BIOENERGETICS Problems to be prepared: 1. Methods of enzymes activity assessment, the role of artificial electron acceptors and donors. 2. Reactions catalyzed by malate dehydrogenase, succinate dehydrogenase, cytochrome oxidase, lactate dehydrogenase and cellular localization of these enzymes. 3. Processes supplied cell in energy. 1. Detection of succinate dehydrogenase activity in liver homogenate using artificial electron acceptors. Reaction catalyzed by succinate dehydrogenase: CH 2 CH + FAD + FADH 2 CH 2 HC Succinate dehydrogenase is a flavoprotein catalyzing reversible conversion (oxidation) of succinate to fumarate (see figure above). It is the only enzyme of Krebs cycle bound to the inner mitochondrial membrane. It is also the component of protein complex named succinate-ubichinon reductase, the element of electron transport chain. Using this complex, electrons and protons from FADH 2 linked with succinate dehydrogenase are transferred onto respiratory chain. colored (blue) oxidized form colorless reduced form Ryc.1. Reaction of methylene blue reduction. Principle of the method Two artificial electron acceptors are used for detection of succinate dehydrogenase activity: methylene blue and dichlorophenoloindophenol (DCPI). Both are colored in oxidized form and colorless in reduced state. Standard red-ox potential for methylene blue is +0.01 V, and for DCPI is +0.22 V. Both red-ox potentials are higher than red-ox potential of FAD/FADH 2, which is -0.06 V. Therefore, protons from FADH 2 can be transferred to either methylene blue or DCPI. The gradual discoloration, which is the effect of methylene blue or DCPI reduction, is the evidence for proceeding the reaction catalyzed by succinate dehydrogenase. Since the reduced form of methylene blue may also be oxidized by molecular oxygen, the in tubes should be covered with a 1
liquid paraffin. Two competitive inhibitors of succinate dehydrogenase are used in the experiment: malonate and pyrophosphate. Malonate has a similar structure to that of succinate, and is the most known competitive inhibitor of the tested enzyme. The less known is fact that pyrophosphate, in spite of its different chemical structure, is a competitive inhibitor too. a) methylene blue as an acceptor Materials and chemicals: 1. 0.01 mol/dm 3 TRIS-HCl 2. 1 mol/dm 3 sodium succinate 3. 0.02 % methylene blue 4. 0.5 mol/dm 3 sodium malonate 5. 5% liver homogenate 6. 5% boiled liver homogenate Add chemicals to five tubes according to table 1. Table 1. 1 2 3 4 5 0.01 mol/dm 3 TRIS-HCl 1 1 1 1 1 1 mol/dm 3 sodium 0.2 0.2 0.2 0.2 succinate 0.02 % methylene blue 0.2 0.2 0.2 0.2 0.2 0.5 mol/dm 3 sodium 0.2 malonate H 2 O 0.4 0.6 0.2 0.4 0.6 5% liver homogenate 0.2 0.2 0.2 0.2 + + boiled liver homogenate After addition of homogenate, shake tubes intensively and immediately put one cm layer of liquid paraffin on the top of the tubes content (after covering with paraffin do not shake tubes!) Incubate tubes at 37 C for 1.5 h. Observe color changes in all tubes after 45 min and 1.5 h of incubation. b) DCPI as an acceptor Materials and chemicals: 1. 0.01 mol/dm 3 TRIS-HCl 2. 1 mol/dm 3 sodium succinate 3. 0.008% DCPI 4. 0.5 mol/dm 3 sodium malonate 5. 5% sodium pyrophosphate 6. 0.1 mol/dm 3 KCN 7. 1% liver homogenate 2
DCPI colored (blue) oxidized form DCPI colorless reduced form Ryc.2. Reaction of DCPI reduction Add chemicals to seven tubes according to table 2. Table 2. 1 2 3 4 5 6 7 0.01 mol/dm 3 TRIS-HCl 1 1 1 1 1 1 1 1 mol/dm 3 sodium 0.2 0.2 0.2 0.2 0.2 0.2 succinate 0.008% DCPI 0.6 0.6 0.6 0.6 0.6 0.6 0.6 5% sodium pyrophosphate 0.5 0.5 mol/dm 3 sodium 0.5 malonate 0.1 mol/dm 3 KCN 0.2 H 2 O 0.5 0.7 0.3 0.5 0.7 1% liver homogenate 0.2 0.2 0.2 0.2 0.2 0.2 + + boiled liver homogenate After addition of homogenate shake tubes and incubate mixtures at room temperature for 60 min. Observe color changes in all tubes after 30 and 60 min. 2. Detection of cytochrome oxidase activity in heart homogenate Cytochrome oxidase is IV protein complex of electron transport chain. It is a hemoprotein consisted of ten polypeptide chains and comprises two cytochromes in the structure: a and a 3. Each cytochrome contains Cu + and Fe 2+ ions. Cytochrome oxidase is the last component of electron transport chain and catalyzes cytochrom c oxidation with direct reduction of oxygen molecule. By the action of cytochrome oxidase, O 2 is reduced at the cost of 2Fe 2+ and 2Cu + oxidation, following by formation of 2 molecules of H 2 O. 3
Reaction catalyzed by cytochrome oxidase: 4 cytochromes c(fe 2+ ) (reduced) + O 2 + 4H + 4 cytochromes c(fe 3+ ) (oxidized) + 2H 2 O Principle of the method P-phenyldiamine is used as an artificial electrons donor. Red-ox potential of p- phenyldiamine is slightly lower than cytochrome c. Thus, when cytochrome oxidase is active and total amount of cytochrom c is sufficient, p-phenyldiamine becomes oxidized in the presence of oxygen. The appearance of a color in the tubes indicates the reaction progress. P-phenyldiamine does not become oxidized without presence of cytochrome oxidase. colorless reduced form colored oxidized form Ryc.3. Reaction of p-phenyldiamine oxidation. Materials and chemicals 1. 0.01 mol/dm 3 Tris-HCl 2. 0.01% cytochrome c 3. 0.1 mol/dm 3 KCN 4. 1% p-phenyldiamine 5. 5% heart homogenate 6. 5% boiled heart homogenate Add chemicals to six tubes according to table 3. Table 3. 1 2 3 4 5 6 0.01 mol/dm 3 Tris-HCl 1 1 1 1 1 1 0.01% cytochrome c 0.1 0.1 0.1 0.1 0.1 mol/dm 3 KCN 3 drops H 2 O 1.9 0.9 0.6 0.6 0.5 0.7 1% p-phenyldiamine 0.5 0.5 0.5 0.5 0.5 0.5 5% heart homogenate 0.3 + 0.3 0.3 0.3 + boiled heart homogenate 4
After addition of all chemicals, shake the tubes content and observe color changes over time. Interpret results. For most of micromolecular metabolites the inner mitochondrial membrane has a very limited permeability, which is conditioned by the presence of specific transporters like ADP-ATP translocase, phosphate translocase etc. Outer mitochondrial membrane acts as molecular bolter (riddle) and allows for passing of compounds with molecular weight not exceeding 10 000 daltons. Thus, exogenous cytochrome c (with molecular weight of 12 000 daltons) does not pass through intact outer membrane. If a part of mitochondria is damaged by too intensive homogenization, exogenous cytochrome c can react with cytochrome oxidase, which is located in inner mitochondrial membrane next to the outer surface. The increase of cytochrome c concentration leads to the stimulation of cytochrome oxidase activity. If a very small amount of mitochondria from heart homogenate is damaged during homogenization, s in tubes 4 and 6 have a similar color. 3. Localization of succinate dehydrogenase, lactate dehydrogenase and cytochrome oxidase in subcellular fractions of the liver. Reaction catalyzed by lactate dehydrogenase (LDH) H C OH + NAD + C = O + NADH + H + CH 3 CH 3 Succinate dehydrogenase and cytochrome oxidase are found exclusively in mitochondria, whereas lactate dehydrogenase is a cytoplasmatic enzyme, participating in glycolysis. LDH catalyzes reversible reaction of oxidation of lactate to pyruvate. In highly aerobic tissues (oxygenated) e.g. in the liver and heart muscle, lactate absorbed from blood is oxidized to pyruvate, what enables its utilization in gluconeogenesis (in liver) or as energetic fuel (in heart muscle). In the cells with anaerobic metabolism (red blood cell, intensively working skeletal muscle cells) reaction catalyzed by lactate dehydrogenase makes glycolysis possible to proceed under anaerobic conditions. Reduction of pyruvate by LDH enables oxidation of NADH + H + when electron transfer chain cannot run. Thus glycolysis, in contrast to other metabolic pathways, can run and generate ATP by substrate level phosphorylation. LDH belongs to a group of indicatory enzymes. Estimation of its activity and detection of isoenzymes presence in blood serum is used in diagnosis of liver and heart diseases. Materials and chemicals 1. 0.01 mol/dm 3 Tris-HCl 2. 0.01 % DCPI 3. 1 mol/dm 3 sodium succinate 4. 1 mol/dm 3 sodium lactate 5. 0.01% cytochrome c 6. 1% p-phenyldiamine 7. 0.5 mmol/dm 3 NAD + (in Eppendorf tubes) 8. 0.5 mol/dm 3 sodium malonate 9. 5% mitochondria suspension 10. 5% cytosolic fraction 5
Add chemicals to nine tubes according to table 4. Table 4. 1 2 3 4 5 6 7 8 9 0.01 mol/dm 3 Tris-HCl 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.01 % DCPI 0.6 0.6 0.6 0.6 0.6 0.6 1 mol/dm 3 sodium lactate 0.3 0.3 0.3 1 mol/dm 3 sodium succinate 0.2 0.2 0.2 0.01% cytochrome c 0.1 0.1 0.1 1% p-phenyldiamine 0.3 0.3 0.3 0.5 mmol/dm 3 NAD + 0.2 0.2 0.2 H 2 O 1.0 1.0 0.5 0.8 0.5 0.5 0.9 0.9 1.1 0.5 mol/dm 3 sodium malonate 0.5 5% mitochondria suspension 0.2 0.2 0.1 0.2 5% cytosolic fraction 0.2 0.4 0.4 + 0.2 + boiled cytosolic fraction Shake the tubes content and observe color changes over time. The positive result in tube 2 may be due to partial degradation of mitochondria during separation and passing the membrane fragments to postmitochondrial supernatant. The positive result in tube 5 may indicate that cytosolic lactate dehydrogenase was absorbed on mitochondrial suspension. 5. Detection of malate dehydrogenase in cytosolic fraction. Effect of ph. Malate dehydrogenase is found in cytosol and mitochondria. In both cellular compartments this enzyme catalyzes the same reaction, but plays different roles. Reaction catalyzed by malate dehydrogenase CHOH CH + NAD + + NADH + H + CH 2 CH 2 In cytosol, malate dehydrogenase participates in transport of hydrogen atoms from NADH + H + of cytosolic dehydrogenases onto respiratory chain (malate/aspartate shuttle). In mitochondria, malate dehydrogenase is one of Krebs cycle enzymes. 6
Reaction catalyzed by malate dehydrogenase is strongly endoergic with very low equilibrium constant (K=10-12 ). Equilibrium of this reaction is markedly moved in the direction of malate formation, thus it is necessary to simultaneously remove the reaction products i.e. oxaloacetate and NADH + H +. In vivo in mitochondria oxaloacetate participates in Krebs cycle and NADH is oxidized, transferring protons and electrons onto ETC. In order to show in vitro that oxaloacetate is formed in this reaction, ph must be increased from 7.0 to 10.0. Under the alkaline conditions, protons (from NADH + H + ) are removed according to the reaction: H + + OH H 2 O and the equilibrium of this reaction shifts into right. Principle of the method Oxaloacetate (produced by malate dehydrogenase) reacts with 2,4- dinitrophenylhydrazine and forms oxaloacetate 2,4-dinitrophenylhydrazone, which is colored under the alkaline conditions. Materials and chemicals 1. 0.5 mol/dm 3 glycine buffer, ph= 10.0 2. 0.02 mol/dm 3 Tris-HCl buffer, ph =8.5 3. 0.02 mol/dm 3 Tris-HCl 4. 0.5 mol/dm 3 sodium malate 5. 0.5 mmol/dm 3 NAD + (in Eppendorf tubes) 6. 0.02 % 2,4-dinitrophenylhydrazine 7. 1.5 mol/dm 3 NaOH 8. 5% cytosolic fraction Add chemicals to four tubes according to table 5. Table 5. 1 2 3 4 0.5 mol/dm 3 glycine buffer, ph= 10.0 0.2 0.2 0.02 mol/dm 3 Tris-HCl buffer, ph= 8.5 0.2 0.02 mol/dm 3 Tris-HCl 0.2 0.5 mmol/dm 3 NAD + 0.1 0.1 0.1 0.1 0.5 mol/dm 3 sodium malate 0.1 0.1 0.1 H 2 O 0.1 5% cytosolic fraction 0.4 0.4 0.4 0.4 After 30 minutes of incubation at 37 C add 0.6 cm 3 of 0.02% 2,4-dinitrophenylhydrazine to all tubes, and next 2 cm 3 of 1.5 mol/l NaOH. After addition of all chemicals, shake the tubes content and observe color changes over time. Interpret results. 7