • Cofactor buffer (see recipe)
  • 1 mM NADPH in BVR assay buffer, pH 8.7 (see recipe for buffer, store on ice; use within 2 hr)
  • 10 mM NADH in cofactor buffer (see recipe for buffer; store on ice; use within 1 hr)
  • Surgical scissors
  • Dounce homogenizer with tight‐fitting Teflon pestle
  • Variable‐speed drill
  • Centrifuge with fixed‐angle rotor and tubes (e.g., Beckman J2 with JA20 rotor)
  • Ultracentrifuge with fixed‐angle rotor (e.g., Beckman L8 with 50Ti rotor)
  • 13 × 100–mm or 12 × 75–mm glass tubes
  • Scanning spectrophotometer
NOTE: Microsomes can be prepared from either fresh or frozen tissue. Enzyme preparation is carried out on ice or at 4°C, and the assay is performed at room temperature.

Basic Protocol 2: Immunoblotting Assay for Biliverdin Reductase

  • Protein samples in 1× SDS sample buffer ( appendix 2A)
  • Prestained or dye‐labeled protein molecular‐weight markers
  • Transfer buffer (see recipe)
  • 1× and 5× PBS (see recipe)
  • Blocking solution (see recipe)
  • Rabbit anti‐BVR antiserum (e.g., StressGen)
  • Antibody diluent (see recipe)
  • 1× PBS containing 0.5% (w/v) sodium cholate (PBS/cholate)
  • Horseradish peroxidase–conjugated anti–rabbit IgG antiserum (e.g., Bio‐Rad or Cappel)
  • 4‐Chloronaphthol
  • Methanol
  • 30% (v/v) H 2O 2
  • 0.2‐µm‐pore nitrocellulose sheets or nylon‐backed equivalent
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; appendix 3A) and electrotransfer of proteins (unit 2.3)

Basic Protocol 3: Two‐Dimensional Gel Isoelectric Focusing

  • Urea
  • IEF acrylamide mix (see recipe)
  • Ampholytes pH 5‐8, pH 4‐6.5, and pH 3.5‐10
  • 10% (w/v) ammonium persulfate
  • Lysis buffer (see recipe)
  • Overlay solution (see recipe)
  • Upper electrode buffer: 0.02 N NaOH
  • Lower electrode buffer: 0.01 M H 3PO 4
  • Sample for analysis
  • Gel equilibration buffer (see recipe)
  • 3‐ml syringe
  • 10‐cm gel tube–loading needle (e.g., Bio‐Rad)
  • IEF tubes, 1.5 mm × 12.5 cm (e.g., Bio‐Rad)
  • IEF apparatus (e.g., Bio‐Rad)
  • Gel tube–extrusion needle (e.g., Bio‐Rad)
  • Glass test tube, ≥15 cm in length
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3A), electrotransfer (unit 2.3), staining proteins in gels ( appendix 3A), and immunodetection (see protocol 2)
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Literature Cited

Literature Cited
   Colleran, E. and O'Carra, P. 1970. Specificity of biliverdin reductase. Biochem. J. 119:16P‐17P.
   Fahkrai, H. and Maines, M.D. 1992. Expression and characterization of a cDNA for rat kidney biliverdin reductase: Evidence suggesting the liver and kidney enzymes are the same transcript product. J. Biol. Chem. 267:4023‐4029.
   Huang, T.J., Trakshel, G.M., and Maines, M.D. 1989. Detection of ten variants of biliverdin reductase in rat liver by two‐dimensional gel electrophoresis. J. Biol. Chem. 264:7844‐7849.
   Kutty, R.K. and Maines, M.D. 1981. Purification and characterization of biliverdin reductase from rat liver. J. Biol. Chem. 256:3956‐3962.
   Maines, M. and Trakshel, G.M. 1993. Purification and characterization of human biliverdin reductase. Arch. Biochem. Biophys. 300:320‐326.
   Maines, M.D., Polevoda, B.V., Huang, T.J., and McCoubrey, W.K., Jr. 1996. Human biliverdin IXα reductase is a zinc‐metalloporphyrin: Characterization of purified and Escherichia coli expressed enzymes. Eur. J. Biochem. 235:372‐381.
   McCoubrey, W.K., Jr. and Maines, M.D. 1994. Site‐directed mutagenesis of cysteine residues in biliverdin reductase: Roles in substrate and cofactor binding. Eur. J. Biochem. 222:597‐603.
   O'Farrell, P.H. 1975. High resolution two dimensional gel electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
   Singleton, J.W. and Laster, L. 1965. Biliverdin reductase of pig liver. J. Biol. Chem 240:4780‐4789.
   Tenhunen, R., Ross, M.E., Marver, H.S., and Schmid, R. 1970. Reduced nicotinamide dinucleotide phosphate dependent biliverdin reductase: Partial purification and characterization. Biochemistry 9:298‐303.
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