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 favorab....

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 t....

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

List of materials used in this article

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