The overall goal of this procedure is to quantify the superoxide/hydrogen peroxide and NADH production rate of purified flavin-containing mitochondrial dehydrogenases using microplate fluorometry. Mitrochondria can contain up to 12 different superoxide/hydrogen peroxide forming sites which have been identified using different substrate and inhibitor combinations with isolated mitochondria. However, some difficulties still remain in regard to accurately quantifying the release capacity of individual sites of production in isolated mitochondria.
And this is associated with unwanted side reactions that also produce reactive oxygen species, but also the presence of antioxidants and the use of inhibitors that lack specificity in addition to the use of additives that can interfere with measurements. Here we present the method for the simultaneous measure of superoxide/hydrogen peroxide and NADH production by purified flavin-dependent mitochondrial dehydrogenases. This method is advantageous since it can allow for the direct and accurate quantification of the ROS-producing potential of individual sites of production by eliminating unwanted side reactions and any reactive oxygen species quenching systems.
The simultaneous measure of both ROS and NADH production also allows for direct comparison of the superoxide/hydrogen peroxide forming potential and activity of individual enzymes which can be valuable for screening inhibitors for ROS production prior to conducting assays with mitochondria. For our purposes here, we will be using pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex of porcine heart origin as examples. Both are flavin-containing enzymes and known sources for reactive oxygen species.
In addition, these two enzymes can be easily purified. The simultaneous measurement of superoxide/hydrogen peroxide and NADH is achieved by tracking changes in florescence. NADH has its own intrinsic florescence properties which allows for easy measurement of its production.
Changes in superoxide/hydrogen peroxide meanwhile requires superoxide dismutatase, horseradish peroxidase and amplex ultrared reagent. In the presence of horseradish peroxidase and hydrogen peroxide, the amplex ultrared is converted from a non-fluorescent molecule to fluorescent resorufin. Superoxide dismutase ensures that any superoxide formed by the dehydrogenase being studied is converted to hydrogen peroxide.
Prior to setting up the assay, working solutions are prepared. To streamline the assay, all reagents are added to the wells of a 96-well black plate in 20-microliter aliquots. The volume for each reaction in the well is 200 microliters, therefore working solutions are 10 times the concentration found in the reaction well.
Next, program the microplate reader to run a kinetic assay for the dual measurement of NADH autoflorescence and resorufin florescence. This is done by first selecting Procedure and setting it to 25 degrees Celsius. Next, click Start Kinetic.
Set the time to run for approximately five minutes followed by selection of a fluorescent read interval of 30 seconds. Next, click Read and set the fluorescent measurement parameters for your assay. Here the position of the probe, the wavelengths used for tracking changes in florescence and the wells that will be read are inputed into the software.
The different channels for the fluorescent reads are also selected with one being dedicated to the autoflorescence of NADH, which is set to 376 nanometers excitation and 450 nanometer emission wavelengths. Changes in resorufin florescence are tracked at 565 nanometers and 600 nanometers. Once the assay conditions have been set up, individual wells are loaded with buffer and different reagents required to measure both NADH and superoxide/hydrogen peroxide production at the same time.
The order for loading the wells with different reagents is as follows. First, the requisite amount of buffer is added to the well followed by the addition of 20 microliters of purified enzyme solution. The enzyme is equilibrated in the buffer for a few minutes inside the instrument.
And then, the remaining reagents are added in the following order. Once the substrate has been added, press Start. The plate carrier will enter the reader and the protocol will be initiated.
During the assay, realtime outputs for raw relative fluorescent units, or RFUs, can be tracked using the software. The user can flip between the different fluorescent reads by using the Data tab above the well layout. Note that curves for the different fluorescent reads can be selected.
In addition, by holding Control on the keyboard and left-clicking on the wells, one can yield a composite graph allowing for the realtime comparisons of the different reactions. Upon completion of the experiment, the microplate carrier will exit the instrument. At this point, results in the representative graph for changes in RFU are exported to Excel.
Raw RFU values are exported by first clicking on Data on the representative RFU graph. This will yield a table of raw RFU values for NADH and resorufin florescence. Not that raw RFU numbers corresponding to changes in NADH or resorufin florescence cannot be exported simultaneously.
Once exported, results are organized for analysis. Using standard curves for NADH and resorufin florescence generated prior to conducting the assay, the amount of NADH in micromole and superoxide/hydrogen peroxide in picomole formed over time can be calculated. This can be followed by calculating the rate of NADH and superoxide/hydrogen peroxide production.
For this procedure, it is vital to check the purity of your isolated flavin-containing dehydrogenase prior to conducting assays. This can be achieved by conducting a simple coomassie stain for proteins or by assaying the activity of the enzyme in the presence of different substrates. Note that it is advised that reducing agents like DTT should be omitted from the assay since it can serve as a substrate for superoxide/hydrogen peroxide production by flavin-linked dehydrogenases.
Overall, this method is highly flexible in that it can be used to screen inhibitors for superoxide/hydrogen peroxide production and also allow for a direct comparison of the ROS-forming capacity of different flavin-linked mitochondrial dehydrogenases. After watching this video, you should have an intimate understanding of how to measure ROS and NADH production by purified dehydrogenases. This assay can serve as a stepping stone for more sophisticated experiments with isolated mitochondria.
