Succinate dehydrogenase or SDH is a vital component in the tricarboxylic acid cycle (TCA) or Krebs cycle (1,2,3). As an important enzyme in the Kreb’s cycle, succinate dehydrogenase oxidizes succinate to fumarate (1,2). Purification of this enzyme is difficult but it has been isolated from organisms such as Bacillus subtilis, E. coli where it presents itself as one of the transmembrane proteins of these organisms (4). It has also been likewise detected and isolated from Rhodospirillum rubrum (4). However, in both mammal and animal tissues, SDH has been purified and the enzyme’s properties in both tissues were seen to be identical (5). Analysis of the structure of SDH (5,6) from those isolated in E. coli revealed that it has two large polypeptide subunits (64.3 kDa and 26 kDa) and two smaller subunits (14.2 kDa and 12.8 kDa) (4, 7). These larger subunits act as the active sites of SDH (7) while the smaller subunits were discovered to be hydrophobic and are attached to the cell membrane of the E. coli (8,9). SDH is unique due to the fact that in the Kreb’s cycle, it is the only single enzyme that is found to be bound to the mitochondria’s inner membrane (8, 9, 10). Existing as a component of complex II in the electron transport chain (10, 11) it functions as a transporter of electrons to uniquinone-10 from succinate(11).
Owing to its importance, it is necessary to be identify and follow laboratory procedures that would elucidate its presence in a mammalian tissue. For this purpose, this laboratory exercise will seek to demonstrate the presence of succinate dehydrogenase in a muscle tissue of a rat.
A muscle section was excised from a freshly frozen block of rat muscle tissue. The section was then incubated in an incubating medium and placed in a humidity chamber with a temperature of 37 degrees celsius for 10 minutes. After 10 minutes, the incubated section was rinsed well with saline solution and fixed in a 10% phosphate buffered formalin for 10 minutes. The section was counterstained with nuclear fast red, dehydrated, cleared and mounted in DPX. Meanwhile, a second section was also excised from the frozen block of rat muscle tissue and incubated in an inhibited medium. This second set-up was used as control for the experiment. After incubating the section in inhibiting medium, the section was also rinsed with saline, fixed with buffered formalin for 10 minutes, counterstained with nuclear fast red and dehydrated, cleared and mounted in DPX.
There were two reagents used in the incubating medium. 0.25 ml of 2.7% sodium succinate (reagent A) and 0.25 ml 1mg/ml Nitro Blue Tetrazolium (N.B.T.) in 0.1M phosphate buffer pH 7.6 (reagent B). In the control medium, reagents A and B were used with an addition of 0.05 ml 1% oxaloacetic acid.
In the first slide, there was a blue localization indicating sites of SDH activity in 54% of the muscle section. There was no reaction in the slide of the inhibitor. Only a light pink color was shown that indicated the absence of SDH activity.
SDH catalyzes the oxidation of succinic acid to fumaric acid (1,2,3) and the activity of this enzyme can be demonstrated histochemically (10) by incubating excised fresh frozen sections of a muscle tissue with a succinate substrate and tetrazolium compound. Hydrogen is released from colored compounds in the presence of enzymatic activities (10, 11). In this laboratory exercise, a tetrazolium was used to indicate whether enzymatic activity involving SDH has occurred in the muscle tissue of rats. Nitro blue served as the substrate and when hydrogen was released during enzymatic activity, this was transferred to tetrazolium (12, 13). As a result, tetrazolium was converted to formazan (11), revealing a deep blue pigment and showing the sites where enzymatic activity was present. The active area also revealed the sites where mitochondria are present in the examined muscle tissue since SDH are integrated in the inner membrane of the mitochondria. The presence of sodium succinate resulted to the injury of the muscle cells and resulted to the increased rate of production of formazan (12).
Revealing the presence of SDH in a muscle tissue indicates that these tissues have abundant mitochondria (13) compared to other tissues that do not use up as much energy. Meanwhile, quantification of SDH is also vital in determining whether there is an overproduction of superoxides that will lead to formation of tumors in humans. As Rustin, P and his colleagues expressed, “SDH plays a specific role in the handling of oxygen in mitochondria” (14). This group of researchers explained that SDH, when it is unable to perform its function in reducing the pool of ubiquinones, can lead to condition where cells and tissues are unable to resist the stress caused by oxygen toxicity. As presented in their research paper (14), SDH not only plays an important role in the Kreb’s cycle and electron transport chain but also serves as an antioxidant enzyme. On the other hand, SDH can also be used in the succinate dehydrogenase inhibition (SDI) test for predicting the heat sensitivity of tumor tissues (14). SDI test will show whether malignant or tumor tissues can be effectively treated with hyperthermia (14, 15).
This experiment is integral in understanding the presence and role of SDH in tissues of animals and humans. In tissues that are highly active, SDH would be present in higher quantities as compared to tissues that are less active and need less energy to carry out their functions. Generally, quantifying SDH would also reveal if there is an abnormality in its production and abundance. For example, in tumor lesions, when SDH is unable to reduce the level of ubiquinones normally, it may lead to inability of tissues and cells to handle oxidative stress. In malignant tissues, the SDI test is also used as a reliable tool to indicate whether these lesions will respond to hyperthermia- one of the important therapies used to treat or contain malignant tissues.