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

Summary

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.

Abstract

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^●-) 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 and side reactions that also form O₂^●-/H₂O₂. Use of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering 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 α-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.

Introduction

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 "downhill" 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^●- and H₂O₂. Although O₂^●- is often considered the proximal ROS formed by mitochondria, it is now evident that sites of production form a mixture of O₂^●- and H₂O₂, which is associated with the free radical chemistry of flavin prosthetic groups^4,5. 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 substrate^3,8,9. In mitochondria, other factors like the concentration of NADH and membrane potential strength also influence ROS production^8,10. For example, the rate of O₂^●-/H₂O₂ production by purified PDHC or KGDHC increases with increasing NADH availability^5,11. 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^●-/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^●-/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^●-/H₂O₂ production¹³. Similar results can be obtained with the chemical S-glutathionylation catalysts, diamide and disulfiram, which almost abolish O₂^●-/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^●-/H₂O₂ production.

Mitochondria can contain up to 12 potential O₂^●-/H₂O₂ sources⁶. Most of these sites are flavin-containing enzymes which generate a mixture of O₂^●- 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^●-/H₂O₂ forming sites in different tissues³. PDHC and KGDHC have been shown to serve as high capacity O₂^●-/H₂O₂ emitting sites in muscle and liver mitochondria^13,15. However, some difficulties remain in regard to the examination of the O₂^●-/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^●-/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^●-/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^●-/H₂O₂ release from individual sites in mitochondria are also difficult to quantify due to the high concentration of O₂^●- and H₂O₂ eliminating enzymes in the matrix and intermembrane space. Therefore, elimination of any competing reactions that can interfere with O₂^●-/H₂O₂ release measurements can be useful when identifying high capacity O₂^●-/H₂O₂ forming sites.

Here, we present a simple method that allows for the simultaneous examination of O₂^●-/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^●-/H₂O₂ production rates for individual flavoenzymes. This method can be used to directly compare the O₂^●-/H₂O₂ forming capacity of different purified dehydrogenases or to screen potential site-specific inhibitors for O₂^●-/H₂O₂ release. Finally, measuring O₂^●-/H₂O₂ and NADH production simultaneously can allow for a real-time assessment of the relationship between enzyme activity and ROS release capacity.

Protocol

1. Chemicals and Purified Enzymes

1. Procure the following materials: PDHC and KGDHC of porcine heart origin (or another purified mitochondrial flavoenzyme); H₂O₂ (30% solution), pyruvate, α-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.

2. Planning the Assay and Reagent Preparation

1. Set up the assay according to Table 1 in a 96-well plate.
   NOTE: The total volume for each reaction is 200 µL. Stock concentrations are adjusted such that 20 µL of reagent is added to each well. This ensures rapid addition of cofactors and substrates to each reaction well prior to starting the assay.
2. Prepare reaction buffer containing 220 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 20 mM HEPES (pH 7.4 with HCl).
   NOTE: This could change depending on the physical properties of different purified enzymes.
3. Examine the purified KGDHC or PDHC for contamination using enzyme activity assays^14,16,17 or by immunoblot or Coomassie stain⁵.
4. Prepare all reagents in the reaction buffer as per the working solution concentrations in Table 1.
5. Store all reagents as 1 mL aliquots at -20 °C. Store pyruvate and α-ketoglutarate at -80 °C in 100-µL aliquots to prevent spontaneous degradation due to auto-oxidation and repeated freeze-thaw.
6. Prepare the AUR reagent master stock in 1 mL of DMSO and then dilute to 100 µM in reaction buffer. Store protected from light.
   NOTE: Here, the 100 µM working solution is made fresh every 2 to 3 weeks and protected from light.

3. Setting Up the Assay Template

1. Set up the assay procedure on a monochromatic microplate reader with dual measurement capabilities.
2. Define the reading procedure by selecting "PROTOCOL". Then click "PROCEDURE".
3. Set "TEMPERATURE" to 25 °C (Figure 1A).
4. Click "START KINETIC" to set read conditions (Figure 1A). Set the time and number of read intervals/experiments.
5. Click "READ" (Figure 1B).
   NOTE: Samples are read from TOP position with a gain of 50. Time: 5 min, read at 30-s intervals. Channel 1: Resorufin fluorescence - excitation/emission = 565 nm:600 nm, wavelength width of 13.5 nm. Channel 2: NADH autofluorescence - excitation/emission = 376 nm:450 nm, wavelength width of 20 nm.
6. Under "READ", select wells to monitor (e.g., A1-A4 and B1-B4).
7. Select "END KINETIC".

4. Standard Curves

1. Thaw the reagents and store on ice until needed.
2. NADH standard curve (Figure 2A)
   1. Prepare the working solutions for NADH in the concentration range of 0.5-10 mM in reaction buffer.
   2. Add 20 µL aliquots from each NADH working solution concentration to wells containing 180 µL reaction buffer. The final concentration of NADH in each well is 0.05-1 mM.
   3. Click "READ" and set excitation/emission = 376 nm:450 nm, wavelength width of 20 nm.
      NOTE: This is an endpoint read only - kinetic parameters are not required.
3. AUR standard curve (Figure 2B)
   1. Prepare the working stocks of hydrogen peroxide in reaction buffer; stock concentrations range from 200-4,000 nM.
   2. Add 120 µL of the reaction buffer to each well.
   3. Add 20 µL of HRP (working stock concentration = 30 U/mL, final concentration in the well = 3 U/mL), 20 µL of SOD (working stock concentration = 250 U/mL, final concentration in the well = 25 U/mL), and 20 µL of AUR (working stock concentration = 100 µM, final concentration in the well = 10 µM) to each well containing reaction buffer.
   4. Add 20 µL of each H₂O₂ working stock solution to each well containing buffer and assay reagents. The final reaction volume is 200 µL and the final concentration of H₂O₂ in each well is 20-400 nM.
   5. Incubate the plates for 1 min in the plate reader at 25 °C.
   6. Click "READ" and set excitation/emission = 565 nm/600 nm, wavelength width of 13.5 nm. Note that this is an endpoint read only - kinetic parameters are not required.

5. Measuring O₂ ^●- /H₂ O₂ Release and NADH Production by KGDHC and PDHC

1. See Table 1 for the order of addition of the different reagents, the concentration of each working solution, and the volume added to each well.
2. Add 40 µL of reaction buffer to each well. For assays using KMV or CPI-613, add 20 µL of buffer to each well.
3. Add 20 µL of PDHC or KGDHC (working stock of 1 U/mL, final concentration per well = 0.1 U/mL) to each well.
4. Incubate PDHC and KGDHC at 25 °C for 2 min.
5. Add 20 µL of KMV (working stock = 100 mM, final concentration = 10 mM) or 20 µL of CPI-613 (working stock concentration = 1.5 mM, final concentration = 150 µM) (Table 2).
   NOTE: This step can be excluded if inhibitors are not being used for the assays.
6. Incubate for 1 min at 25 °C.
   NOTE: This step can be excluded if inhibitors are not being used for the assays.
7. Add 20 µL of HRP (working stock concentration = 30 units/mL, final concentration in the well = 3 units/mL) and 20 µL of SOD (working stock concentration = 250 units/mL, final concentration in the well = 25 units/mL) to each well.
8. Add 20 µL of coenzyme A (working stock concentration = 1 mM, final concentration = 0.1 mM), 20 µL of TPP (working stock concentration = 3 mM, final concentration = 0.3 mM), and 20 µL of NAD⁺ (working stock concentration = 10 mM, final concentration = 1 mM) to each well.
9. Remove the AUR reagent from protective tinfoil covering and add 20 µL to each well.
10. Add 20 µL of pyruvate (working stock concentration ranging from 1-100 mM, final concentration for assays = 0.1-10 mM) to wells containing PDHC and α-ketoglutarate (working stock concentration ranging from 1-100 mM, final concentration for assays = 0.1-10 mM) to wells containing KGDHC.
11. Click "READ" to start kinetic measurement.

6. Data Analysis

1. Hold down the 'CTRL' button on the keyboard and click wells to generate one graph containing all kinetic measurements corresponding to channel 1 (Figure 3A).
2. Click "data" in the view icon in the upper right corner for raw values for relative fluorescence units (RFU) measured at the different time points (Figure 3B).
3. Export the values for analysis (Table 3).
4. Click the "Graph" drop down menu on top right (Figure 3B) to access RFU values associated with the fluorescence channel 2.
5. Repeat steps 6.1 to 6.3 for channel 2.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Pyruvate dehydrogenase complex	SIGMA	P7032-10UN	purified flavoenzyme
alpha-ketoglutarate dehydrogenase complex	SIGMA	K1502-20UN	purified flavoenzyme
30% hydrogen peroxide solution	SIGMA	HX0640-5	reagent, standard curves
NAD+	SIGMA	N0632-1G	reagent, activity/ROS release assay
NADH	SIGMA	N4505-100MG	reagent, standard curves
pyruvate	SIGMA	P2256-5G	reagent, activity/ROS release assay
alpha-ketoglutarate	SIGMA	75892-25G	reagent, activity/ROS release assay
CoASH	SIGMA	C3019-25MG	reagent, activity/ROS release assay
thiamine pyrophosphate	SIGMA	C8754-1G	reagent, activity/ROS release assay
mannitol	SIGMA	M4125-100G	buffer component
Hepes	SIGMA	H3375-25G	buffer component
sucrose	SIGMA	S7903-250G	buffer component
EGTA	SIGMA	E3889-10G	buffer component
KMV	SIGMA	198978-5G	reagent, ROS release inhibitor
CPI-613	Santa Cruz	sc-482709	reagent, ROS release inhibitor
SOD	SIGMA	S9697-15KU	reagent, ROS release detection
horseradish peroxidase	SIGMA	P8375-1KU	reagent, ROS release detection
Amplex Ultra Red	Thermofisher	A36006	reagent, ROS release detection
Biotech Synergy 2 microplate reader	BioTek Instruments		microplate reader for assays
Gen5 software	BioTek Instruments		software, used for collection of raw RFU
Graphpad Prism	Graphpad software		software, data analysis
Microsoft EXCEL	Microsoft		software, data analysis