An enzyme assay to determine the rate of succinate dehydrogenase (complex II) activity in Brassica oleracea and the effects of malonate on complex II indirectly using dichlorophenol indophenol
Abstract
In this study, the rate of succinate dehydrogenase activity in the mitochondria of Brassica oleracea was assessed using an enzyme assay. Isolated mitochondria and mitochondria-free fractions were collected using differential centrifugation using isolation buffer (300 mM D-mannitol, 5.88mM KH2PO4, 13.8 mM K2HPO4) at 600 relative centrifugal force (RCF) for 10 minutes, then at 8500 RCF for 45 minutes while the temperature was maintained at 4°C. The mitochondrial fraction was collected from the final pellet, and the mitochondria-free fraction from collected from the supernatant. The electron transport chain was inhibited by the addition of sodium azide to isolate complex II, as it inactivates the antioxidant systems that prevent the accumulation of reactive oxygen species. The addition of malonate, a competitive inhibitor of succinate dehydrogenase (SDH) was used to assess its effect on SDH and mitochondrial membrane potential. The reduction of succinate to fumarate was quantified indirectly by the spectrophotometric measurement of DCIP concentration, an artificial electron acceptor. It found was found the addition of malonate to an isolated mitochondrial fraction caused a maximum decrease in the rate of DCIP concentration by 0.22 µM/minute, this decrease in DCIP concentration was the greatest rate observed in this study. The mitochondrial fraction had a similar yet less severe decrease in DCIP concentration with maximum rate of 0.13 µM/minute. The sharp decrease in DCIP concentration observed in the mitochondria fraction with malonate was concluded to be caused by a mitochondrial membrane potential collapse induced by the depolarization of the mitochondrial electric potential.
Introduction
In all organisms, the mitochondrion is responsible for the production of adenosine triphosphate (ATP) to power other cellular processes. The mitochondrion carries out numerous metabolic processes such as intermediate metabolism, and steroid metabolism (Fernandez-Gomez et al., 2004). The synthesis of ATP occurs in the mitochondria through cellular respiration from the catabolism of macromolecules; the inner and outer membranes, and intermembrane space of mitochondria are used to carry out this process. Cellular respiration is divided into glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. Glycolysis is the breakdown of glucose, a six-carbon sugar, into two pyruvate molecules, a three-carbon sugar, for every one glucose molecule, and two molecules of ATP are also produced. The products of glycolysis are utilized in the TCA cycle. The TCA cycle and oxidative phosphorylation are both aerobic and require enzymes to catalyze the oxidation-reduction reactions present in both processes. Together, the TCA cycle and oxidative phosphorylation produce 36 ATP molecules from a single glucose molecule. There are four enzymatic complexes in oxidative phosphorylation, the complex of interest to this study is complex II, succinate dehydrogenase (SDH), SDH is located on the inner mitochondrial membrane. SDH catalyzes the oxidation of succinate to fumarate in the matrix and the reduction of ubiquinone to ubiquinol in the inner membrane to release electrons that are captured by the electron acceptor, flavine adenine dinucleotide (FAD) to reduce it to FADH2 (Jones and Hirst, 2013). The protons and electrons released by the oxidation of succinate are transferred to ubiquinone to form ubiquinol, this transfer of electrons links the TCA cycles and ETC. Complex II does not pump protons into the intermembrane space of the mitochondria, and the positioning of SDH is localized to the inner mitochondrial membrane. The remaining electrons from oxidative phosphorylation are transferred from the ETC to oxygen (O2), the terminal electron acceptor.
The transfer of electrons to oxygen in aerobic respiration can be experimentally blocked by the addition of sodium azide, a powerful poison, by blocking the transfer of electrons in the ETC, it allows them to be taken up by another electron acceptor. Sodium azide functions by inhibiting the antioxidant enzymes that prevent the accumulation of reactive oxygen species (ROS) produced at the FAD-binding site, these ROS act as signalling molecules that regulate plant development (Jardim-Messeder et al., 2015). In this study, dichlorophenol indophenol (DCIP) was used as an artificial electron acceptor to retrieve the electrons produced by SDH. DCIP undergoes a colour change when it reduced to DCIPH2 by the acceptance of electrons from SDH, this colour change can be spectrophotometrically measured to assess the concentration of DCIP present. The use of malonate, a structural analog of succinate, can be utilized to competitively inhibit SDH activity, thus inhibiting the overall ETC (Rikhvanov et al., 2003). Malonate and coenzyme A (CoA) can be utilized to produce malonyl-CoA, an important metabolite in fatty acid synthesis, and it is suspected that malonate may be produced by the decarboxylation of oxaloacetate (Chen and Yan, 2013, Bowman and Wolfgang, 2018). Malonate is also known to cause mitochondrial potential collapse by causing the rapid depolarization of the mitochondrial electric potential and increases in the rate of ROS production (Fernandez-Gomez et al., 2004). Collapse of the mitochondrial membrane potential and increased ROS causes the transfer of electrons from the ROS to DCIP as well as the release of proapoptotic factors (Fernandez-Gomez et al., 2004). The release of proapoptotic factors leads to cell death, as the mitochondrial membrane collapse overwhelms mitochondrial antioxidant systems by the rapid increase of ROS (Fernandez-Gomez et al., 2004).
In this study, the activity of SDH in B. oleracea was assessed using the artificial electron acceptor DCIP, and the inhibition of the ETC using sodium azide to isolate SDH activity specifically. The electrons released from the oxidation of succinate were picked up by DCIP to enable the indirect measurement of SDH through the qualitative colour change of DCIP upon reduction. B. oleracea was chosen for this study because it does not contain chlorophyll, which would interact with the spectrophotometric measurements. The study was conducted using mitochondrial fractions and mitochondrial-free fractions, collected using differential centrifugation, to assess the rate of SDH activity, and the effects of malonate on SDH. Additional succinate was added to the samples to ensure the complete catalysis of succinate to fumarate by SDH. The measurement of SDH activity enables the assessment of the overall efficiency of the enzyme and the ETC at the reduction of molecules to synthesize ATP (Jones and Hirst, 2013). An understanding of the rate of production and retrieval of ROS in mitochondria enables further research into mechanisms to induce targeted cell death and the impacts of environmental stress on mitochondrial health.
Methods
A. Isolation of Mitochondria and Mitochondria-Free Fractions
The methods for the experiment were taken from the University of Victoria Biology 225 Lab Manual (Curry et al., 2018). Mitochondria and mitochondria-free fractions of B. oleracea were isolated by mechanical disruption using 25 g of freshly grated samples, 6 g of chilled sea sand and 20 mL of isolation buffer (300 mM D-mannitol, 5.88mM KH2PO4, 13.8 mM K2HPO4). Cell paste was maintained at 4°C throughout the experiment. An additional 20 mL of isolation buffer was added and the cell paste underwent further mechanical disruption. The cell paste was filtered using 4 layers of cheese cloth and 5 mL of isolation buffer was used to clean mortar used for mechanical disruption. Filtrate was centrifuged for 10 minutes at 600 relative centrifugal force (RCF) in a refrigerated centrifuge set to 4°C. Supernatant was removed and centrifuged once more for 45 minutes at 8500 RCF at 4°C. Following centrifugation, top portion of supernatant was removed as the mitochondria-free fraction (MFF). Remaining supernatant was discarded and 5 mL assay buffer (300mM D-mannitol, 5.88 mM KH2PO4, 13.8 mM K2HPO4, 10.7 mM KCl, 4.9 mM MgCl2) was added to pellet to resuspend it to form the mitochondria fraction (MF). The experiment was replicated 140 times and the average of these trials was used to calculate the rate of change in the DCIP concentration.
B. Preparation of a DCIP Standard Curve
A standard curve of 2,6-dichlorophenol indophenol (DCIP) was prepared using 6 samples of DCIP and assay buffer as varying concentrations. The six concentrations used ranged from 0.0 µM to 10.0 µM. The standard curve was produced using a NovaSpec III+ spectrophotometer set to 600 nm.
C. Determination of DCIP Concentration as a Measure of SDH Activity
Eight tubes were prepared at 22ºC according to the concentration listed below; the samples were prepared using varied concentrations of assay buffer, and a set concentration of sodium succinate, sodium azide, malonate, DCIP, and MF or MFF fractions with each present or not. The control-blank was made using 20 µM sodium succinate and 4 mM sodium azide; the control with 20 µM sodium succinate, 4 mM sodium azide, 10 mM malonate, and 5 µM DCIP; the mitochondria-free fraction (MFF) blank with 40 µM sodium succinate, 4 mM sodium azide, and 1/10 dilution of MFF; the MFF with 40 µM sodium succinate, 4 mM sodium azide, 5 µM DCIP, and 1/10 of MFF; the mitochondria fraction (MF) blank with 40 µM sodium succinate, 4 mM sodium azide, and 1/10 of MF; the MF with 40 µM sodium succinate, 4 mM sodium azide, 5 µM DCIP, and 1/10 of MF; the mitochondria fraction with malonate (MF-M) blank with 40 µM sodium succinate, 4 mM sodium azide, 10 mM malonate and 1/10 of MF fraction, 40 µM sodium succinate, 4 mM sodium azide, 5 µM DCIP, 10 mM malonate, and 1/10 of MF. Once the values were obtained, the initial concentrations of DCIP at time zero in each samples were adjusted to be equivalent to the concentration present in the control due to the inability to measure accurately at time zero.
Results
In this experiment, the concentration of DCIP in the isolated MFF, MF, and MF-M from B. oleracea were assessed at 5-minute intervals, over 20-minutes at 22°C. The results on the experiment are presented in Figure 1. In Table 1, the overall and maximum rates of change in DCIP concentration are presented. At time zero, the concentration of DCIP in the control, MFF, MF, and MF-M were adjusted to 4.26±0.12 µM. In the MFF, the concentration at five minutes declined slightly, while the concentration dropped significantly in the MF and MF-M. The concentrations of DCIP in the control and MFF remained relatively constant at the 10-minute mark. At the 15-minute mark, a small decrease in concentration was seen in the control and MFF. The concentration of DCIP in the MF and MF-M fractions at 15-minutes were once again significantly lower than earlier readings. Overall, the concentration of DCIP in the control and MFF decreased slightly over the 20-minute period with an overall rate of 0.01 µM/minute, and 0.015 µM/minute, respectively. The maximum rates of change in the control and MFF were slightly more than the overall rates of change of with values of 0.01 µM/minute, and 0.04 µM/minute, respectively. The concentration in the MF and MF-M decreased significantly to almost half of the starting concentration over the 20-minute time interval. The concentration of DCIP was reduced most significantly and rapidly in the MF-M with a maximum rate of 0.22 µM/minute between zero and five minutes and an overall rate of 0.089 µM/minute respectively. The MF maximum and overall rates DCIP concentration changes were 0.13 µM/minute and 0.073 µM/minute, respectively, showing the most similar reduction in concentration to the MF-M. The maximum rates of decrease in DCIP concentration in the MF and MF-M occurred between time zero and five minutes, while in the control and MFF the maximum rate occurred between 15 and 20 minutes. The standard error in the control and MFF were relatively small compared to the standard error of the MF and MF-M. The MFF and control fractions showed a slight decrease which was outside of the expected concentration.
Figure 1. Concentration of DCIP in samples of isolated MFF, MF, and MF-M fractions as well as a control of B. oleracea at 22°C. DCIP concentrations were obtained from a standard curve of DCIP using the equation of the line at 5-minute intervals over 20-minutes.
Concentration of DCIP is used as an indirect measure of succinate dehydrogenase (SDH) activity in B. oleracea to determine effects of malonate and sodium azide.
Table 1. Rates of change in the concentration of DCIP in isolated mitochondria fractions from B. oleracea of MFF, MF, and MF-M. Rates of change were calculated from the values presented in Figure 1, values were collected at five minute intervals over a span of 20 minutes.
Sample
Maximum rate of change ( µM/minute )
Overall rate of change ( µM/minute)
Control
-0.012
-0.0030
MFF
-0.040
-0.015
MF
-0.13
-0.073
MF-M
-0.22
-0.089
Discussion
In this study, the enzymatic activity of SDH in B. oleracea was evaluated using the indirect measurement of DCIP concentration by qualitative spectrophotometric assay. DCIP was used as an artificial electron acceptor, which picked up electrons from the reduced succinate, as the ETC was inhibited by the addition of sodium azide. The activity of SDH was assayed in the presence of malonate, a known competitive inhibitor of SDH. Malonate is known to cause an increase in ROS production (Fernandez-Gomez et al., 2004). The objectives of this study were to analyze the rate at which SDH oxidizes succinate, and the impacts of malonate on the rate of SDH activity. The major results noted were the slight decrease in DCIP concentration in the MFF, and the sharp decreases in the MF and MF-M samples. The DCIP concentration decreased in the MFF with an overall rate of 0.015 µM/minute. This unexpected decrease may be a result of the inherent inconsistencies in differential centrifugation therefore some mitochondria may have remained in the sample. As expected, the MF samples showed a strong decrease in DCIP concentration occurred with a maximum rate of 0.13 µM/minute. Surprisingly, the MF-M samples showed the most rapid and significant decrease in DCIP concentration with a maximum rate of 0.22 µM/minute. This result may have occurred due to the mitochondrial membrane collapse caused by the addition of malonate. The addition of malonate was expected to cause little to no change in DCIP concentration because malonate is a competitive inhibitor of SDH.
Other relevant research in this field focused similarly on SDH, yet they alternatively looked at the production of ROS and the effects of malonate on mitochondrial membrane potential. In their research, Fernandez-Gomez et al. (2004) evaluated the effects of malonate as a competitive inhibitor of complex II (SDH) using a Percoll gradient to isolate mitochondria from the brain of Sprague-Dawley rats, and autofluorescence to detect NAD(P)H as a quantification of SDH activity instead of DCIP. Fernandez-Gomez et al. (2004) measured cell viability to determine the effects of malonate through quantification of lactate dehydrogenase activity. It was concluded that malonate caused a mitochondrial membrane potential collapse as shown by the release of tetramethylrhodamine ethyl ester, a membrame-permeant fluorescent probe in a concentration-dependent manner of malonate (Fernandez-Gomez et al., 2004). The results of Fernandez-Gomez et al. (2004) indicated the relation between the addition of malonate and a mitochondrial membrane collapse due to the rapid release of ROS. Our study demonstrated similar results indicated by the rapid decrease in DCIP concentration in the MF-M samples. Our results demonstrate that malonate causes a mitochondrial membrane potential collapse implied by the rapid reduction of DCIP concentration following the addition of malonate. The methods used in the study conducted by Fernandez-Gomez et al. (2004) were more far more complex and precise than the methods used to conduct our study, although the results of our study appear to be conclusive with their results. While they did use an animal model, mitochondria function is generally conserved between eukaryotic organisms, although it may have affected the ability to compare the results of our study to theirs.
In the research conducted by Jardim-Messeder et al. (2015), the production of ROS by SDH was analyzed in Arabidopsis thaliana and Oryza sativa, and the effects of malonate on ROS production were studied. ROS production was assessed using the effects of competitive and noncompetitive inhibitors of SDH. Quantification of SDH activity was also measured using DCIP and spectrophotometric measurements, although they utilized the molar absorption coefficient of reduced DCIP to calculate the rate of SDH activity rather than using a standard curve of DCIP as used in our study. The plant species utilized in their study may have affected the results due to the presence of chlorophyll in the cells, which may have caused disproportionate data dependent on which region of the plant was used. Jardim-Messeder et al. (2015) also used several competitive and noncompetitive inhibitors of SDH, including malonate to conduct their study while we only used malonate. The results of our study provide conclusive results that are in agreement with the results of Jardim-Messeder et al. (2015), due to the observed decrease in DCIP concentration in the MF samples. From their results, Jardim-Messeder et al. (2015) concluded that SDH is a site of ROS production. The implications of ROS production by SDH on the regulation of plant growth and stress responses may be significant. The results of their study indicated that the inhibition of SDH may cause some decreases in plant growth.
The results of our study may have been effected by several factors, such as the temperature at which our experiment was conducted, the concentration of mitochondria in the MFF and MF samples, and the health of the cells collected from B. oleracea. The temperature of our experiment was not carefully regulated as it was conducted on ice, thus some cellular degradation may have occurred due to a higher temperature than optimal for the B. oleracea. The concentrations of mitochondria present in the fractions may not been absolutely ideal, due to the inherent impurities of differential centrifugation, thus causing the slight decrease in DCIP concentration that occurred in the MFF samples due to some residual mitochondria in the MFF. The viability of the B. oleracea cells was not calculated before testing which have had some effect on the observed impact of malonate, as healthy cells may not have been affected as greatly by the addition of malonate due to a greater abundance of antioxidant systems such as FAD.
In this study, the activity of SDH was analyzed in varying sample conditions of MFF, MF and MF-M to assess the rate of SDH through the reduction of succinate to fumarate. The MF-M sample was used to test the effects of malonate on SDH activity as a competitive inhibitor. The results collected demonstrate that malonate causes a mitochondrial membrane potential collapse indicated by the rapid decrease in DCIP concentration from the release of ROS. The DCIP concentration in the MF-M fraction decreased overall at a rate of 0.089 µM/minute with the most drastic decrease occurring between time zero minutes and five minutes with a rate of 0.22 µM/minute. The MF samples showed a significant decrease as well with an overall and maximum rate of 0.13 µM/minute and 0.073 µM/minute, respectively, this decrease was not as rapid as the one observed in the MF-M. A slight decrease in DCIP concentration occurred in the MFF, decreasing with an overall rate of 0.015 µM/minute. The inhibitory effects of malonate on SDH could be further studied using varying concentrations to assess when it becomes toxic to the cell. The varying concentrations of malonate would allow further understanding of malonate concentration decreases SDH activity and the correlation between the two. An understanding of the prevalence of malonate in plant cells is currently lacking and could contribute to the research on regulation of plant growth and responses to environmental stress. Research into how the rate of SDH activity is determined by health of cells may be useful in determining the optimal rates of ROS production and cellular respiration in healthy cells.