The citric acid cycle occurs within mitochondria.

Lab 6 - CELLULAR RESPIRATION: THE CITRIC ACID CYCLE REACTIONS In chapter 6 (A Tour of the Cell) you learned that the function of mitochondria is to produce energy in the form of ATP for cell use. The process that converts the energy in food molecules into the energy of ATP is known as cellular respiration. Our current understanding of the respiration process suggests that there are several independent but interconnected stages involved. These include: 1. Glycolysis: food molecules (typically considered to be the sugar glucose) are converted to three-carbon molecules called pyruvate. 2. An Intermediate Step: a complex reaction that essentially results in 1) the oxidation of pyruvate to generate acetate, 2) the attachment of coenzyme A to acetate to produce the molecule known as acetyl CoA, and 3) the release of CO 2 and NADH (a reduced electron carrier). Acetyl CoA is the starting point for the citric acid cycle. 3. The Citric Acid Cycle (or Krebs Cycle or TCA Cycle): a series of reactions in which the acetyl CoA is broken down one step at a time to eventually produce carbon dioxide and in the process generate reduced electron carriers (e.g. NADH and FADH 2 ). 4. The Electron Transport System (ETS): a series of oxidation-reduction reactions that use the transport of electrons to generate energy needed to phosphorylate ADP to produce ATP. Your text book (see Chapter 9) reports the location where each of the major stages of cellular respiration occurs. Glycolysis occurs in the cytoplasm of cells, while the others occur in mitochondria. Mitochondria are a double membrane-bound organelle. The interior membrane is folded into repeated structures called cristae, and it is here that the ETS is located. The citric acid cycle occurs largely within the mitochondrial matrix (the liquid region created/surrounded by the inner membrane). In this lab exercise, you will perform a study similar to that performed by scientists several years ago to explore the cellular location of the citric acid cycle. A protocol involving differential centrifugation is available for isolating mitochondria from plant tissues. Succinate dehydrogenase is an enzyme used to catalyze a reaction in the citric acid cycle. The study that you will perform will test the following hypothesis: The citric acid cycle occurs within mitochondria. Again, to test this hypothesis you will look for the presence of one of the enzymes that functions in this cycle, namely succinate dehydrogenase (SDH). The initial reaction catalyzed by succinate dehydrogenase is summarized as follows: Succinate + FAD -----> fumarate + FADH2 In vivo, the reduced form of FAD (e.g. FADH 2 ) is covalently attached to SDH, and transfers the electrons through iron sulfur centers to ubiquinone and the electron transport system where their free energy is used to generate ATP. 1

The above reaction is difficult to detect directly because succinate and fumarate are not colored molecules, and only small amounts of FAD are present. However, we can indirectly measure the amount of FADH 2 produced by coupling the above reaction to a second reaction. The reduced form of the coenzyme (FADH 2 ) transfers electrons to the electron transport system to be used in the generation of ATP. The second reaction involves the use of an artificial electron acceptor, 2, 6- dichlorophenolindophenol (DCIP) to accept electrons from FADH 2. This acceptance of electrons results in a corresponding color change from blue to colorless. Thus, the activity of succinate dehydrogenase will be assayed by replacing the electron acceptor in the electron transport system with an artificial electron acceptor whose reduction can be measured spectrophotometrically. To ensure that electrons are passed to the dye and not funneled to the electron transport system, sodium azide or potassium cyanide is added. This chemical inhibits the action of cytochrome oxidase, the terminal component in the electron transport chain, and effectively bottlenecks the chain, thereby preventing the acceptance of electrons from FADH 2. Rx. 1: To summarize: Succinate dehydrogenase Succinate + FAD ----------------------------> fumarate + FADH2 Rx. 2: FADH 2 + DCIPox --------------------------> FAD + DCIPred (blue) (colorless) Again, a successful reaction can be easily monitored using a spectrophotometer and will be indicated by a decrease in units (measured as Abs 620nm ) over time. Mitochondria will be isolated from cauliflower tissue by differential centrifugation. Upon completion of this step, you will have samples of the original crude homogenate, the first pellet and second (final) pellet and supernatant. Previous studies using other marker molecules and electron microscopy indicate that the differential centrifugation steps outlined here will yield a concentration of mitochondria in the second pellet; however this will not be monitored. The class or the lab assistants will prepare a crude plant homogenate. Each group will receive a portion of this homogenate for use in preparing a mitochondrial fraction by differential centrifugation. Each group will then use this fraction to look for the presence of succinate dehydrogenase activity. The activity of an enzyme varies greatly with conditions, with the purity of the samples, and with the source of the enzyme. Therefore, any fair comparison of enzyme activity must compare all test samples to the same standard scale. The International Union of Biochemistry has set a standard for reporting enzyme activity called the Specific Activity (SA). It involves a measure of the purity of the enzyme (amount of protein that has enzyme activity versus the total amount of protein in the stock solution) and a measure of the amount of activity under specified, standard conditions called the Enzyme Unit or EU. An EU is defined as the amount of enzyme necessary to catalyze the formation of 1 micromole (1 x 10-6 moles) of product in one minute at 30 o C. Specific Activity (SA) is defined as the number of Enzyme Units per milligram of total protein in the stock solution. In your analysis, you will need to convert all your raw data to SA in order to make good comparisons between your tests. 2

The prediction for this lab will be: If the citric acid cycle occurs within the mitochondria then the fraction with mitochondria will have the highest SA values. So, if the fraction that contains the mitochondria has the greatest SDH activity it has the greatest presence of the enzyme. Why might the other fractions contain SDH if it is normally found in the mitochondrion? A. Preparation of Cauliflower Homogenate (THESE PROCEDURES WILL BE DONE FOR YOU BEFORE YOU COME TO LAB. Please familiarize yourself with the procedures you will be responsible for them on your lab exam.) Procedure: Cauliflower heads have been obtained fresh from the grocery. Selected tissue will be removed from the head and homogenized in a grinding buffer. The grinding medium is composed of 0.5 M sucrose and 0.1 M KH 2 PO 4, ph 7.3. PERFORM ALL PROCEDURES IN AN ICE BATH TO PREVENT DETERIORATION OF THE MATERIAL. 1. Shave off the top 2-3 mm of the cauliflower head with a razor blade (2-3 heads per 5 groups. 2. Weigh the sliced cauliflower. All weights and volumes in the instructions below are based on approximately 25 g of tissue. If more cauliflower tissue is used adjust the amounts accordingly. KEEP THIS AND ALL ISOLATION MATERIALS ON ICE. 3. Place the cauliflower into a chilled blender. Add 20 ml of cold grinding buffer and blend for about 10 seconds until a uniform slurry is achieved. Over blending will not only rupture cells, but may damage mitochondria. 4. Pour the slurry through four layers of cheesecloth. Collect the filtrate in cold polycarbonate centrifuge tubes. Wash the blender with 5-10 ml of cold grinding buffer and pour the wash through the cheesecloth as well. Squeeze out all of the juice into cold tubes. This slurry is the homogenate. 5. Transfer 2 ml of the crude homogenate to a clean tube and label as H. KEEP THIS AND ALL SUBSEQUENT FRACTIONS ON ICE TO PRESERVE MITOCHONDRIAL/ENZYME ACTIVITY. B. Isolation of Cauliflower Mitochondria by Differential Centrifugation (THESE PROCEDURES WILL BE DONE FOR YOU BEFORE YOU COME TO LAB. Please familiarize yourself with the procedures you will be responsible for them on your lab exam.) Procedure: 1. Using the pre-chilled, high-speed refrigerated centrifuge, centrifuge the rest of the homogenate at 500 x g for 10 minutes at 0-4 o C to remove large cellular debris. The procedure for using the high speed, refrigerated centrifuge is as follows: a. Use the SS-34 rotor and polycarbonate tubes. 3

b. Make sure that the temperature of the rotor and the centrifuge is at 0 o C. c. Remove the rotor cover by loosening the black screw and then the silver screw. d. Check for rubber stoppers at the bottom of each well to be used. e. Place the weight-balanced tubes into wells directly opposite one another. f. Replace the rotor cover by first tightening the silver screw and then the black screw. g. Close the door and hit the BRAKE button. h. Set angular velocity for the correct rpm reading. i. Set timer for the correct time. j. Set rate = 0. k. Hit START button. 2. Upon completion of this centrifugation step, you should have a visible pellet and supernatant (containing mitochondria). Carefully transfer the supernatant (S1) to a clean, cold centrifuge tube. Save for step 4. 3. Resuspend the pellet in 4 ml of cold suspending medium (NOT grinding buffer). Label this sample/cellular fraction as P1 (the first pellet material). Store fraction P1 on ice. It may be necessary to use this fraction later in the exercise. 4. Weigh balance the tube containing the supernatant (from step 2) and centrifuge at 20,000 x g for 30 minutes (0-4 o C) to pellet the mitochondria. 5. Decant the supernatant into a clean graduated centrifuge tube. It should contain ribosomes, pieces of the endoplasmic reticulum, and other small or soluble cellular components. Label this fraction S2. Save fraction S2 on ice. It may be necessary to use this fraction later in the exercise. 6. The pellet should appear cream colored. Resuspend the mitochondrial pellet in 4 ml of suspending medium. Label this fraction P2. Save fraction P2 on ice. Do not write on or place tape on the Cuvettes!!!! C. Assay for Succinate Dehydrogenase (THESE PROCEDURES ARE TO BE COMPLETED BY YOU IN LAB). Procedure: 1. Set the wavelength of the spectrophotometer to 620 nm. 2. Designate two cuvettes 1 and 2. They will be prepared as follows: 4

Hint: For tubes with DCIP and succinate, do not add DCIP or succinate until the very last minute. Cuvette 1 Cuvette 2 Phosphate buffer 1.0 ml 1.0 ml MgSO4 (0.5 M) 0.1 0.1 KCN (0.1 M) 0.2 0.2 HCl (5%, v/v) 0.1 0.1 Water 3.8 4.0 Sodium succinate (0.25 M) 0.4 0.4 P2 suspension 0.4 none DCIP none 0.2 Total volume 6.0 ml 6.0 ml Prepare Cuvette 1 first. Seal the cuvette with parafilm and then gently mix contents by inverting cuvette. Use this to blank the spectrophotometer (follow the instructions that are located on the spectrophotometer for achieving 0% absorbance). Record the absorbance at 1 minute intervals for 15 minutes. Prepare Cuvette 2. Mix contents. Place cuvette in spetrophotometer and record absorbance in the data table provided. This is your time 0 recording for Tube 2. Record the absorbance at 1 minute intervals for 15 minutes. Notice that the contents of Tube 2 are identical to that of Tube 4, with the exception that the P2 suspension has been omitted. What is the purpose of this tube? 3. In the meantime, prepare the third and fourth cuvettes as follows: Cuvette 3 Cuvette 4 Phosphate buffer 1.0 ml 1.0 ml MgSO4 (0.5 M) 0.1 0.1 KCN (0.1 M) 0.2 0.2 HCl (5%, v/v) 0.1 0.1 Water 4.0 3.6 Sodium succinate (0.25 M) 0.0 0.4 P2 suspension 0.4 0.4 DCIP 0.2 0.2 Total volume 6.0 ml 6.0 ml For cuvette 3: Mix contents. Place cuvette in spetrophotometer and record absorbance in the data table provided. This is your time 0 recording for Tube 3. Record the absorbance at 1 minute intervals for 15 minutes. Notice that the contents of Tube 3 are identical to that of Tube 4, with the exception that the sodium succinate has been omitted. What is the purpose of this tube? For cuvette 4: NOTE: work quickly in preparing and using this cuvette. If mitochondria are present and active (with working succinate dehydrogenase), the reduction of DCIP will begin immediately upon the addition of sodium succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and record the absorbance in the data table provided. This is your time 0 recording for Tube 2. Record the absorbance at 1 minute intervals for 15 minutes. 5

4. Now, prepare the fifth and sixth cuvettes as follows: Cuvette 5 Cuvette 6 Phosphate buffer 1.0 ml 1.0 ml MgSO4 (0.5 M) 0.1 0.1 KCN (0.1 M) 0.2 0.2 HCl (5%, v/v) 0.1 0.1 Water 3.8 3.6 Sodium succinate (0.25 M) 0.4 0.4 P1 suspension 0.4 0.4 DCIP none 0.2 Total volume 6.0 ml 6.0 ml Cuvette 5 is the blank for cuvette 6. Prepare this tube first and blank the spetrophotometer before preparing Cuvette 6. For cuvette 6: NOTE: work quickly in preparing and using this cuvette. If mitochondria are present and active (with working succinate dehydrogenase), the reduction of DCIP will begin immediately upon the addition of sodium succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and record the absorbance in the data table provided. This is your time 0 recording for Tube 6. Record the absorbance at 1 minute intervals for 15 minutes. 5. Finally, prepare the fifth and sixth cuvettes as follows: Cuvette 7 Cuvette 8 Phosphate buffer 1.0 ml 1.0 ml MgSO4 (0.5 M) 0.1 0.1 KCN (0.1 M) 0.2 0.2 HCl (5%, v/v) 0.1 0.1 Water 3.8 3.6 Sodium succinate (0.25 M) 0.4 0.4 S2 suspension 0.4 0.4 DCIP none 0.2 Total volume 6.0 ml 6.0 ml Cuvette 7 is the blank for cuvette 8. Prepare this tube first and blank the spetrophotometer before preparing Cuvette 8. For cuvette 8: NOTE: work quickly in preparing and using this cuvette. If mitochondria are present and active (with working succinate dehydrogenase), the reduction of DCIP will begin immediately upon the addition of sodium succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and record the absorbance in the data table provided. This is your time 0 recording for Tube 8. Record the absorbance at 1 minute intervals for 15 minutes. 6

Title: Date: Time (min) Cuvette 1 (no DCIP) Cuvette 2 (no P2) Cuvette 3 (no succinate) Cuvette 4 (P2) Cuvette 5 (P1) Cuvette 6 (S2) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Avg. change in /min A. Determine the slope for the linear range of each plot. This is the average change in /min or an estimate of reaction rate. These will be used in calculating SA. B. Prepare a graph of and time (x-axis) for all fractions and solutions tested. All plots should be placed on one graph. 7

C. Standardization Procedures: Enzyme Units and Specific Activity Enzyme Units (How much enzyme activity was needed to completely catalyze each tube of DCIP?) The amount of AP'ase enzyme needed to convert 2 moles of PNPP into two moles of PNP should be twice as much as the amount of AP'ase needed to convert 1 mole of PNPP into 1 mole of PNP. This will be true PROVIDED ALL OTHER PHYSICAL CONDITIONS AND THE TIME ARE CONSTANT. Therefore, it is always necessary to control the conditions under which a reaction is taking place. In order to ensure uniformity in reporting results, the International Union of Biochemistry has set up a standard based on the Enzyme Unit (EU). AN EU IS DEFINED AS THE AMOUNT OF ENZYME NECESSARY TO CATALYZE THE FORMATION OF 1 MICROMOLE (1 X 10-6 MOLES) OF PRUCT IN ONE MINUTE AT 30 o C. Consider the following steps for calculating EU: 1. The absorbency of one mole of DCIP under standard conditions has been reported as 19.1 units. If this is accurate, then the absorbency of one micromole of PNP under standard conditions corresponds with a change of units. 2. Since 1 EU of enzyme catalyzes one micromole of DCIP under standard conditions, then 1 EU must also correspond with a change of units (this is a very small number, which will result in a large number in Step 5). 3. The average increase per minute is calculated as: at 15 minutes - at 0 minutes 15 minutes 4. The total dilution of the enzyme is determined as follows: DILUTION FACTOR FROM ADDING SUBSTRATE (Step 7 of spec ) 5. EU can now be calculated as: Average /minute (from Step 3) x Total Dilution = micromoles catalyzed DCIP = EU /micromoles DCIP (from Step 4) minute (from Step 2) Specific Activity (How much enzyme activity is in each mg of enzyme solution?) Once an enzyme is extracted from a specific tissue or cell type, the complete separation of that enzyme from all other proteins is a difficult and often unnecessary task. Usually the total amount of protein in a cell extract is much greater than the amount of enzyme present. Since the relative purity of an enzyme extract is often critical to researchers, the International Union of 8

Biochemistry has established a measurement of purity called the Specific Activity (SA). THE SA IS DEFINED AS THE NUMBER OF ENZYME UNITS (EU) PER MILLIGRAM OF TOTAL PROTEIN IN THE SAMPLE. In order to calculate SA, the concentration of total protein (mg/ml) in the original fraction must be determined. In Laboratory 3, the method for determining total protein was introduced -- the Biuret test. Use the protocol for the Biuret test in Laboratory #3 and the Standard Curve of Protein Concentration to determine the concentration of total protein in the solution. TUBE 1: 4.5 ml of Biuret reagent + 0.5 ml distilled water TUBE 2: 4.5 ml of Biuret reagent + 0.5 ml of P1 TUBE 3: 4.5 ml of Biuret reagent + 0.5 ml of P2 TUBE 4: 4.5 ml of Biuret reagent + 0.5 ml of S2 Use the directions from Lab #3 to complete this test. What is the dilution factor for the protein at this step? Fraction Reading Protein Concentration from Graph Concentration corrected for dilution (multiply by df) P1 P2 S2 D. Calculation of Enzyme Units and Specific Activities for This Data Fraction Average /minute /micromoles DCIP Total Dilution EU SA = EU/ total protein concentration (from above) P1 P2 S2 9