Name: simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases
Uploaded: 2018-02-24T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). Video production was carried out in collaboration with the Center for Innovation in Teaching and Learning (CITL) at Memorial University of Newfoundland.

1. Chemicals and Purified Enzymes
<ol>
	<li>Procure the following materials: PDHC and KGDHC of porcine heart origin (or another purified mitochondrial flavoenzyme); H2O2 (30% solution), pyruvate, &#945;-ketoglutarate, NAD+, NADH, CoASH, thiamine pyrophosphate (TPP), mannitol, HEPES, sucrose, EGTA, 3-methyl-2-oxo valeric acid (KMV), superoxide dismutase (SOD), horseradish peroxidase (HRP), 10-Acetyl-3,7-dihydroxyphenoxazine reagent (AUR), and CPI-613.</li>
</ol>
2. ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mitochondrial+reactive+oxygen+species+production+by+GSH+mediated+S-glutathionylation+of+2-oxoglutarate+dehydrogenase.">Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase.</a> Redox Biol. 8, 285-297 (2016).</li><li>Mailloux, R. J., Gardiner, D., O'Brien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Oxoglutarate+dehydrogenase+is+a+more+significant+source+of+O2(.-)/H2O2+than+pyruvate+dehydrogenase+in+cardiac+and+liver+tissue.">2-Oxoglutarate dehydrogenase is a more significant source of O2(.-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.</a> Free Radic Biol Med. 97, 501-512 (2016).</li><li>Ambrus, A., Adam-Vizi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dihydrolipoamide+dehydrogenase+(E3)+deficiency:+Novel+insights+into+the+structural+basis+and+molecular+pathomechanism.">Human dihydrolipoamide dehydrogenase (E3) deficiency: Novel insights into the structural basis and molecular pathomechanism.</a> Neurochem Int. , (2017).</li><li>Young, A., Gardiner, D., Brosnan, M. E., Brosnan, J. T., Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+levels+of+formate+activate+mitochondrial+superoxide/hydrogen+peroxide+release+from+mouse+liver+mitochondria.">Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.</a> FEBS Lett. 591 (16), 2426-2438 (2017).</li><li>Fisher-Wellman, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+glutathione+depletion+reveals+a+novel+role+for+the+pyruvate+dehydrogenase+complex+as+a+key+H2O2-emitting+source+under+conditions+of+nutrient+overload.">Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.</a> Free Radic Biol Med. 65, 1201-1208 (2013).</li><li>Kussmaul, L., Hirst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+superoxide+production+by+NADH:ubiquinone+oxidoreductase+(complex+I)+from+bovine+heart+mitochondria.">The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.</a> Proc Natl Acad Sci U S A. 103 (20), 7607-7612 (2006).</li><li>Huang, L. 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This protocol is advantageous since, 1) it eliminates any competing reactions that may otherwise interfere with H2O2 detection (e.g., antioxidant systems or other sources of ROS), 2) provides a direct assessment of the native rate of ROS release by a flavin-containing mitochondrial dehydrogenase, 3) allows the comparison of the native ROS release rates of two or more purified flavin-based dehydrogenases, 4) can allow for a direct comparison of the rate of ROS release and enzyme activity, an...

The ultimate goal of nutrient metabolism is to make adenosine triphosphate (ATP). In mammalian cells, this occurs in mitochondria, double-membraned organelles that convert the energy stored in carbon into ATP. The production of ATP begins when carbon is combusted by mitochondria forming two electron carriers, NADH and flavin adenine dinucleotide (FADH2)1. NADH and FADH2 are then oxidized by multi-subunit respiratory complexes I and II, respectively, and the liberated electrons are ferried to the terminal electron acceptor molecular oxygen (O2) at complex IV1. The thermodynamically favorable &#34;downhill&#34; transfer of electrons to O2 at the end of the chain is coupled to the export of protons into the intermembrane space by complexes I, III, and IV. This creates a transmembrane electrochemical gradient of protons (proton-motive force), a temporary form of Gibbs free energy that is tapped by complex V to make ATP2. Electron transfer reactions in mitochondria are not perfectly coupled to ATP production. At various points in the Krebs cycle and the respiratory chain, electrons can prematurely interact with O2 to form ROS3. The most biologically relevant ROS generated by mitochondria are O2&#9679;- and H2O2. Although O2&#9679;- is often considered the proximal ROS formed by mitochondria, it is now evident that sites of production form a mixture of O2&#9679;- and H2O2, which is associated with the free radical chemistry of flavin prosthetic groups4,5. At high levels, ROS can be dangerous, damaging biological constituents required for cell function resulting in oxidative distress6. However, at low amounts, mitochondrial ROS fulfill vital signaling functions. For instance, H2O2 release from mitochondria has been implicated in controlling T-cell activation, stress signaling (e.g., induction of Nrf2 signaling pathways), the induction of cell proliferation and differentiation, insulin signaling and release, and feeding behavior7. Considerable progress has been made in understanding the signaling function of ROS. However, important questions still remain in regard to which enzymes in mitochondria serve as the most important sources and how production is controlled.
ROS release by a site of production depends on several factors: 1) the concentration of the electron donating site, 2) the redox state of the electron donating site, 3) access to oxygen, and 4) the concentration and type of oxidation substrate3,8,9. In mitochondria, other factors like the concentration of NADH and membrane potential strength also influence ROS production8,10. For example, the rate of O2&#9679;-/H2O2 production by purified PDHC or KGDHC increases with increasing NADH availability5,11. In this scenario, electrons are flowing backwards from NADH to the FAD center in the E3 subunit of PDHC or KGDHC, the site for O2&#9679;-/H2O2 production12. Similarly, the provision of NAD+ has the opposite effect, decreasing ROS release from KGDHC12. Thus, controlling entry or exit of electrons from sites of ROS production can alter how much O2&#9679;-/H2O2 is formed. For instance, blocking the E2 subunit of PDHC or KGDHC with CPI-613, a lipoic acid analog, results in an almost 90% decrease in O2&#9679;-/H2O2 production13. Similar results can be obtained with the chemical S-glutathionylation catalysts, diamide and disulfiram, which almost abolish O2&#9679;-/H2O2 production by PDHC or KGDHC via the conjugation of glutathione to the E2 subunit14. The trade-off for blocking electron flow through the E2 subunit of PDHC and KGDHC is a decrease in NADH production which diminishes ROS formation by the electron transport chain (e.g., complex III). This can also decrease ATP output by the electron transport chain. Overall, blocking electron entry to sites of ROS production can be a highly effective means of controlling O2&#9679;-/H2O2 production.
Mitochondria can contain up to 12 potential O2&#9679;-/H2O2 sources6. Most of these sites are flavin-containing enzymes which generate a mixture of O2&#9679;- and H2O2. Use of different substrate and inhibitor combinations have allowed for the identification of which respiratory complexes and mitochondrial dehydrogenases serve as high capacity O2&#9679;-/H2O2 forming sites in different tissues3. PDHC and KGDHC have been shown to serve as high capacity O2&#9679;-/H2O2 emitting sites in muscle and liver mitochondria13,15. However, some difficulties remain in regard to the examination of the O2&#9679;-/H2O2 forming potential of individual sites in mitochondria and the impact that different substrate and inhibitor combinations have on the activities of the enzymes. This is due to the presence of unwanted side reactions (e.g., formation of O2&#9679;-/H2O2 by sites other than the enzyme of interest), contaminating endogenous nutrients (e.g.,fatty acids) or organelles (e.g., peroxisomes which also form O2&#9679;-/H2O2), and use of inhibitors that lack selectivity, and/or use of compounds that do not fully inhibit ROS production. Certain inhibitors can also alter the mitochondrial bioenergetics and direction of electron flow, and this alters ROS release from other sites of production and confounds results. Absolute rates for O2&#9679;-/H2O2 release from individual sites in mitochondria are also difficult to quantify due to the high concentration of O2&#9679;- and H2O2 eliminating enzymes in the matrix and intermembrane space. Therefore, elimination of any competing reactions that can interfere with O2&#9679;-/H2O2 release measurements can be useful when identifying high capacity O2&#9679;-/H2O2 forming sites.
Here, we present a simple method that allows for the simultaneous examination of O2&#9679;-/H2O2 production and NADH formation by purified flavin-dependent dehydrogenases. By using purified enzymes, unwanted ROS forming side reactions and ROS degrading enzymes can be eliminated allowing for a more accurate measure of native O2&#9679;-/H2O2 production rates for individual flavoenzymes. This method can be used to directly compare the O2&#9679;-/H2O2 forming capacity of different purified dehydrogenases or to screen potential site-specific inhibitors for O2&#9679;-/H2O2 release. Finally, measuring O2&#9679;-/H2O2 and NADH production simultaneously can allow for a real-time assessment of the relationship between enzyme activity and ROS release capacity.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pyruvate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P7032-10UN</td>
			<td style="width:167px;">purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">alpha-ketoglutarate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">K1502-20UN</td>
			<td>purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30% hydrogen peroxide solution</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">HX0640-5</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD+</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N0632-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NADH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N4505-100MG</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pyruvate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P2256-5G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">alpha-ketoglutarate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">75892-25G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CoASH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C3019-25MG</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">thiamine pyrophosphate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C8754-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mannitol</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">M4125-100G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">H3375-25G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S7903-250G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGTA</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">E3889-10G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KMV</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">198978-5G</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPI-613</td>
			<td style="width:71px;">Santa Cruz</td>
			<td style="width:119px;">sc-482709</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SOD</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S9697-15KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">horseradish peroxidase</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P8375-1KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amplex Ultra Red</td>
			<td style="width:71px;">Thermofisher</td>
			<td style="width:119px;">A36006</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotech Synergy 2 microplate reader</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>microplate reader for assays</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Gen5 software</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>software, used for collection of raw RFU</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Graphpad Prism</td>
			<td style="width:71px;">Graphpad software</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft EXCEL</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Figure 3A provides a representative trace for the RFU collected during the simultaneous measurement of H2O2 and NADH production by purified KGDHC. The raw RFU data for each time interval is depicted in Figure 3B. The raw RFU data are then exported for analysis. By extrapolating from standard curves presented in Figure 2, the absolute amount of NADH and H2O2...

Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...