CELL RESPIRATION. 3.) An enzymatic reaction of the Krebs cycle will be studied. substrates.

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1 Introduction to cell respiration laboratory This laboratory consists of three sets of experiments that illustrate the oxidation of glucose for the production of ATP. 1.) The photosynthesis vs. respiration experiment illustrates the importance of respiration to plants. 2.) Fermentation products will be tested for to demonstrate the importance of yeast in the fermentation process. 3.) An enzymatic reaction of the Krebs cycle will be studied to show how enzyme activity may be manipulated by changing substrates.

2 Introduction to comparison of photosynthesis and respiration It is well known that in the presence of light plants perform photosynthesis. It is less well understood that at the same time plants are also performing cell respiration. To demonstrate this we will determine whether CO 2 is consumed 2 or produced as Elodea is placed in either a light or dark environment. The change in CO 2 will be detected by the ph indicator phenol red. Think about the chemical equations for respiration and photosynthesis. Which one releases CO 2 causing an increase and which one uses up CO 2 which will cause a decrease? p 2

3 Introduction to comparison of photosynthesis and respiration (cont.) Phenol red is yellow under acidic conditions (high H ion concentration), pink under basic or alkaline conditions (low H ion concentration) and orange under neutral conditions. ion. A change in CO 2 will cause a directly yproportional p change in H If the CO 2 concentration decreases, the H ion concentration will also decrease and the solution will change to pink. If the CO concentration increases the H ion concentration will If the CO 2 concentration increases, the H ion concentration will also increase and the solution will change to yellow.

4 Experimental procedure for comparison of photosynthesis and respiration Step 1: Fill four test tubes approximately 2/3 full with tap water and add 15 drops of phenol red. What tap water should turn pink. Is Glendora tap water acidic or basic? Step 2: Adjust the ph of the tap water to neutral, orange by gently (remember this word gently) blowing into the solution with iha straw. Repeat this procedure with ihall llfour tubes. It is important that all four tubes are the same orange color at the beginning of the experiment.

5 Experimental procedure for comparison of photosynthesis and respiration (cont.) Step 3: Place a piece of Elodea (cut end up) in two of the tubes. Each set of tubes will contain one with Elodea and a control without.

6 Experimental procedure for comparison of photosynthesis and respiration (cont.) Step 4: Place two tubes, one with Elodea and one without between the lights on the counter.

7 Introduction to fermentation CELL RESPIRATION Fermentation is a pathway for the oxidation of glucose that produces ATP in the absence of oxygen. Fermentation is similar to respiration because it begins with glycolysis. Fermentation differs from respiration because it does not involve the Krebs cycle or an electron transport tchain. Fermentation yields waste products other than CO 2 and H 2 O. In humans, fermentation yields lactic acid and in yeast fermentation yields ethyl alcohol in addition to CO 2. Since fermentation lacks a Krebs cycle and an electron transport chain, the only energy products come from glycolysis. The 2 NADH are used to produce the alcohol or lactic acid. The total amount of ATP produced from one molecule of glucose is 2.

8 Experimental procedure for fermentation Tests performed by the Instructor: Test for carbon dioxide (CO 2 )production: The instructor will demonstrate the reaction between CO 2 and barium carbonate by using a straw to breath into a tube of barium carbonate. Note the reaction when the instructor performs this activity. Students will not perform this activity because barium carbonate is toxic. The instructor will then place a tube from a beaker containing yeast in a sucrose solution into a second tube of barium carbonate to test for CO 2 production by the yeast.

9 Experimental procedure for fermentation (cont.) Tests performed by the Instructor (cont.): Test for ethyl alcohol production: The instructor will assemble a distillation apparatus to collect alcohol from both flasks. The flasks will be heated to separate the alcohol from the water. The distillate will be tested for the presence of alcohol as described in the lab manual. This flask contains sucrose alone. =====> This flask contains yeast and sucrose. <=====

10 Experimental procedure for fermentation (cont.) Tests performed by the Student: Controls for alcohol production: You will prepare two tubes (Tube 1 and Tube 2) that will be used as controls for the presence of alcohol. Tube 1 will be a negative control because it will not contain alcohol. This tube will give a negative result for the presence of alcohol. Tube 2 will contain alcohol and will give a positive result for the presence of alcohol. You will use these tubes to interpret the results of the alcohol tests performed by the instructor.

11 Introduction to the Krebs Cycle and the Action of Succinic (Acid) Dehydrogenase. Succinic acid dehydrogenase is and enzyme that oxidizes succinic acid into fumaric acid as part of the Krebs cycle. During this reaction the coenzyme FAD is reduced to FADH 2. This reaction is shown as Figure 74i 7.4 in your lab manual. This reaction represents a typical enzymatic reaction that involves a substrate (succinic ii acid), an enzyme (succinic ii acid iddhd dehydrogenase), and a product (fumaric acid). We will use methylene blue as an indicator that this reaction is taking place. Methylene blue will remove the electrons from FADH 2 and turn from blue to colorless. Thus we will be able to determine the rate of the reaction by noting how long it takes the methylene blue to turn from blue to clear.

12 Introduction to the Krebs Cycle and the Action of Succinic (Acid) Dehydrogenase. We will use methylene blue as an indicator that this reaction is taking place. Methylene blue will remove the electrons from FADH 2 and turn from blue to colorless. Thus we will be able to determine the rate of the reaction by noting how long it takes the methylene blue to turn from blue to clear. The methylene blue will start out blue. ===> The methylene blue will clear as FADH 2 is produced. <====

13 Experimental procedure for The Krebs Cycle and the Action of Succinic (Acid) Dehydrogenase. Tube 1: Place approximately 2 cm of liquid hamburger into a test tube. Then add 10 drops of succinic acid and 3 drops of methylene blue to the hamburger and mix. Note the time as you mix. This is a timed experiment and diti is important tto know the time the reaction started. t Once the reagents are mixed do not mix them again (Your instructor will explain why). Not the time when the only blue remaining is a thin band at the surface of the reagents. Tube 2: Place approximately 2 cm of liquid hamburger into a test tube. Then add 10 drops of distilled water instead of succinic acid and 3 drops of methylene blue to the hamburger and mix. Note the time as before and watch for the color to change.

14 Experimental procedure for The Krebs Cycle and the Action of Succinic (Acid) Dehydrogenase (cont.). Tube 3: Place approximately 2 cm of liquid hamburger into a test tube. Then add 10 drops of malonic acid instead of succinic acid and 3 drops of methylene blue to the hamburger and mix. Note the time as bf before and watch thfor the color to change. Tube 4: Add 5 ml of water to the test tube instead of liquid hamburger. Then add 10 drops of succinic i acid and 3 drops of methylene blue to the hamburger and mix. Note the time as before and watch for the color to change. Suggestions: Rinse the mixing rod between tubes. Mix all the tubes at the same time. Do wait for tube 1 to finish reacting before starting tube 2, tube 3, or tube 4.

15 Experimental procedure for The Krebs Cycle and the Action of Succinic (Acid) Dehydrogenase (cont.). Results: Three of the four tubes should clear. The time required for each of the three tubes should be different. To determine why the times are different you should consider the only variable used in this part of fthe experiment. One of fthe tubes should not change. Can you guess which one and why? Your results should look like this picture.