Measurement of Cytochrome P‐450

John B. Schenkman1, Ingela Jansson1

1 University of Connecticut Health Center, Farmington, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.1
DOI:  10.1002/0471140856.tx0401s13
Online Posting Date:  November, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The family of cytochrome P‐450 enzymes are involved in the metabolism of xenobiotics. This unit provides protocols for the three most common ways to measure cytochrome P‐450. Spectrophotometric detection of the Soret band gives a quantitative measure of total cytochrome P‐450 in microsomes, liver homogenate, or mitochondria, but lacks information about the specific P‐450 forms. Immunochemical methods such as immunoblotting provide very sensitive semiquantitative identification of specific P‐450 enzymes Finally, enzymatic assays depend on substrate specificity and also provide information about specific P‐450 enzymes, although some members of this group of enzymes have broad and sometimes overlapping specificities.

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

Table of Contents

  • Basic Protocol 1: Spectrophotometric Measurement of Cytochrome P‐450
  • Alternate Protocol 1: Spectrometric Measurement of Cytochrome P‐450 in Liver Homogenates
  • Alternate Protocol 2: Spectrometric Measurement of Cytochrome P‐450 in Mitochondria
  • Basic Protocol 2: Immunological Measurement of Cytochrome P‐450
  • Basic Protocol 3: Enzymatic Analyses of Cytochrome P‐450 Forms
  • Support Protocol 1: Subfractionation of Different Tissues for Analysis of Cytochrome P‐450 Content
  • Support Protocol 2: Rapid Preparation of Microsomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Spectrophotometric Measurement of Cytochrome P‐450

  Materials
  • Microsomal preparation containing 20 mg/ml protein (see protocol 6Support Protocols 1 and protocol 72)
  • 0.05 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • Oxygen‐free carbon monoxide in tank with two‐stage valve
  • Dithionite: crystalline sodium hydrosulfite (Na 2S 2O 4)
  • 4% (w/v) NADH in 1% KHCO 3 (prepared fresh daily; optional)
  • Spectrophotometric cuvettes with 1‐cm light path for spectral analyses between 400 and 500 nm
  • Dual‐beam UV/VIS spectrophotometer or spectrophotometer capable of optically or electronically producing a difference spectrum
  • Plastic microspatulas for stirring cuvette contents
CAUTION: When wet, dithionite will release hydrogen. It is also hygroscopic and decomposes with time. For convenience, when opening the jar for the first time, separate contents into a number of smaller vials, such as scintillation vials, 1 g into each. Tape covers with plastic electrical tape to ensure sealed jars stay dry.

Alternate Protocol 1: Spectrometric Measurement of Cytochrome P‐450 in Liver Homogenates

  • Liver tissue
  • 0.9% NaCl ( appendix 2A)
  • Buffered sucrose: 0.25 M sucrose/0.05 M Tris⋅Cl, pH 7.4 (see appendix 2A for Tris⋅Cl recipe)
  • 0.6 M sodium ascorbate (optional)
  • 0.3 M sodium succinate in distilled water (optional)
  • 0.6 M sodium cyanide in distilled water (optional)
  • Potter‐Elvehjem glass homogenizer with motor‐driven Teflon pestle
CAUTION: Before discarding sodium cyanide, add a few milliliters of alkali. HCN can form if the pH drops.

Alternate Protocol 2: Spectrometric Measurement of Cytochrome P‐450 in Mitochondria

  • Mitochondrial preparation containing 10 mg/ml protein (see protocol 6, step )
  • Buffered sucrose: 0.25 M sucrose/0.05 M Tris⋅Cl, pH 7.4 (see appendix 2A for Tris⋅Cl recipe)
  • 0.6 M sodium cyanide in distilled water
CAUTION: Before discarding sodium cyanide, add a few milliters of alkali. HCN can form if the pH drops.

Basic Protocol 2: Immunological Measurement of Cytochrome P‐450

  Materials
  • Samples for analysis
  • Individual forms of cytochrome P‐450 as positive controls
  • Ponceau S solution (Sigma; dilute the solution—obtained as 2% Ponceau S in 30% TCA/30% sulfosalicylic acid—10‐fold with distilled water for use)
  • Blocking buffer: 2.5% (w/v) BSA in PBS (with 0.1% ovalbumin, if chicken antibodies are used; appendix 2A for PBS)
  • Primary antibodies: antibodies to individual forms of cytochrome P‐450
  • Secondary antibodies: alkaline phosphatase conjugated IgG antibodies (e.g., Sigma)
  • Transfer buffer: 25 mM Tris base in 192 mM glycine
  • PBS ( appendix 2A)
  • 5‐Bromo‐4‐chloro‐3‐indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) phosphatase substrate reagent (Kirkegaard & Perry; sold ready for use)
  • Molecular weight standards (e.g., Sigma, Amersham, Pharmacia, Biotech; 10 to 100 kDa, premixed)
  • Additional reagents and equipment for SDS‐PAGE gel ( appendix 3A) and electroblotting (unit 2.3)

Basic Protocol 3: Enzymatic Analyses of Cytochrome P‐450 Forms

  Materials
  • 0.1 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • 0.15 M MgCl 2
  • Substrates for specific forms of cytochrome P‐450 (e.g., 10 mM benzphetamine⋅HCl)
  • 0.15 mM glucose‐6‐phosphate
  • Glucose‐6‐phosphate dehydrogenase
  • Microsomal suspension containing 6 mg/ml protein (see protocol 6Support Protocols 1 and protocol 72)
  • 10 mg/ml NADPH in 1% KHCO 3
  • 12.5% trichloroacetic acid (TCA)
  • Nash reagent (see recipe)
  • 2 mM HCHO
  • Water bath, 58°C

Support Protocol 1: Subfractionation of Different Tissues for Analysis of Cytochrome P‐450 Content

  Materials
  • Tissue to be fractionated
  • 0.9% NaCl ( appendix 2A), ice cold
  • 0.25 M sucrose/10 mM Tris⋅Cl, pH 7.4 ( appendix 2A for Tris⋅Cl)
  • 1.15% KCl/10 mM Tris⋅Cl, pH 7.4 (optional; see appendix 2A for Tris⋅Cl recipe), ice cold
  • 0.15 M KCl
  • Potter‐Elvehjem glass homogenizer with motor‐driven Teflon pestle (optional)
  • Liquid nitrogen (optional)
NOTE: Carry out all steps at 0° to 4°C.

Support Protocol 2: Rapid Preparation of Microsomes

  • 80 mM CaCl 2
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Burke, M.D., Prough, R.A., and Mayer, R.T. 1977. Characteristics of a microsomal cytochrome P‐448‐mediated reaction. Ethoxyresorufin O‐de‐ethylation. Drug Metab. Dispos. 5:1‐8.
   Cammer, W. and Estabrook, R.W. 1967. Spectrophotometric studies of the pigments of adrenal cortex mitochondria. Arch. Biochem. Biophys. 122:735‐747.
   Cinti, D.L. and Schenkman, J.B. 1972. Hepatic organelle interaction. 1. Spectral investigation during drug biotransformation. Mol. Pharmacol. 8:327‐338.
   Cohn, J.A., Alvares, A.P., and Kappas, A. 1977. On the occurrence of cytochrome P‐450 and aryl hydrocarbon hydroxylase activity in rat brain. J. Exp. Med. 145:1607‐1611.
   Favreau, L.V., Malchoff, D.M., Mole, J.E., and Schenkman, J.B. 1987. Responses to insulin by two forms of rat hepatic microsomal cytochrome P‐450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes. J. Biol. Chem. 262:14319‐14326.
   Hook, G.E.R., Bend, J.R., Hoel, D., Fouts, J.R., and Gram, T.E. 1972. Preparation of lung microsomes and a comparison of the distribution of enzymes between subcellular fractions of rabbit lung and liver. J. Pharmacol. Exp. Ther. 182:474‐490.
   Juchau, M.R. and Smuckler, E.A. 1973. Subcellular localization of human placental aryl hydrocarbon hydroxylase. Tox. Appl. Pharmacol. 26:163‐179.
   Matsubara, T., Touchi, A., and Ogawa, A. 1982. Heterogenous distribution of the cytochrome P‐450 monooxygenase system in rat liver lobes. Jpn. J. Pharmacol. 32:999‐1011.
   Moloney, S.J., Fromson, J.M., and Bridges, J.W. 1982. Cytochrome P‐450 dependent deethylase activity in rat and hairless mouse skin microsomes. Biochem. Pharmacol. 31:4011‐4018.
   Mukhtar, H. and Bickers, D.R. 1981. Drug metabolism in skin. Comparative activity of the mixed function oxidases, epoxide hydratase, and glutathione S‐transferase in liver and skin of the neonatal rat. Drug Metab. Dispos. 9:311‐314.
   Omura, T. and Sato, R. 1964. The carbon monoxide–binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. 239:2379‐2385.
   Sasame, H.A., Ames, M.M., and Nelson, S.D. 1977. Cytochrome P‐450 and NADPH cytochrome c reductase in rat brain: Formation of catechols and reactive catechol metabolites. Biochem. Biophys. Res. Commun. 78:919‐926.
   Schenkman, J.B. and Cinti, D.L. 1978. Preparation of microsomes with calcium. Methods Enzymol. 52:83‐89.
   Schenkman, J.B., Remmer, H., and Estabrook, R.W. 1967. Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3:113‐123.
   Schneider, W.C. 1948. Intracellular distribution of enzymes III. The oxidation of octanoic acid by rat liver fractions. J. Biol. Chem. 176:259‐266.
   Walther, B., Ghersi‐Egea, J.F., Minn, A., and Siest, G. 1986. Subcellular distribution of cytochrome P‐450 in the brain. Brain Res. 375:338‐344.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library