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An HPLC Method to Detect Heme Oxygenase Activity

Stefan W. Ryter¹, Rex M. Tyrrell²

¹Southern Illinois University School of Medicine, Springfield, Illinois
²University of Bath, Bath, United Kingdom

Publication Name:

Current Protocols in Toxicology

Unit Number:

Unit 9.6

DOI:

10.1002/0471140856.tx0906s05

Online Posting Date:

May, 2001

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Abstract

This unit presents a method to calculate heme oxygenase enzymatic activity from the formation of bilirubin equivalents [biliverdin-Ixalpha (BV) and bilirubin-IXalpha (BR)]. The BV and BR generated in the reaction are separated by reversed-phase HPLC and detected using visible absorbance spectroscopy. Since both metabolites of heme degradation are directly quantifiable, the assay eliminates the requirement for biliverdin reductase supplementation.

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Unit Introduction
Basic Protocol: Determining Heme Oxygenase Activity Using HPLC
Support Protocol: Preparation of Microsomal Protein Extracts from Cultured Cells
Alternate Protocol: Detection of BVR Activity
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol: Determining Heme Oxygenase Activity Using HPLC

Materials

Microsomal protein extract from cultured cells (see Support Protocol)
Solution A (see recipe)
Partially purified biliverdin reductase (BVR; optional)
20 mM -NADPH (see recipe)
40 mM glucose 6-phosphate (see recipe)
1 U/µl glucose-6-phosphate dehydrogenase (see recipe)
2.5 mM protoporphyrin IX (Sn PPIX; see recipe; optional)
2.5 mM hemin stock solution (see recipe)
Stop solution with mesoporphyrin internal standard (see recipe)
HPLC buffer A (see recipe)
HPLC buffer B: methanol (HPLC grade; Fisher; degassed 2 min with compressed He)
20 µM biliverdin and bilirubin standards (see recipes)
1.5- and 0.5-ml microcentrifuge tubes
High performance liquid chromatography (HPLC) system with two solvent reservoirs and pumps (e.g., Kontron or equivalent)
Data acquisition system (e.g., MT 450 MS-DOS or higher; Kontron or equivalent)
Autosampler (e.g., HPLC 360; Kontron, or equivalent)
Visible detector (e.g., UVIKON 720 LC microdetector; Kontron or equivalent)
Reversed-phase C18 steel cartridge HPLC column (e.g., Novapak 3.9 mm × 15 cm) or phenyl reversed-phase column (both available from Waters Chromatography)
Compressed helium tank

Support Protocol: Preparation of Microsomal Protein Extracts from Cultured Cells

Materials

Cultures of cells
Phosphate-buffered saline (PBS), pH 7.4 (appendix 2A), ice-cold
PBS/EDTA solution (see recipe), ice-cold
Solution A (see recipe)
1000× protease inhibitor stock solutions (see recipe)
Rubber cell scrapers
15-ml centrifuge tubes (e.g., 16 × 125–mm tissue culture tubes with screw caps; Corning)
Refrigerated tabletop centrifuge (e.g., GPK centrifuge; Beckman, or equivalent)
Sonicator (e.g., Branson sonifier B-12 sonicator; Branson Ultrasonics, or equivalent)
Ultracentrifuge (e.g., Centrikon T20-60 ultracentrifuge; Kontron, or equivalent)
Fixed-angle rotor capable of 105,000 × g (e.g., TFT65.13 rotor; Kontron, or equivalent)
Polyallomar ultracentrifuge tubes, (e.g., 13.5-ml; Kontron)
Additional reagents and equipment for determination of protein concentration (appendix 3A)

NOTE: Perform all steps at 4°C.

Alternate Protocol: Detection of BVR Activity

Additional Materials (also see Basic Protocol)

Protein sample to be analyzed for BVR activity
2.5 µM biliverdin (BV) solution in DMSO (see recipe for 20 µM stock)

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Figures

Figure 9.6.1

HPLC chromatogram showing the resolution of tetrapyrrole standards in an alcohol extract of sonicated microsomal protein solution. Final concentrations were 2 µM biliverdin (BV), 2 µM bilirubin-IX (BRIX), 0.35 µM BRIII, 0.27 µM BRXIII, 12.5 µM hemin, 0.4 µM mesoporphyrin (MP), corresponding to injected amounts of 240 pmol BV, 240 pmol BRIX, and 48 pmol MP. The tetrapyrroles eluted with the following typical retention times: (1) biliverdin (BV), 5.29 min; (2) hemin, 9.8 min; (3) bilirubin structural isomer (III or XIII), 10.39 min; (4) bilirubin-IX (BRIX), 10.9 min; (5) bilirubin structural isomer (III or XIII), 11.38 min; and (6) mesoporphyrin (MP), 12.54 min. Optical density was detected at 405 nm. Reprinted from Ryter et al. (1998), with permission from Elsevier Science.

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Literature Cited

Literature Cited
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	Durante, W. and Schafer, A. 1998. Carbon monoxide and vascular cell function (review). Int. J. Mol. Med. 2:255-262.
	Keyse, S.M. and Tyrrell, R.M. 1989. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. U.S.A. 86:99-103.
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	Maines, M.D. 1992. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, Fla.
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	Ryter, S., Kvam, E., Richman, L., Hartmann, F., and Tyrrell, R. M. 1998. A chromatographic assay for heme oxygenase activity in cultured human cells: Application to artificial heme oxygenase over-expression. Free Radic. Biol. Med. 24:959-971.
	Ryter, S., Kvam, E., and Tyrrell, R.M. 2000. The heme oxygenases: Current methods and applications. Methods Mol. Biol. 99:369-391.
	Schacter, B. 1978. Assay for microsomal heme oxygenase in liver and spleen. Methods Enzymol. 52:367-372.
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	Tenhunen, R., Ross, M.E., Marver, H.S., and Schmid, R. 1970. Reduced nicotinamide-adenine dinucleotide phosphate dependent biliverdin reductase: Partial purification and characterization. Biochemistry. 9:298-303.
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Key References
	Ryter, S., Kvam, E., and Tyrrell, R.M. 1999. Determination of heme oxygenase activity by high performance liquid chromatography. Methods Enzymol. 300:322-336.
This paper also provides detailed methodology for the analysis of HO activity using high performance liquid chromatography.
	Ryter et al., 1998. See above.
This paper shows graphic examples of the application of the HPLC technique to the detection of heme oxygenase activity.
	Maines, 1992. See above.
This book provides a comprehensive account of the basic background information related to the heme oxygenase system.