The Complex IV (cytochrome c oxidase)

PlayBook

MitoSciences

Tips and

techniques

for research

into Complex IV

(cytochrome c oxidase)

Edited by Dr. Roderick CapaldiOriginal studies from the

MitoSciences Research Team

“The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.” -Sir William Bragg

Complex IV (cytochrome c oxidase) is a key component of oxidative phosphorylation. In recent years, as the workings of this exquisite enzyme have become better understood, the focus of research has moved away from structure-function studies toward examination of the role that this complex plays in health and disease. Not only is cytochrome c oxidase deficiency found in genetic mitochondrial disease, but levels and functioning of the enzyme are altered as a part of neurodegenerative conditions, including Alzheimer’s disease and Parkinson’s disease. Moreover, there is a mounting evidence of regulation of cytochrome c oxidase by diet and exercise; thus a link to obesity.

As researchers try to unravel the role of cytochrome c oxidase in these wide range of conditions, there are four major questions they must ask:

1) How much of this enzyme is present in a cell or tissue samples?

2) What is the activity level of the enzyme in these cells or tissues?

3) Has the enzyme been fully assembled?

4) Has the complex been modified in any way (e.g. through phosphorylation or oxidative damage)?

MitoScienceS provideS approacheS to anSwer each of theSe queStionS.

Complex IV1 PlayBook

1a) Quantitating CytoChrome C oxidase LeveLs and mosaiCism by

immunoCytoChemistry and immunohistoChemistry:

It is often the case, particularly in cytochrome c oxidase deficiency due to genetic causes and with drug-induced lesions, that enzyme levels differ from cell to cell. This mosaicism can be evaluated by immunocytochemistry (ICC) in cells or by immunohistochemisty (IHC) for tissues. Levels can be quantitated with ICC when flow cytometry is available.

Figure 1 shows an ICC image of fibroblasts from a patient with MELAS (myoclonus, epilepsy, lactic acidosis, and stroke-like episodes) where a mutation in a mitochondrially-encoded tRNA gene alters the biogenesis of Complex IV. This mutation is heteroplasmic, thus only a fraction of the cells are affected; moreover, cells that are affected are altered in amount of Complex IV to varying extents.

The mosaicism is clearly evident with the Complex IV subunit I monoclonal antibody (green), MitoSciences catalog number MS404, used to detect cytochrome c oxidase and a Complex V antibody (red), MS507, used to detect total mitochondria. Red cells are cytochrome c oxidase deficient while the yellow cells are normal for the enzyme.

Mosaicism is a clear indication that the contributing mutation is in mtDNA and does not involve a nuclear-encoded gene. Figure 2 shows the IHC image of biopsy liver stained for cytochrome c oxidase with MS404 using horseradish peroxidase. Note the different levels of cytochrome c oxidase in different cell types. MS404 works well for studies of human, mouse, and rat tissue.

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Figure 2Courtesy of Hannes Vogel (Stanford U.)

Figure 1

1b) anaLyzying CytoChrome C oxidase LeveLs by

Western bLotting

Besides a cell by cell analysis, overall cell or tissue levels of cytochrome c oxidase can be quantitated by Western blotting. Figure 3 shows an example of cytochrome c oxidase deficiency in cell lines evaluated by Western blotting. In this case, levels of Complex IV were related to those of the other OXPHOS complexes by using MitoSciences OXPHOS Detection Kit (MS601). This kit uses the Complex IV monoclonal antibody against subunit I (MS404) to evaluate the levels of cytochrome c oxidase. In the example provided, cell extract is used, not isolated mitochondria, thereby reducing the amount of material and time needed for analysis. Lane 1 is a control fibroblast cell line while lane 2 is a rho0 fibroblast cell line that has no mtDNA, and therefore does not contain cytochrome c oxidase or functional complex I or III. The F1 part of the ATP synthase is assembled, hence the presence of the α subunit of the complex. Lane 3 and 4 are fibroblast samples from patients with mtDNA mutations, causing reduced Complex IV and Complex I assembly. Lane 5 is a patient with a specific cytochrome c oxidase deficiency. The bar graph portion of figure 3 quantitatively compares Complex IV (cytochrome oxidase) and Complex V (ATP synthase) derived from the corresponding Western blot.

Sometimes, cytochrome c oxidase deficiency can result from mutations in the machinery needed to assemble Complex IV. In the case of the assembly factor SURF1, the partially assembled but inactive complex contains high levels of subunit I. SURF1 is involved in copper insertion into subunit II. Therefore, mutants in SURF1 are characterized by low levels of subunit II, which can be detected with MitoSciences Complex IV subunit II monoclonal antibody (MS405) (See Figure 4). MitoSciences also provides a monoclonal antibody specific to SURF1 (MS403). In figure 4, lane 1 is a control fibroblast cell line, lane 2 are fibroblast rho0 cells from a patient. (Also note that there is upregulation of SURF1). Lane 3 shows cells from a patient with a SURF1 mutation leading to reduced levels of subunit II.

Figure 3

Figure 4 Capaldi et al. Mitochondrion 2004, (4) ),p417-426

Complex IV3 PlayBook

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1C) high-throughput miCropLate assays

of CytoChrome C oxidase LeveLs

Western blotting is useful when the number of samples is relatively low, but often high-throughput analysis is required; for instance, when examining drug effects on cells as part of a drug discovery or drug toxicity screening. Moreover, it is sometimes useful to be able to multiplex quantity measurements with activity determinations in order to obtain specific activities in a study. In these cases, MitoSciences Microplate Assay Kits provide a useful platform for the analyses (includes Activity, Quantity, and Multiplexing kits for human, mouse, and rat samples, MS421 through MS427 respectively).

The basis of the microplates is as follows: cytochrome c oxidase is immunocaptured from a sample of cell or tissue extract (no mitochondrial isolation is necessary) by a monoclonal antibody attached to a 96-well plate. The amount of enzyme captured is determined by the reaction of a second monoclonal antibody against a different epitope in the protein complex with the signal reported by an appropriate secondary antibody, which is conjugated with alkaline phosphatase. In the case of activity microplates, the enzyme is immunocaptured by the plate-bound monoclonal antibody, then its activity measured by following the oxidation of cytochrome c (see page 6).

Figure 5 shows the use of Microplate Assay Kits for Complex IV to screen the levels of enzyme in cells grown in the presence of the antiviral drug ddC, which is known to inhibit mtDNA replication. In the experiment shown here, fibroblasts were grown in 4µM ddC, collected after various doubling times and the amount of cytochrome c oxidase measured with the Microplate Assay Kit for Human Complex IV Activity (MS421). The activity of the enzyme was then measured again using a 96-well plate assay. The results of the plate assays demonstrate that mtDNA depletion by the drug reduces both amount and activity proportionally (i.e. the specific activity remains the same).

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Figure 5From Murray, Schilling, Yoo, Gibson, Marusich, and Capaldi. Biotechnology and Applied biochemistry 2007. (in press)

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1d) Quantitating CytoChrome C oxidase LeveLs in smaLL amounts of

tissue inCLuding CLiniCaL sampLes using dipstiCk assay kits

Sample amount is often an issue in studies of cytochrome c oxidase deficiency, particularly when cell culture material is being examined, or in experiments using mice or other rodents when specific areas of an organ are to be microdissected for analysis.

In such cases, MitoSciences Dipstick Assay Kits become invaluable (MS430, MS431, MS432). The basics of the dipstick method are shown in figure 6. The method is rapid, as data can be obtained in 20-30 minutes. Figure 7 shows analysis of cytochrome c oxidase levels after various drug treatments using HepG2 cells grown in single wells of a 96-well plate (i.e. around 20,000 cells). In this study the level of Complex IV, with its requirement for mitochondrial-encoded subunits, was compared

with that of frataxin, a nuclear-encoded protein, for which MitoSciences also has a Dipstick Assay Kit (MSF31) (MSF01 Frataxin monoclonal antibody also available). The dipsticks are easily read and levels of enzyme quantitated using a flatbed scanner, although more accurate and reproducible data is obtained using MitoSciences’ Hamamatsu CA-1000 Immunochromatographic Reader.

The value of dipstick assaying of cytochrome c oxidase is further demonstrated in figures 8 and 9, which show Complex IV quantity determined from human cheek swabs and a single drop of whole blood respectively. The ease of use and the speed of obtaining results makes the Dipstick Assay Kits the ideal platform for clinical studies of Complex IV deficiencies due to genetic cause.

Complex IV5 PlayBook

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2) measuring CompLex iv aCtivity in sampLes by miCropLate and dipstiCk methods

MitoSciences provides Complex IV activity assays in two formats: the Microplate Assay and the Dipstick Assay. The plate assay measures the oxidation of reduced cytochrome c oxidase spectrophotometrically, while the dipstick assay determines Complex IV turnover by precipitation of DAB (di-amino benzidine) on the dipsticks. The plate assay is particularly useful in screening for the effects of drugs as shown earlier.

Cytochrome c oxidase can be immunocaptured from a tissue of interest and dosed with compounds. Figure 10 shows an IC50 measurement of the effect of Tamoxifen on Complex IV activity using bovine heart mitochondria. The inhibition of cytochrome c oxidase activity by this drug is surprising and it is not clear whether the effect represents unwanted toxicity, or a desired reduction of OXPHOS that enhances apoptotic events. The immunocapture approach is useful not only because it allows pre-animal testing, but also because the enzyme can be isolated after animal dosing and toxic in-vivo inhibitory effects of drugs identified. Both the Microplate Assay Kits, or more simply the DipStick Assay Kits, can be used to examine cytochrome

c oxidase deficiency in patients with suspected mitochondrial disease.

Figure 11 shows the Complex IV activity of fibroblasts measured by both the plate and the dipstick methods, as well as with Western blotting for a series of patients with defects in this enzyme. Lane 1 is a control fibroblast cell line while lanes 2, 3, 4, 5, and 6 are patient cell lines with varying degrees of Complex IV deficiency.

3) CytoChrome C oxidase subunit anaLysis for

assembLy CharaCterization

MitoSciences has monoclonal antibodies against twelve of the thirteen subunits of mammalian Complex IV, facilitating detailed studies of cytochrome c oxidase assembly in Western blotting. MitoSciences also has an antibody against one of the several known assembly factors for the enzyme, SURF1 (MS403). Many of these monoclonal antibodies have excellent cross-reactivity with human, mouse, and rat, while some react with C. elegans, yeast, and zebrafish.

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S. Nadanaciva, A. Bernal, R. Aggeler, R. Capaldi, Y. Will. Toxicolgy in Vitro. 2007

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4) monitoring post-transLationaL modifiCation of CytoChrome C oxidase in heaLth & disease

The key to being able to identify and further characterize post-translational modifications of Complex IV, either as a part of enzyme regulation (e.g. phosphorylation), or as an unwanted consequence of oxidative stress, is to be able to purify the enzyme cleanly and rapidly for such analyses. MitoSciences provides an immunocapture kit that makes possible the rapid isolation of enzyme from very small amounts of material (MS401 and MS402 Complex IV Immunocapture Kit for Human and Rodent, respectively).

Figure 12 details an experiment involving carbonyl modification as a result of Fe2+-induced oxidative damage. Part A shows immunocapture of the enzyme from mitochondria using MS401 that have been exposed to Fe2+ plus ascorbate for different lengths of time. Part B shows carbonyl modification of subunits (via oxyblot).

Cytochrome c oxidase is remarkably resistant to oxidative damage with subunit IV being the major site of

modification. In this connection, it may be significant that the enzyme is the only one of the OXPHOS complexes to to evolve since organisms began to inhabit an oxygen-rich environment. Mass spectometry can be used to identify the precise modification and localize sites. MitoSciences has conducted extensive mass spectometry of the subunits separated by gel electrophoresis after immunocapture, in order to establish the extent of coverage obtained by protease digestion and mass spectometry.

As an alternative to Western blotting, modification can be measured using the microplate assay format. Figure 13 shows an experiment in which peroxynitrite oxidation of Complex IV has been followed in this way. Interestingly, incorporation of nitrotyrosine occurs in several subunits (II, Va, VIIab) with little effect on activity.

Complex IV7 PlayBook

Figure 13

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MitoSciences Products for Complex IV Research:

catalog # deScriptive naMe SpecieS reactivity* application(S)**

MS401 Complex IV Immunocapture Kit H, B IP

MS402 Complex IV Immunocapture Kit for Rodent M, R IP

MS421 Microplate Assay Kit for Human Complex IV Activity H MP

MS422 Microplate Assay Kit for Human Complex IV Quantity H MP

MS423Microplate Assay Kit for Human Complex IV Activity & Quantity (Multiplexing) H MP

MS424 Microplate Assay Kit for Mouse Complex IV Activity M MP

MS425 Microplate Assay Kit for Mouse Complex IV Quantity M MP

MS426Microplate Assay Kit for Mouse Complex IV Activity & Quantity (Multiplexing) M MP

MS427 Microplate Assay Kit for Rat Complex IV Activity R MP

MS430 Dipstick Assay Kit for Human Complex IV Activity H, B DS

MS431 Dipstick Assay Kit for Human Complex IV Quantity H, B DS

MS432 Dipstick Assay Kit for Rodent Complex IV Activity M, R DS

MS601 Human Total OXPHOS Complexes Detection Kit H WB

MS602 OXPHOS/ PDH Immunocytochemistry Kit H ICC, IHC

MS602a OXPHOS/ PDH Immunocytochemistry Kit H ICC, IHC

MS604 Rodent Total OXPHOS Complexes Detection Kit H, B, M, R WB

MS401c Complex IV Immunocapture monoclonal antibody H, B IP

MS403 Complex IV subunit SURF1 monoclonal antibody H WB

MS404 Complex IV subunit I monoclonal antibody H, B, M, R, Z, Ce WB, ICC, IHC

MS405 Complex IV subunit II monoclonal antibody H WB, ICC, IHC

MS406 Complex IV subunit III monoclonal antibody H, B, M, R, Y WB

MS407 Complex IV subunit IV monoclonal antibody H, B, M, R, Z WB

MS408 Complex IV subunit IV monoclonal antibody H, B WB, ICC, IHC

MS409 Complex IV subunit Va monoclonal antibody H, B, M, R, Z WB, ICC, IHC

MS410 Complex IV subunit Vb monoclonal antibody H, B, M, R WB

MS411 Complex IV subunit VIaH monoclonal antibody B WB

MS412 Complex IV subunit VIaL monoclonal antibody H, B, R WB

MS413 Complex IV subunit VIb monoclonal antibody H, B, R WB

MS414 Complex IV subunit VIc monoclonal antibody H, B, R WB

MS415 Complex IV subunit VIIaHL monoclonal antibody B, R WB

MS416 Complex IV subunit VIIaHL monoclonal antibody B, R WB

MS418 Complex IV subunit I monoclonal antibody for yeast Y WB

MS419 Complex IV subunit II monoclonal antibody for yeast Y WB

MS420 Complex IV subunit IV monoclonal antibody for yeast Y WB

*Species Reactivity Codes: Human (H), Bovine (B), Mouse (M), Rat (R), C. elegans (Ce), Zebrafish (Z), Yeast (Y)

**Application Codes: Western Blotting (WB), Immunocapture (IP), Immunocytochemistry (ICC), Immunocytochemistry (ICC), Immunohistochemistry (IHC), Microplate Assay (MP), Dipstick Assay (DS)

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Complex IV Products for Non-Mammalian Species:

The level of conservancy of the Complex IV subunits, particularly the mitochondrial-encoded components allows the use of MitoSciences antibodies in studies involving non-mammalian species, including yeast, C. elegans, and zebrafish. A full table of cross reactivities is available on our website at www.mitosciences.com/species_table.html .

catalog # naMe non-MaMMalian SpecieS reactivity

MS404 Complex IV subunit I mAb C. elegans, Zebrafish

MS406 Complex IV subunit III mAb Yeast

MS407 Complex IV subunit IV mAb Zebrafish

MS409 Complex IV subunit Va mAb Zebrafish

MS418 Complex IV subunit I mAb for yeast Yeast

MS419 Complex IV subunit II mAb for yeast Yeast

MS420 Complex IV subunit IV mAb for yeast Yeast

Ancillary Products of use in Complex IV Research:

catalog # deScriptive naMe

MS801 Human heart mitochondria for Western blotting control

MS804 Human liver mitochondria for Western blotting control

MS802 Bovine heart mitochondria for immunocapture control

MS811 Rat liver mitochondria for Western blotting control

MS817 Rat liver mitochondria for immunocapture control

MS812 Rat heart mitochondria for Western blotting control

MS818 Rat heart mitochondria for immunocapture control

MS813 Rat brain mitochondria for Western blotting control

MS819 Rat brain mitochondria for immunocapture control

MS814 Mouse liver mitochondria for Western blotting control

MS820 Mouse liver mitochondria for immunocapture control

MS815 Mouse heart mitochondria for Western blotting control

MS821 Mouse heart mitochondria for immunocapture control

MS816 Mouse brain mitochondria for Western blotting control

MS822 Mouse brain mitochondrial for immunocapture control

MS850 Mitochondria Isolation Kit for Rodent Tissue

MS851 Mitochondria Isolation Kit for Rodent Tissue (with Dounce Homogenizer

MS852 Mitochondria Isolation Kit for Cultured Cells

MS853 Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer

Complex IV9 PlayBook

catalog # deScriptive naMe

MS901 Goat anti-Mouse IgG-TXRD

MS902 Goat anti-Mouse IgG-FITC

MS903 Goat anti-Mouse IgG1-TXRD

MS904 Goat anti-Mouse IgG1-FITC

MS905 Goat anti-Mouse IgG2a-TXRD

MS906 Goat anti-Mouse IgG2a-FITC

MS907 Goat anti-Mouse IgG2b-TXRD

MS908 Goat anti-Mouse IgG2b-FITC

MS910 n-dodecyl-β-D-maltopyranoside (Lauryl maltoside 10%)

MS911 n-dodecyl-β-D-maltopyranoside (Lauryl maltoside 15%)

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MitoSciences is the world leader in products and services for mitochondrial testing.

MitoSciences has produced and continues to add to the world’s largest collection of anti-mitochondrial monoclonal antibodies. These antibodies are being sold for life sciences research, developed into kits and services for drug discovery, and developed for a broad range of diagnostic and therapeutic applications.

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