Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Polarography and Spectrophotometric Enzyme Assays

Antoni Barrientos1, Flavia Fontanesi1, Francisca Díaz1

1 University of Miami Miller School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.3
DOI:  10.1002/0471142905.hg1903s63
Online Posting Date:  October, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, the assays describe methods that form a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues. Curr. Protoc. Hum. Genet. 63:19.3.1‐19.3.14. © 2009 by John Wiley & Sons, Inc.

Keywords: electron transport chain; mitochondria; OXPHOS; polarography; spectrophotometry

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Polarographic Oxygen Consumption Studies Using Intact Cells
  • Basic Protocol 2: Oxygen Consumption Studies in Isolated Mitochondria
  • Basic Protocol 3: Enzymatic Activity Studies
  • Support Protocol 1: Normalization of OXPHOS Activities
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Polarographic Oxygen Consumption Studies Using Intact Cells

  Materials
  • Exponentially growing intact, non‐permeabilized cells
  • Respiration buffer (RB; see recipe)
  • Inhibitors (Sigma): 2 mM rotenone, 1 M malonate, 0.2 mM antimycin A, 80 mM potassium cyanide (KCN), 1.5 mM oligomycin
  • Digitonin (high purity; Calbiochem)
  • Bovine serum albumin (BSA; Sigma)
  • Adenine nucleotides (Sigma): 40 mM ADP, 40 mM ATP
  • Substrates (Sigma): 50 mM pyruvate, 175 mM malate, 175 mM L‐glutamate, 50 mM succinate, 50 mM duroquinone, 50 mM ascorbate, 500 mM L‐glycerol 3‐phosphate (G3P) (all substrates are dissolved in water and pH adjusted to pH 7.4 with 5M NaOH)
  • 6 mM N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD)
  • Graduated syringe (e.g., Hamilton)
  • Polarographic chamber with adjustable chamber volume (i.e., from 0.25 ml up to 1 ml) (Hansatech Instruments)
  • Clark oxygen electrode in a water‐jacketed microcell, magnetically stirred at 37°C (Hansatech Instruments)

Basic Protocol 2: Oxygen Consumption Studies in Isolated Mitochondria

  Materials
  • Mitochondria preparation or whole cells
  • Hypotonic medium (see recipe)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 2D)
  • Bovine serum albumin (BSA)
  • Electron donors and acceptors (Sigma): 40 mM NADH, 20 mM decylubiquinone, 50 mM succinate, 0.2 M 2,6‐dichlorophenolindophenol (DCPIP), 400 mM phosphoenolpyruvate (PEP), 2 mM cytochrome c
  • Inhibitors (Sigma): 0.2 mM antimycin A (AA), 80 mM KCN, 2 mM rotenone, 100 mM thenoyltrifluoroacetone (TTFA), 1 M malonate
  • SQR/SCCR/QCCR medium (see recipe)
  • Adenine nucleotides (Sigma): 40 mM ADP, 40 mM ATP
  • Lithium borohydrate
  • Concentrated HCl
  • Mitochondrial isotonic medium (see recipe)
  • 10 M lauryl maltoside
  • Isosmotic COX medium (see recipe)
  • MgCl 2
  • KCl
  • Uncouplers (Sigma): carbonyl cyanide m‐chlorophenylhydrazone (CCCP)
  • Enzymes (Sigma): lactate dehydrogenase, pyruvate kinase
  • Oligomycin
  • 30° and 37°C incubators
  • Spectrophotometer with heated chamber
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 3: Enzymatic Activity Studies

  Materials
  • 10 mM Tris⋅Cl, pH 7.5 ( appendix 2D)
  • 10% (w/v) Triton X‐100
  • Electron donors and acceptors (Sigma): 50 mM oxaloacetic acid (adjust to pH 7.4), 10 mM acetyl CoA, 0.1 M 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB)
  • Mitochondrial proteins
  • 30°C incubator
  • Spectrophotometer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Acin‐Perez, R., Bayona‐Bafaluy, M.P., Fernandez‐Silva, P., Moreno‐Loshuertos, R., Perez‐Martos, A., Bruno, C., Moraes, C.T., and Enriquez, J.A. 2004. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell. 13:805‐815.
   Acin‐Perez, R., Fernandez‐Silva, P., Peleato, M.L., Perez‐Martos, A., and Enriquez, J.A. 2008. Respiratory active mitochondrial supercomplexes. Mol. Cell. 32:529‐539.
   Barrientos, A. 2002. In vivo and in organello assessment of OXPHOS activities. Methods 26:307‐316.
   Birch‐Machin, M.A. and Turnbull, D.M. 2001. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell. Biol. 65:97‐117.
   Chretien, D., Bourgeron, T., Rotig, A., Munnich, A., and Rustin, P. 1990. The measurement of the rotenone‐sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem. Biophys. Res. Commun. 173:26‐33.
   Chretien, D., Rustin, P., Bourgeron, T., Rotig, A., Saudubray, J.M., and Munnich, A. 1994. Reference charts for respiratory chain activities in human tissues. Clin. Chim. Acta. 228:53‐70.
   Chretien, D., Gallego, J., Barrientos, A., Casademont, J., Cardellach, F., Munnich, A., Rotig, A., and Rustin, P. 1998. Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age‐related changes. Biochem. J. 329:249‐254.
   Chretien, D., Slama, A., Briere, J.J., Munnich, A., Rotig, A., and Rustin, P. 2004. Revisiting pitfalls, problems and tentative solutions for assaying mitochondrial respiratory chain complex III in human samples. Curr. Med. Chem. 11:233‐239.
   DiMauro, S. and Schon, E.A. 2003. Mitochondrial respiratory‐chain diseases. N. Engl. J. Med. 348:2656‐2668.
   Fernandez‐Vizarra, E., Tiranti, V., and Zeviani, M. 2009. Assembly of the oxidative phosphorylation system in humans: What we have learned by studying its defects. Biochim. Biophys. Acta. 1793:200‐211.
   Hartwig, S., Feckler, C., Lehr, S., Wallbrecht, K., Wolgast, H., Muller‐Wieland, D., and Kotzka, J. 2009. A critical comparison between two classical and a kit‐based method for mitochondria isolation. Proteomics. 9:3209‐3214.
   Ingle, J.D.J. and Crouch, S.R. 1988. Spectrochemical Analysis. Prentice Hall, New Jersey.
   Krahenbuhl, S., Talos, C., Wiesmann, U., and Hoppel, C.L. 1994. Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans. Clin. Chim. Acta. 230:177‐187.
   Medja, F., Allouche, S., Frachon, P., Jardel, C., Malgat, M., de Camaret, B.M., Slama, A., Lunardi, J., Mazat, J.P., and Lombès, A. 2009. Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis. Mitochondrion. May 9. [Epub ahead of print]
   Munnich, A. and Rustin, P. 2001. Clinical spectrum and diagnosis of mitochondrial disorders. Am. J. Med. Genet. 106:4‐17.
   Pallotti, F. and Lenaz, G. 2001. Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 65:1‐35.
   Ragan, C.I. and Cottingham, I.R. 1985. The kinetics of quinone pools in electron transport. Biochim. Biophys. Acta 811:13‐31.
   Reeve, A.K., Krishnan, K.J., and Turnbull, D. 2008. Mitochondrial DNA mutations in disease, aging, and neurodegeneration. Ann. N.Y. Acad. Sci. 1147:21‐29.
   Rustin, P., Chretien, D., Bourgeron, T., Wucher, A., Saudubray, J.M., Rotig, A., and Munnich, A. 1991. Assessment of the mitochondrial respiratory chain. Lancet 338:60.
   Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A., Saudubray, J.M., and Munnich, A. 1994. Biochemical and molecular investigations in respiratory chain deficiencies. Clin. Chim. Acta 228:35‐51.
   Villani, G., Greco, M., Papa, S., and Attardi, G. 1998. Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types. J. Biol. Chem. 273:31829‐31836.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library