LAB 6: Cellular Respiration

Transcription

1 LAB 6: Cellular Respiration Introduction Cellular respiration is the process of the controlled, biological combustion of glucose, (CH 2 O) 6 + O 2, the energy produced being used to add 36 phosphate molecules onto 36 ADPs (adenosine diphosphate) making 36 ATPs (adenosine triphosphate), a process called phosphorylation. Carbon dioxide, CO 2, and water, H 2 O, are given off as waste products. ATP is the high energy (low stability) molecule responsible for powering most cellular processes in all cells, including those of plants and animals. Note that this is the exact reverse of photosynthesis (see Lab 7). These two processes are mutually dependent on one another; without one, the other could not exist. The biochemical reactions making up cellular respiration are grouped into three distinct processes: glycolysis, which occurs mostly in the cytoplasm (some glycolysis enzymes are associated with the smooth endoplasmic reticulum), the Kreb s cycle (also called the citric acid or tricarboxylic acid cycle), which occurs in the matrix (juice) of mitochondria, and the electron transport chain, which occurs through integral proteins and enzymes set in and on the membrane of the inner membrane folds of the mitochondria called the cristae. The quantitative balanced chemical equation for respiration is: (CH 2 O) 6 + 6O 2 6CO 2 + 6H 2 O + 36 ATP net Note that the two gases involved in respiration are oxygen and carbon dioxide. If you could measure either the amount of carbon dioxide produced or the amount of oxygen consumed, you d have a way to directly measure respiration rate. If respiration is carried out in an enclosed container, and if all carbon dioxide is removed from the container as it is being produced, then the rate of respiration would be directly related to the amount of oxygen consumed, in other words, to the change in gas pressure. Potassium hydroxide, KOH, chemically combines with carbon dioxide, producing solid potassium carbonate, K 2 CO 3, thereby removing the carbon dioxide from the atmosphere in a closed system: CO 2 + 2KOH K 2 CO 3 + H 2 O Thus, if you soak an absorbent cotton ball in KOH and place it in the bottom of an closed container, the KOH will remove the CO 2 from the system, leaving only O 2 (and other atmospheric gases that are unaffected by respiration). The Respirometer The device we use to measure the changes in gas pressure is called a respirometer. Respirometers come in many shapes and sizes, but they all work by having a closed container in which the experimental biological system (organism, tissue, cell culture, or organelles) is placed, with a calibrated tube or pipette to measure the change in gas volume: 6.1

2 absorbant cotton soaked with KOH non-absorbant cotton germinating peas Figure 6.1. A respirometer. lead ring (weight) calibrated pipette water bath open end Respirometers are often placed in water baths as temperature affects the rate of respiration and water baths allow us to control the temperature of the system. This also allows for a convenient way to measure decreases in gas pressure. Initially, positive pressure within the respirometer keeps water from the water bath from entering the open end of the calibrated pipette (see Fig. 6.1). But as oxygen is consumed, and with a cotton ball of KOH absorbing any carbon dioxide produced, the gas pressure inside the test tube decreases, allowing a proportional amount of water to enter the calibrated pipette. Respirometers placed in water baths must be weighted so that they remain submerged. Respiration Rates and Temperature Considerations Rate is the occurrence of some phenomenon over a given amount of time, for instance, the kilometers traveled per hour or the number of hot dogs eaten per five minutes (if you re into that sort of thing!). A convenient measurement for respiration is mmolo 2 min -1, which is read, millimoles of oxygen consumed per minute. This figure is calculated from the number of liters of oxygen consumed. In chemistry, you learned that gas volume is directly proportional to temperature: as temperature rises, so does gas volume. So, in order to accurately measure true changes in the volume of a gas, including oxygen, temperature must be taken into consideration. Recall the ideal gas law equation, PV = nrt, where P is atmospheric pressure (mm Hg), V is volume (liters), n is the number of moles of the gas, R is the ideal gas law constant (62.4 L mmhg/(mole K), and T is temperature, in K. (Remember: To convert C to K, add to C.) Since the volume of a given number of moles of a gas is different at different temperatures (more at higher temperatures, less at lower temperatures), temperature must be corrected for when determining the number of moles of oxygen consumed. So, we need to rewrite the ideal gas law equation to solve for the number of moles of gas produced: n = PV RT For example, in our closed system, how many moles of O 2 gas are actually absorbed, considering an atmospheric pressure of 755 mmhg, a measured volume of O 2 gas absorbed of 0.58 ml and a temperature of 23.4º C? (755 mmhg)(0.58 ml x 1 L/1000 ml) n = = 2.4 x 10-5 moles O 2 (62.4 L-mmHg/mole.K)( º K) 6.2

3 Then to convert the number of moles, n, per time in minutes, t, into mmolo 2 min -1, use unit analysis: n moles O 2 t min 1000 mmol x = mmolo 2 min -1 1 mol In our above example, if we ran our experiment for 20 minutes, the mmolo 2 min -1 would be: 2.4 x 10-5 moles O 2 20 min 1000 mmol x 1 mol = 1.2 x 10-3 mmolo 2 min -1 Temperature affects enzyme rates. Hence, temperature affects the rate of respiration as all of the complex biochemical reactions that make up cellular respiration are catalyzed by enzymes. Exactly how does temperature affect the rate of cellular respiration? Does it speed it up or slow it down or have no effect? We ll be trying to answer these questions during this lab. Comparing Rates The respiration rate of any biological system can be studied, although some are easier to study than others. One easy system to study is germinating peas, beans or other legumes. By soaking legumes in water for a day then allowing them to germinate, you have obtained a rapidly-respiring organism that is easy to handle and fits conveniently into a respirometer. But how do you compare the rates of cellular respiration in germinating legumes that are of different size? The answer is, you weigh them, in grams, then calculate the number of mmol of oxygen consumed per milligram of germinating pea per unit time, mmolo 2 mg -1 min -1, as your final measurement of respiration rate: mmolo 2 min 1 grams x 1 gram = mmolo 2 mg -1 min mg Using the above example, suppose 5.6 grams of germinating peas consumed 1.2 x 10-3 mmolo 2 min -1 ; what would be the respiration rate? 1.2 x 10-3 mmolo 2 min grams x 1 gram = 2.1 x 10-7 mmolo 2 mg -1 min mg Applications of These Data Quite a bit of information can be extrapolated from these data. For instance, given a specific rate of respiration, we can tell the rate at which glucose is being consumed. Who cares? Well, you should, if you like to eat food. Seeds of all kinds have only a certain amount of glucose storage to get them from germination to the stage at which chlorophyll is produced and they can begin to produce their own glucose. Temperature affects this. So, planting seeds at the wrong temperature might spell disaster for a germinating crop or, at best, produce weak seedlings that might result in an inferior crop. 6.3

4 So, how do you calculate the rate glucose is being consumed, in mmol glucose mg -1 min -1, given mmolo 2 g -1 min -1 consumed? Using the balanced chemical equation for cellular respiration and a little stoichiometry, we have: mmoles O 2 1 mole O x 2 1 mol C x 6 H 12 O mmol x = mgrams min 1000 mmol 6 mol O 2 1 mol C 6 H 12 O 6 mmol C 6 H 12 O 6 mg min Continuing with our previous example, the rate of glucose consumption is: 2.1 x 10-1 mmolo 2 mg -1 min -1 x 1 mole O 2 x 1 mol C 6 H 12 O 6 x 1000 mmol C 6 H 12 O 6 = 1000 mmol 6 mol O 2 1 mol C 6 H 12 O x 10-2 mmol glucose mg -1 min -1 We could also ask, what is the mass, in milligrams, of the glucose being consumed per mg of legume per minute. First, we need the gram molecular mass of glucose, which is 180 g mole -1 (C: 6 x 12; H: 12 x 1.0; O: 6 x 16). Then use unit analysis and our example: 2.1 x 10-1 mmol glucose mg -1 min -1 1 mol C x 6 H 12 O g mg x x = 1 mmol C 6 H 12 O 6 1 mol C 6 H 12 O 6 1 g 3.8 x 10-2 mg glucose mg -1 min -1. Laboratory Objectives After mastery of this laboratory, including doing the assigned readings and required laboratory work, the student should be able to: 1. State the equation for cellular respiration and explain its relationship to that of photosynthesis. 2. Explain the components of and demonstrate an experiment using a respirometer and apply gas laws to obtain quantitative rates of cellular respiration of germinating pea seeds at various temperatures. 3. Given the ideal gas law equation, atmospheric pressure in mmhg, volume O 2 absorbed, temperature in ºC and R, calculate the moles O 2 consumed, mmolo 2 absorbed min -1, mmol O 2 consumed mg -1 of germ plant min -1, rate and mass of glucose consumption gram -1 of germinating plant min -1. Materials and Methods The hypothesis we are testing in this experiment is: Rates of respiration increase with increased temperature. 6.4

5 Before you proceed, read and understand the lab safety advisory below! Lab safety advisory: Biohazard! Potassium hydroxide, KOH, is a powerful base. Do not get it in your eyes or on your skin. Wear safety glasses. If you get KOH in your eyes, immediately go to the eyewash station and irrigate your eyes for several minutes, while calling for help. 1. Each lab table (group of two lab stations) should have three water baths, one for cold water, one for room temperature water, and one for warm water. One water bath should already have room-temperature water in it; if there are other lab sections after yours, please don t dump this water out when finished! There should be thermometers in each water bath. Make sure there is at least 4 cm of water in each water bath, enough to cover the respirometers. 2. Each lab table should also have a hot plate, 600 ml beaker and a beaker mitt. Fill the beaker ¾ full of tap water and put the water on to boil. When the water has come to a boil, using the beaker mitt, pour the water into one of the water baths, along with an equal amount of cooler water this bath will be your warm water bath. Add more water to the beaker and set it on the burner to boil, just in case more hot water is needed. Monitor the temperature of this bath and try to keep it as close to 50 C as possible by adding boiling water or tap water. 3. At each lab table there should be a large (~1000 ml) beaker or plastic container. Take this container, fill it with ice and put the ice in the remaining water bath; do this two times this bath will be your cold water bath. Monitor the temperature of this bath and try to keep it as close to 10 C as you can by either adding ice or tap water. 4. Each lab station should have 6 respirometers consisting of glass vials or test tubes, weights, one-holed stoppers and calibrated pipettes. With grease pencils, mark the respirometer test tubes with your lab station number and respirometer number; for instance, if you are lab station 3, mark your respirometers 3-1, 3-2, 3-3, 3-4, 3-5 and 3-6. Each lab station should also have 6 absorbant cotton balls, 6 nonabsorbant cotton balls, forceps, and access to a dropper bottle of 15% KOH solution. 5. Obtain 30 germinating peas (or 15 germinating beans) and 30 dry peas (or 15 dry beans). Divide the germinating legumes into three groups and the nongerminating (dry) legumes into three groups. Zero your scale, then put a square of paper towel on the scale. Tare this piece of paper towel. Then weigh your legumes by placing them on the paper towel. (Never place anything directly on the pan of a scale!) Record these values in your lab report (Data Table 6.1). 6. Using forceps, place an absorbant cotton ball in the bottom of each respirometer test tube. Next, add 20 drops of 15% KOH directly onto each ball; make sure the KOH drops squarely onto the cotton and does NOT touch the sides of the container! If it does, you have to rinse the test tube out, dry it, and start over again. With the forceps, place a ball of nonabsorbant cotton over the KOH-soaked cotton. 7. Put the groups of germinating legumes into respirometers 1, 3 and 5, making sure you have 6.5

6 recorded the weights of each group with the corresponding respirometer! Then put the groups of nongerminating legumes into respirometers 2, 4 and 6, again making sure you have recorded the weights of each group with the corresponding respirometer. 8. Stopper each respirometer securely. Place respirometers 1 and 2 into the cold bath, 3 and 4 into the room temperature bath, and 5 and 6 in the warm bath and weight them down. Check to make sure the respirometers don t leak; water should not enter the main respirometer chamber and no bubbles should be released. If water does enter the chamber, get your instructor to help you. Also, the respirometers should not float; if they do, add more weights. Allow the respirometers to equilibriate in their respective water baths for 10 minutes before beginning to take measurements. In the meanwhile, monitor the temperatures of the cold and warm baths and try to keep the temperatures stable. 9. After 10 minutes, a small amount of water should have entered the open end of the calibrated pipettes into where the calibrations are (look for the meniscus); you may have to wait a little longer for this to happen. Note this value for each respirometer at time zero. Note this value again at the end of 20 minutes, or as directed by your instructor. During those 20 minutes, try to keep the water bath temperatures stable! 10. At the end of 20 minutes, or as directed by your instructor, clean up! Take the respirometers out of the water baths, dump the water, and make sure the hot plates are turned off and unplugged. Return the legumes to your instructor. With your forceps, put the cotton balls in the waste container designated by your instructor. Rinse all glassware with tap water, followed by three good rinses of DW (or as directed by your instructor). 11. Record the actual temperatures of each water bath in your lab report (Data Table 6.2). In the same table, record the calibrated pipette readings at time 0, in ml, and at time 20 minutes, in ml, for each respirometer. Subtract the time 0 from time 20 min reading to get the volume change, in ml. Divide the volume change, in ml, by the mass, in grams, of legumes in each treatment. Then subtract the germinating vol. change (ml) gram -1 from the nongerminating vol. change (ml) gram -1 to get the actual volume change (ml) gram -1 at each of the three temperatures. 12. Obtain the atmospheric pressure, in mmhg, and record it in your lab report. 13. Convert the water bath temperatures into K [by adding to your recorded K] and record in your lab report (Data Table 6.3). Convert the actual volume changes, in mlg -1, into Lg -1 and record in the same table. Using these data, and the information in the introduction, calculate the number of moles of oxygen consumed per gram and record in Data Table Finally, using the data you have recorded, and the information in the introduction, calculate the number of milimoles of oxygen consumed per milligram per minute and record in Data Table 6.3. This is the respiration rate. *If you are confused, go back to the beginning and proceed one step at a time! After doing this, and you re still confused, have your instructor help you before you turn in your lab report! 6.6

7 Biol 160 Lab 6: Cellular Respiration Prelab (5 points) Name: Date: Lab Section: ~Complete this prelab before coming to lab; it is due at the beginning of lab! 1. Write out the balanced, quantitative equation for cellular respiration. a. What gas is consumed? b. What gas is produced? 2. What is the function of KOH in this lab? 3. If we removed all of the CO 2 from our closed system, the respiration rate would be directly equal to what? 4. What is the safety concern in this lab? 5. What is the function of the water bath in this exercise? 6.7

8 6. What is the total mg of glucose consumed by 7.8 g of germinating bean seedlings at 25.2º C, if 0.89 ml O 2 is consumed at 760 mmhg atmospheric pressure in 20 minutes? (Show all work!) [Hint: Determine number of moles of O 2 used first using gas law equation, convert from moles O 2 to moles glucose, then to grams glucose, then to mg glucose; you don t need to deal with mmol O 2 for this problem!] 6.8

9 Biol 160 Lab 6: Cellular Respiration Report (20 points) Name: Lab Date: Lab Section: Results 1. Data Table 6.1: Masses of legumes. Germinating Masses of Legumes (g) Resp. 1 Resp. 2 Resp. 3 Resp. 4 Resp. 5 Resp. 6 Nongerminating 2. Data Table 6.2: Gas volume changes. Temperatures (Record actual values): cold water bath: ºC Gas Volumes (ml): room temperature: ºC warm water bath: ºC time 0 time 20 min vol. chng vol. chng mlg -1 time 0 time 20 min vol. chng vol. chng mlg -1 time 0 time 20 min vol. chng vol. chng mlg -1 Germinating Nongerminating Actual Volume Change, mlg

10 Analysis 3. Atmospheric pressure: mmhg 4. Data Table 3: MmolO 2 g -1 min -1 consumed. Water Bath Temperatures, in K Actual Volume Changes, Lg -1 Number moles O 2 consumed cold bath room temperature warm bath mmolo 2 g -1 min -1 consumed Discussion 5. If temperature is not considered, will the determination of the number of moles of oxygen consumed be too low or too high? Why? 6. Why did we need to use nongerminating legumes in our study of germination rates? 7. Do your data support or reject the stated hypothesis, or are your data inconclusive? Why? 6.10

11 8. What was the rate of stored glucose consumption, in mmol glucose mg -1 min -1, at each of the three temperatures? (Show all work!) a. Warm temperature: b. Room temperature: c. Cold temperature: 6.11

12 9. Of what significance would the answer to question 8 be to terrestrial plant communities affected by quick changes in temperature, as in global warming? 6.12