There are two kinds of succinate dehydrogenase, one takes Ubiquinone as the receptor, and the other acts on all receptors.
Succinate dehydrogenase
Common Name : succinate dehydrogenase English: Successful dehydrogenase (ubiquinone)
catalytic reaction ： Succinic acid +Ubiquinone= Fumaric acid +Panthenol (with two-way catalytic function) System name: succinic acid: ubiquinone Oxidoreductase Note: It is iron sulfur centered Flavoprotein 。 Exists in mitochondrion Medium. Succinate dehydrogenase
Common name: succinate dehydrogenase
English: Successful dehydrogenase
Catalytic reaction: succinic acid+receptor=fumaric acid+reduced receptor
System name: succinic acid: (receptor) oxidoreductase
CAS No.: 9002-02-2
Note: It is iron sulfur centered Flavoprotein Succinate dehydrogenase is linked Oxidative phosphorylation And electron transport One of the hubs of Eukaryotic cell mitochondrion And multiple Prokaryotic cell Oxygen demand and capacity respiratory chain It provides electrons, and its activity can generally be used as an indicator to evaluate the running degree of tricarboxylic acid cycle. The enzyme uses FAD as its receptor for electron removal instead of NAD+. The relationship between succinate dehydrogenase and FAD is based on covalent bond It is linked to each other, so it is an enzyme and Cofactor Relationship. It is very special, because generally FAD and enzyme Noncovalent bond Combination of forms. Although the function of succinate dehydrogenase is specific Malonic acid (Similar to the substrate in structure) can combine with this enzyme, but the enzyme cannot catalyze it dehydrogenation Therefore, malonic acid is Succinic acid Strong of inhibitor 。 The dehydrogenation of succinate catalyzed by succinate dehydrogenase has strict stereospecificity. Succinate dehydrogenase and Citric acid cycle Other enzymes in the Mitochondrial intima The enzyme is an important part of the mitochondrial inner membrane, and other enzymes mostly exist in mitochondrion Matrix. Succinate dehydrogenase mitochondrial tricarboxylic acid cycle
Succinate dehydrogenase (SDH), Flavin Enzymes, which belong to the inner membrane of mitochondria Conjugating enzyme , a membrane binding enzyme, is linked Oxidative phosphorylation And electron transport One of the hubs of Eukaryotic cell Mitochondria and many Prokaryotic cell Oxygen demand and capacity respiratory chain It provides electrons and is a kind of mitochondria Marker enzyme 。 Succinate dehydrogenase is one of the marker enzymes reflecting the function of mitochondria. Its activity can generally be used as an indicator to evaluate the running degree of tricarboxylic acid cycle, which is useful for evaluating the function of sperm mitochondria and studying the ketopenia in cows Pathological process And so on. Extraction of succinate dehydrogenase and its physiological significance
Succinate dehydrogenase is present in all aerobic respiratory cells, and mitochondrial membrane Firm combination, yes Tricarboxylic acid cycle The enzymes that bind to the intima are dehydrogenase The most important enzyme in. So far, it has changed from Prokaryote and Eukaryote In organization Separation and purification The production of this enzyme laid a foundation for the enzymology research of this enzyme. As a membrane Endoenzyme , succinate dehydrogenase and Lipid bilayer The mitochondrial membrane of the structure is tightly bound and difficult to dissolve, and once it leaves the membrane, it will be exposed to the air quickly Inactivation Therefore, its purification is very difficult. Chinese biochemists in 1950 Wang Yinglai 、 Zou Chenglu , Wang Jingying, etc Membrane protein Succinate dehydrogenase, after repeated experiments N-butanol Extraction method Succinate dehydrogenase was successfully dissolved from the mitochondrial membrane of rat liver tissue to obtain high-purity Water solubility The activity of succinate dehydrogenase is more than twice as high as that reported abroad in the same period, thus establishing China's leading position in the field of succinate dehydrogenase research. Later, Davis et al. (1971, 1977) used NaClO4 to Ox heart mitochondrion Dissolve the enzyme; Tsukaho Hattori and Tadashi Asahi (1982) choose ionic type Detergent Sodium deoxycholate SDH was extracted from mitochondria of sweet potato root; Suraveratum et al. (2000) Plasmodium falciparum Succinate dehydrogenase purification ； Xia Yufeng et al. (2000) took maize etiolated seedlings as biological materials , via Differential centrifugation Get mitochondria, use ultrasonic wave to crush them, use 2% TritonX-100 Dissolving membrane, Ultracentrifugation ， Ammonium sulfate precipitation , DEAE-C32 chromatography was used to purify succinate dehydrogenase; Xin Mingxiu et al. (2004) Enzymology method From psychrophilic yeast (Y18) Separation and purification Not available catalytic activity Succinate dehydrogenase. Succinate dehydrogenase is directly linked to Electron transfer chain The hydrogen receptor is FAD instead of NAD +, which makes Succinic acid Oxidation to Fumaric acid Produced in the process FADH2 Combined with the enzyme, two electrons from FADH2 are directly transferred to Fe3+of the enzyme. Existing research shows that succinate dehydrogenase is composed of α、β Two Subunit form, α Subunit relative molecular mass 70.0 × 103, containing FAD and two iron sulfur clusters; β The subunit has a relative molecular weight of 27.0 × 103 and contains an iron sulfur cluster. Succinate dehydrogenase Vitality assay There are five main methods. Methyl sulfate Phenazine (PMS) reaction method Succinate dehydrogenase can pass through a series of artificial electron acceptor , such as PMS (phenazine dimethyl ester sulfate ）， DCPIP （2，6- Dichlorophenol indophenol ）Etc Succinic acid With the help of Intermediate product Of Color change , via Spectrophotometer The detection can be reflected quantitatively, and the reaction formula is: ① Successful+PMS → Fumarate+PMSH2; ② PMSH2+DCPIP→PMS+DCPIPH2。 DCPIP is blue, standard absorption spectrum At 600nm, this color can gradually fade due to its reduction, so that optical density The activity of SDH can be calculated by measuring the reduction rate of 2.6-DPIP. Monomolecular DCPIP It is reduced, that is, it represents a molecule Succinic acid Is oxidized. Therefore, the activity of SDH can be calculated by measuring the absorbance change of this reaction system at 600nm. SDH activity calculation: (standard determination)/standard= μ mol/min/mg。 Potassium ferricyanide [K3Fe (CN) 6] reduction method with iron Potassium cyanide [K3Fe (CN) 6] and Sodium succinate As substrate, succinate dehydrogenase catalytic reaction Reduction of potassium ferricyanide to Potassium ferrocyanide [K4Fe (CN) 6], K4Fe (CN) 6 are generated by interaction with Fe3+ prussian blue The absorbance value is measured at the wavelength of 700nm to detect the production of Prussian blue, which is used as the Reducibility The higher the absorbance value, the stronger the succinate dehydrogenase activity. TTC Tetrazole ）Law
Colorless TTC (2,3,5-chlorinated Triphenyl Tetrazole ）As an artificial hydrogen acceptor, it receives hydrogen during cell respiration and is reduced to triphenyl methylate (TF). The latter is in red crystal The form of Organic solvent (such as toluene acetic acid Acetic vinegar Trichloromethane 、 acetone or ethanol Etc.). Extractant Determination of 485 nm absorbance After, expressed as TTC reduction amount dehydrogenase Activity according to standard curve The production of TF was calculated, and the activity of TTC dehydrogenase was calculated. MTT is a yellow color dyestuff 。 Living cell mitochondrion Succinate dehydrogenase can metabolize and reduce MTT Cytochrome C Under the action of Isopropanol The action particles dissolve and develop color. Under normal circumstances, the amount of formazan is proportional to the number of living cells, so it can be determined according to optical density At 570nm OD value The number of living cells was estimated. NBT is easily soluble in water, and it is light yellow. NBT is used as hydrogen acceptor Sodium succinate The hydrogen removed from the salt by the action of enzymes forms a violet blue precipitate, and PMS is added to the reaction system, Evenly mix , holding at 37 ℃ for 30min, and then adding TCA to terminate the reaction. Add isopropanol to dissolve and develop color, mix well, and then measure the OD value at 548nm. It is specified that the amount of enzyme is 1 when the OD value is 1.0 at 548nm within 30min Dynamic unit 。 Specific activity =Vitality units/mg protein. 
