Foreword
This laboratory involves the use of an enzyme that will react with hydrogen peroxide. The enzyme is catalase, and the substrate is hydrogen peroxide (H2O2). The reaction is as follows: catalase
2H2O2 2H2O + O2
Ingested hydrogen peroxide is a poison, while external use of this substance is not. However, the generation of oxygen gas requires careful handling due to the potential combustion hazard oxygen presents when handled near a heat source or spark potential.
During this lab all data will be based upon in vitro (outside the organism) observations as opposed to in vivo (inside the organism). Also, we are assuming that the amount of gas produced is indicative of enzyme activity. Hydrogen peroxide is a poisonous by-product of reactions in cells andmust broken down quickly into harmless substances.
Reaction Activation Energy
no catalyst
- 2H2O2 2H20 + O2 EA1 = 72 kcal/mol
iron (Fe)
- 2H2O2 2H20 + O2 EA2 = 52 kcal/mol
catalase
- 2H2O2 2H20 + O2 EA3 = 20 kcal/mol
Purpose: The purpose of this enzyme lab is to be able to measure and record the total amount of oxygen made during the decomposition/consumption of H2O2 by catalase which is in the liver. Additionally, I will be correlating catalase concentration to the concentration of O2. The independent value of this lab is the amount of filter paper discs to liver tissue. In the lab,
the variable is changed to analyze the effect on the reaction rate. The dependent variable in the lab is the volume of oxygen gas produced which is measured over the span of every 10 seconds in a total time of 2 minutes.
Hypothesis: I hypothesize that if the amount of filter paper discs being used in the lab increases, there will be an increase in oxygen production as well. This is because the filter paper creates more catalytic sites for the enzyme catalase to break the hydrogen peroxide into more oxygen and water. Essentially, the reaction time will increase which also increases the amount of volume of oxygen gas within the time frames and the gas production rates will increase over time as the amount of filter discs increase as well however, once there is a certain amount of filter discs, there will be no further increase in gas production rates due to all the sites being saturated already.
Materials and Apparatus: 1 pneumatic trough Safety goggles Thermometer 1 Tweezer 50 ml beaker Waste container 3% Hydrogen peroxide 100 ml graduated cylinder Bottle brush Pipette pump 10 ml graduated cylinder 3 reaction vessels Ground liver 30 filter paper disks 1 rubber stopper/tubing
Procedure: (refer to Enzyme Lab handout and make note of changes only) Preparing the Water Displacement Apparatus
- Place a plastic pneumatic trough in a lab sink and fill it with tap water to a level just below the overflow hole.
- Submerge a 100 ml graduated cylinder in the trough so that it is completely filled with water. Invert the graduated cylinder, making sure that no air bubbles have been trapped in it.
Preparing the Reaction Vessel
- Obtain a 50 ml beaker containing a sample of the ground liver tissue.
- Using a pair of tweezers, dip a filter paper disc into the ground liver tissue and wipe off excess tissue on the lip of the beaker. The disc should be saturated but no excess tissue should be dripping from the disc.
- Holding the reaction vessel horizontally, carefully place the saturated disc onto the upper wall of the vessel (be sure that the disc sticks to the wall and stays in place). 6. Wearing safety goggles, use a 10 ml graduated cylinder, measure 10.0 ml of 3% hydrogen peroxide and carefully deliver the hydrogen peroxide into the horizontal reaction vessel. Do not allow the hydrogen peroxide to come into contact with the saturated filter paper (if any bubbles are observed, repeat the procedure with a clean reaction vessel).
- Use the rubber stopper with extended plastic tubing to seal the vessel.
Measuring the Volume of Oxygen (O2) Gas Produced
- While one member of the group lifts the inverted graduated cylinder slightly, another group member positions the end of the plastic tubing below the opening of the graduated cylinder. 9. Rotate the reaction vessel so that the hydrogen peroxide comes in contact with the saturated filter paper disc.
- Measure the volume of O2 gas produced every 10 seconds for 2 minutes. Your measurements should be made to the 10th of a ml (0.1ml).
- Empty the contents of the reaction vessel into the waste container and wash the reaction vessel with soap and water. Invert the clean reaction vessel on some paper towel to drip dry.
- Repeat steps 2-11 using 3 filter paper discs saturated with liver tissue.
- Repeat steps 2-11 using 5 filter paper discs saturated with liver tissue.
- Complete up to three trials of the above procedure (as time permits).
Observations:
Display your raw data for the 1 disc trials in a well-labelled table (similar to the one shown in Table 1 below). Produce similar tables for the 3 disc and 5 disc trials. Include qualitative observations below the raw data table.
Table 1: The Amount of water displaced after the liver enzyme is in contact with hydrogen peroxide
| Time (s) | Trial 1 (ml) | Trial 2 (ml) | Trial 3 (ml) | Trial 4 (ml) |
| 10 | 5 | 5 | 5 | 3 |
| 20 | 6 | 12 | 10 | 5 |
| 30 | 9 | 20 | 15 | 8 |
| 40 | 13 | 26 | 21 | 13 |
| 50 | 18 | 31 | 26 | 16 |
| 60 | 21 | 35 | 31 | 20 |
| 70 | 26 | 40 | 35 | 23 |
| 80 | 29 | 43 | 39 | 26 |
| 90 | 32 | 47 | 43 | 29 |
| 100 | 35 | 50 | 48 | 34 |
| 110 | 38 | 54 | 52 | 36 |
| 120 | 43 | 58 | 57 | 39 |
Table 2: The amount of displaced water after the liver enzyme of 3 discs comes in contact with the hydrogen peroxide
| Time (s) | Trial 1 | Trial 2 | Trial 3 | Trial 4 |
| 10 | 9 | 11 | 9 | 8 |
| 20 | 19 | 22 | 22 | 24 |
| 30 | 30 | 32 | 34 | 35 |
| 40 | 40 | 42 | 44 | 40 |
| 50 | 49 | 50 | 56 | 57 |
| 60 | 60 | 59 | 65 | 64 |
| 70 | 68 | 66 | 75 | 73 |
| 80 | 75 | 73 | 81 | 83 |
| 90 | 80 | 82 | 86 | 90 |
| 100 | 83 | 88 | 89 | 99 |
| 110 | 87 | 93 | 95 | 105 |
| 120 | 89 | 98 | 97 | 107 |
Table 3: The amount of displaced water after the liver enzyme of 5 discs comes in contact with the hydrogen peroxide
| Time (s) | Trial 1 | Trial 2 | Trial 3 | Trial 4 |
| 10 | 13 | 15 | 22 | 9 |
| 20 | 18 | 23 | 36 | 20 |
| 30 | 22 | 29 | 46 | 27 |
| 40 | 35 | 35 | 57 | 32 |
| 50 | 50 | 44 | 65 | 41 |
| 60 | 65 | 57 | 74 | 51 |
| 70 | 72 | 72 | 80 | 59 |
| 80 | 82 | 83 | 85 | 67 |
| 90 | 91 | 94 | 90 | 79 |
| 100 | 99 | 100 | 99 | 87 |
| 110 | 102 | 104 | 108 | 96 |
| 120 | 105 | 107 | 112 | 105 |
Qualitative Observations:
The filter paper discs used in the lab discoloured when the wet ground liver was added on to it for the lab. It additionally obtained a moist texture. When the hydrogen peroxide was added to the reaction vessel, it made bubbles instantly. This indicated the enzyme catalase started to break down the hydrogen peroxide immediately explaining the bubbles which was a release of oxygen gas as a product of the reaction. However, overtime, the amount of bubbles slowly decreased which means the enzymes were running out of hydrogen peroxide to break down and resulting in a decrease in production of oxygen.
Sample Calculations For 10 – 20s intervals for 5 discs:
V(Trial 1) = 18ml – 13ml = 5ml V(Trial 2) = 23ml – 15ml = 8ml V(Trial 3) = 36ml – 22ml = 14ml V(Trial 4) = 20ml – 9ml = 11ml
Avg = 5 + 8 + 14 + 11 = 38ml / 4 = 9.5ml
Avg Rate of O2 Production = 9.5ml / 10 s = 0.95 ml/s
Table 4: Average rate of oxygen gas produced every 10 s for 2 min for one disc of liver tissue containing catalase.
| Time (s) | Change in Volume [V] of O2 (ml) | Average V of O2 (ml) | Average Rate of O2 Production (ml/s) | |||
| Trial 1 | Trial 2 | Trial 3 | Trial 4 | |||
| 10 | 5 | 5 | 5 | 3 | 4.5 | 0.45 |
| 20 | 1 | 7 | 5 | 2 | 3.8 | 0.38 |
| 30 | 3 | 8 | 5 | 3 | 4.8 | 0.48 |
| 40 | 4 | 6 | 6 | 5 | 5.3 | 0.53 |
| 50 | 5 | 5 | 5 | 3 | 4.5 | 0.45 |
| 60 | 3 | 4 | 5 | 4 | 4.0 | 0.40 |
| 70 | 5 | 5 | 4 | 3 | 4.3 | 0.43 |
| 80 | 3 | 3 | 4 | 3 | 3.3 | 0.33 |
| 90 | 3 | 4 | 4 | 3 | 3.5 | 0.35 |
| 100 | 3 | 3 | 5 | 5 | 4.0 | 0.40 |
| 110 | 3 | 4 | 4 | 2 | 3.3 | 0.33 |
| 120 | 5 | 4 | 5 | 3 | 4.3 | 0.43 |
Table 5: Average rate of oxygen gas produced every 10 s for 2 min for three discs of liver tissue containing catalase.
| (s) | Trial 1 | Trial 2 | Trial 3 | Trial 4 | V of O2 (ml) | of O2 Production (ml/s) |
| 10 | 9 | 11 | 9 | 8 | 9.3 | 0.93 |
| 20 | 10 | 11 | 13 | 16 | 12.5 | 1.25 |
| 30 | 11 | 10 | 12 | 11 | 11.0 | 1.10 |
| 40 | 10 | 10 | 12 | 5 | 9.3 | 0.93 |
| 50 | 9 | 8 | 12 | 17 | 11.5 | 1.15 |
| 60 | 11 | 9 | 9 | 7 | 9.0 | 0.90 |
| 70 | 8 | 7 | 10 | 9 | 8.5 | 0.85 |
| 80 | 7 | 7 | 6 | 10 | 7.5 | 0.75 |
| 90 | 5 | 9 | 5 | 7 | 6.5 | 0.65 |
| 100 | 3 | 6 | 3 | 9 | 5.3 | 0.53 |
| 110 | 4 | 5 | 6 | 6 | 5.3 | 0.53 |
| 120 | 2 | 5 | 2 | 2 | 2.8 | 0.28 |
Table 6: Average rate of oxygen gas produced every 10 s for 2 min for five discs of liver tissue containing catalase.
| Time (s) | Change in Volume [V] of O2 (ml) | Average V of O2 (ml) | Average Rate of O2 Production (ml/s) | |||
| Trial 1 | Trial 2 | Trial 3 | Trial 4 | |||
| 10 | 13 | 15 | 22 | 9 | 11.8 | 1.18 |
| 20 | 5 | 8 | 14 | 11 | 9.5 | 0.95 |
| 30 | 4 | 6 | 10 | 7 | 6.8 | 0.68 |
| 40 | 13 | 6 | 11 | 5 | 11.8 | 0.88 |
| 50 | 15 | 9 | 8 | 9 | 9.5 | 1.03 |
| 60 | 15 | 13 | 9 | 10 | 6.8 | 1.18 |
| 70 | 7 | 15 | 6 | 8 | 11.8 | 0.90 |
| 80 | 10 | 11 | 5 | 8 | 9.5 | 0.85 |
| 90 | 9 | 11 | 5 | 12 | 6.8 | 0.65 |
| 100 | 8 | 6 | 9 | 8 | 11.8 | 0.78 |
| 110 | 3 | 4 | 9 | 9 | 9.5 | 0.63 |
| 120 | 3 | 3 | 4 | 9 | 6.8 | 0.48 |
Make a scatter plot graph to show the average rate of oxygen production (mL/s) over the 2 minutes time period for each of the three liver quantities (1,3, and 5 discs) on the same graph. Choose an appropriate legend so that the data points for each liver quantity can be easily distinguished. Produce smooth “lines of best fit” or “curves of best fit” (rather than connecting the points) for each liver quantity (use a legend to distinguish the lines/curves from each other).
Discussion Questions:
- Explain the difference between an enzyme and other catalysts.
Enzymes and catalysts can change and affect the reaction times that they correspond to. However, as enzymes may be catalysts, some catalysts aren’t enzymes. Enzymes are organic, developed naturally and are catalysts for biochemical processes (Robinson). Enzymes are additionally high molecular globular proteins as well. Whereas, catalysts can be inorganic or organic. The inorganic ones are mainly low in molecular mass and not all catalysts are biochemical catalysts as well.
- What name is used to describe the energy needed to initiate a chemical reaction? Why would organisms that can produce catalase have an advantage over those that cannot?
The energy used to initiate a chemical reaction is called activation energy. Activation energy is energy that needs to be generated to essentially kickstart the chemical reaction process. In this case, hydrogen peroxide needs to be broken down and released as oxygen, this is a chemical reaction.
However, the enzyme catalase lowers the activation energy needed and the time it takes for the reaction to complete. Organisms that produce catalase will have a significant advantage over organisms that do not produce catalase because the organisms that do not will have to use up a lot more energy to get the reaction to initiate and it will take a lot more time for the reaction to complete.
- Explain two specific reasons that in vitro analysis may not give an accurate indication of the rate of enzyme activity, in vivo.
Temperature and pH Influence: Enzymes are extremely particular and sensitive proteins where the slightest increase of temperature and pH can affect its speed and even denature it. Vitro experiments can somewhat control and monitor these factors. However, it doesn’t completely replicate the enzyme’s environment and precise conditions. In vivo, temperature and pH will change due to factors like circulatory systems and metabolic activities that will affect the enzyme’s health. Differences and inconsistent data like will tamper with the overall data, giving an inaccurate of enzyme activity therefore, confirming that temperature and pH is a reason.
No Cellular Context: Vitro experiments simulate enzyme activity while it is completely isolated and on its own when that isn’t a reality for enzymes. Enzymes live within crowded environments with many other cells and molecules therefore, inaccurately depicting the environment will inaccurately depict the enzyme activity, leaving important environmental details for enzymes.
- Describe in detail, one way in which the experimental procedure could be improved to yield more accurate results. Be sure to point out the problem and suggest a solution that could actually be implemented. Do not include human error.
Firstly, there was an inability to consistently mix the reaction vessel manually after adding the hydrogen peroxide. Consistent mixing would’ve made a great difference with a better reaction rate and better activity from the enzymes and better results. The magnetic stirring bar can be a solution due to this problem being a manual problem whereas, the magnetic stirring bar will automatically stir the reaction vessel consistently with accuracy as well.
Conclusion:
This lab’s overall goal was to observe, analyze and understand how the number of filter paper discs with the enzyme catalase affects the amount of oxygen gas production from breaking down hydrogen peroxide. The final data obtained from the lab affirmed my hypothesis stating that the more filter paper discs there were, the more oxygen gas production there will be. Due
to the discs creating more catalytic sites to increase the production rate (Robinson). In our lab, we immediately saw a demonstration of our hypothesis as the production rate increased drastically by 0.45 ml/s and proved it again when it dropped back to 0.43 ml/s. As stated in the hypothesis, the filter discs will initially increase the rate of production greatly however, when all the sites have been saturated, it will decelerate.
The more discs added, the more the initial increase will accelerate but additionally, the more discs, the faster the rate decelerates. Essentially, I observed the dramatic factors of catalytic sites and freeing how production rates will be determined off of those factors including the factor of there being a limit to the overall reaction speed due to the finite amount of catalytic sites. In conclusion, this lab confirmed our hypothesis and aligned with the collision theory as well.
