IB Enzyme Lab
Many organisms can decompose hydrogen peroxide (H202) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities of living organisms. They act as catalysts, substances that speed up chemical reactions without being destroyed or altered during the process. Enzymes are extremely efficient and may be used over and over again. One enzyme may catalyze thousands of reactions every second. Both the temperature and the pH at which enzymes function are extremely important. Most organisms have prefered temperature range in which they survive, and their enzymes most likely function best within that temperature range. If the environment of the enzyme is too acidic or too basic, the enzyme may irreversibly denature, or unravel, until it no longer has the shape necessary for proper functioning.
H202 is toxic to most living organisms. Many organisms are capable of enzymatically destroying the H202 before it can do much damage. H202 can be converted to oxygen and water, as follows:
2 H202 2 H20 + O2
Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least two different enzymes are known to catalyze this reaction: catalase, found in animals and protists, and peroxidase, found in plants. A great deal can be learned about enzymes by studying the rates of enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a number of ways including:
- measuring the pressure of the product as it appears (in this case, O2)
- measuring the rate of disappearance of substrate (in this case, H202)
- measuring the rate of appearance of a product (in this case, O2 which is given off as a gas)
In this experiment, you will measure the rate of enzyme activity under various conditions, such as different enzyme concentrations, pH values, and temperatures. It is possible to measure the pressure of the oxygen gas formed as H202is destroyed and a plot can be made to visualize the process.
At the start of the reaction, there is no product, and the pressure is the same as the atmospheric pressure. After a short amount of time, oxygen accumulates at a rather constant rate. The slope of the curve at this initial time is constant and is called the initial rate. As the peroxide is destroyed, less of it is available to react and the O2 is produced at lower rates. When no more peroxide is left, O2 is no longer produced.
In this experiment, you will:
- Use a computer and Gas Pressure Sensor to measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various enzyme concentrations.
- Measure and compare the initial rates of reaction for this enzyme when different concentrations of enzyme react with H202.
- Measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various temperatures.
- Measure and compare the initial rates of reaction for the enzyme at each temperature.
- Measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various pH values
- Measure and compare the initial rates of reaction for the enzyme at each pH value.
Write a hypothesis for each of the following experimental conditions.
- Effect of Enzyme Concentration
- Effect of Enzyme Temperature
- Vernier computer interface
- Logger Pro Software
- Vernier Gas Pressure sensor
- 1-hole rubber stopper assembly
- 10 ml beaker of water
- 250 ml beaker of water
- 3% H2O2
- 600 ml beaker
- Enzyme suspension
- Two 18 x 150 mm test tubes
- pH buffers
- Test tube rack
- Vernier Thermometer
- Three dropper pipettes
- Obtain and wear goggles
- Write hypotheses for each conditions: enzyme concentration, temperature, and pH.
- Connect the Gas Pressure Sensor to the computer interface. Prepare the computer for data collection by opening the file “06B Enzyme (Pressure)” from the Biology with Vernier folder of Logger Pro.
- Connect the plastic tubing to the valve on the Gas Pressure Sensor.
Part A: Testing the Effect of Enzyme Concentration
- Partially fill a beaker with Distilled water for use in step 5.
- Add 3 mL of water and 3 drops mL of 30-35% H2O2 to each test tube.
- Using a clean dropper pipette, add 1 drop of enzyme suspension to Test Tube 1. Note: Be sure not to let the enzyme fall against the side of the test tube.
- Stopper the test tube and gently swirl to thoroughly mix the contents. The reaction should begin. The next step should be completed as rapidly as possible.
- Connect the free-end of the plastic tubing to the connector in the rubber stopper. Click Green “Go” Arrow to begin data collection. Data collection will end after 3 minutes.
- If the pressure exceeds 130 kPa, the pressure inside the tube will be too great and the rubber stopper is likely to pop off.
- When data collection has finished, disconnect the plastic tubing connector from the rubber stopper. Remove the rubber stopper from the test tube, discard the contents into the trash and rinse the test tube.
- Find the rate of enzyme activity:
- Move the mouse pointer to the point where the data values begin to increase. Hold down the mouse button. Drag the mouse pointer to the point where the pressure values no longer increase and release the mouse button; if the rubber stopper does come off during the experiment don’t include this portion of the graph in your calculation of rate of enzyme activity.
- Click the Linear Fit button to perform a linear transgression. A floating box will appear with the formula for a best-fit line.
- Record the slope of the line, m, as the rate of enzyme activity. This value represents the rate of enzyme activity which can be compared between conditional variables to assess the effect of different conditions, i.e. comparing the enzyme activity of different temperatures.
- Close the linear regression floating box.
- Find the rate of enzyme activity for the following:
- Add 2 drops of the enzyme solution to test tube 2. Repeat steps 7-11
- Add 3 drops of the enzyme solution to test tube 3. Repeat steps 7-11
- Add 4 drops of the enzyme solution to test tube 4. Repeat steps 7-11
- Add 0 drops of the enzyme solution to test tube 4. Repeat steps 7-11
Part B: Testing the Effect of Temperature
- Add 3 mL of 3% H2O2 and 3 mL of water to each test tube.
- Measure the enzyme activity at 10 degrees Celsius:
- Place the test tube in a beaker containing ice and water until the temperature of the water/ice in the beaker reaches a temperature of 10 degrees; maintain this temperature for the duration of the 3 minute analysis and measure by using a Vernier Temperature Probe connected to a second computer. Do not record the temperature of the contents within the test tube, this would be impossible once the stopper is added.
- Add 2 drops of the enzyme solution and a combination of ice or hot water to reach 20 degrees Celsius. Repeat steps 7-11.
- Repeat steps 13 & 14 for the remaining temperature variations: “20” “30” “40” and “50.”
Part C: Testing the Effect of pH
- Add 3 mL of 3% H2O2 and 3 mL of pH 3 buffer to each test tube.
- Repeat steps 7-11, 14 & 15 to find the rate of enzyme activity for pH 4, 7 (distilled water), 10 & 12.
Write below a hypothesis for each condition being tested
Data Collection & Processing:
- Create data tables for each of the different sections of the lab (substrate concentration, temperature, and pH); this should include both qualitative and quantitative. This is your raw data
- Submit your individual group data to the following form; class data can be found here.
- Use the class data for all statistical analysis to analyze the raw data; calculated data should be included in a separate data tables for each respected lab section on a separate data table.
- Create a graphs for each of the different lab sections to present your data; you should have three different graphs.
- Perform statistical tests/analysis for each graph.
Insert Data Tables & Graphs Here
- Using your graphs and statistical analysis of data of the class data, analyze the experiment results and your results given the Conclusion & Evaluation rubric.
Insert Evaluation Here
Turn in via Google Classroom.
Lab Standard: Analysis
Lab Standard: Evaluation
A full explanation of the lab standard rubric can be found here