Procedures

Experiment 1: Effect of Temperature on Catalase Activity

1. Take a constant temperature bath, thermometer, and gas syringe from the Instruments shelf and place them onto the workbench.
2. Set the temperature bath to 10 °C.
3. Take a small test tube from the Containers shelf and place it in the constant temperature bath.

1. Add 5 mL of 3% hydrogen peroxide from the Materials shelf to the test tube.
2. Add 5 mL of water from the Materials shelf to the test tube.
3. Move the thermometer from the workbench into the test tube.

1. Wait until the temperature stabilizes at the same temperature as the bath.
2. Move the gas syringe from the workbench into the test tube.
3. Click the gas syringe and note the initial volume of gas in the syringe.

1. Add 1 mL of catalase solution from the Materials shelf to the test tube and immediately note the time on the lab clock, particularly the seconds.
2. After 15 seconds elapse, click the gas syringe again to determine the volume of gas released from the reaction. Record the two volumes (initial and after 15 seconds) to reference later.
3. Move the thermometer and gas syringe to the workbench.
4. Empty the test tube in the waste bin, and place it in the sink.
5. Repeat steps 2 – 13 four additional times, using temperatures of 21.5, 40, 60, and 80 °C instead of 10 °C. Record the optimum temperature for catalase activity based on your results.
6. Clear the workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

Experiment 2: Effect of Substrate Concentration on Catalase Activity

1. Take four constant temperature baths off the Instruments shelf and place them side to side onto the workbench. Space is limited, so place them as close to the left wall and each other as possible.
2. Set all four constant temperature baths to the optimum temperature for catalase activity that you determined in Experiment 1.
3. Take four small test tubes from the Containers shelf and place one into each constant temperature bath. Double-click on each test tube and rename them: 1, 2, 3, and 4.
4. Take four thermometers from the Instruments shelf and place one into each test tube.
5. Using items found on the Materials shelf, fill the four test tubes as follows.

undefined

Test Tube	Water (mL)	3% Hydrogen Peroxide (mL)
1	8	2
2	5	5
3	2	8
4	0	10

undefined

1. Wait until the temperature stabilizes and the tubes reach the same temperature as the bath.
2. Take four gas syringes from the Instruments shelf and place one in each test tube.
3. Click the gas syringe in test tube 1 and note the initial volume of gas in the syringe.
4. Add 1 mL of catalase solution to test tube 1 and immediately note the time on the lab clock, particularly the seconds.
5. After 15 seconds elapse, click the gas syringe in test tube 1 again to determine the volume of gas released from the reaction. Record the two volumes (initial and after 15 seconds) to reference later.
6. Repeat steps 8 – 10 for the three other test tubes. Record the concentration of hydrogen peroxide that produced the most gas.
7. Clear the workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

Experiment 3: Effect of pH on Catalase Activity

1. Take three constant temperature baths from the Instruments shelf and place them onto the workbench.
2. Set all three constant temperature baths to the optimum temperature for catalase activity that you determined in Experiment 1.
3. Take three small test tubes from the Containers shelf and place one into each constant temperature bath.
4. Take three thermometers from the Instruments shelf and place one into each test tube.
5. Add 5 mL of 3% hydrogen peroxide from the Materials shelf to each test tube.
6. Add 5 mL of pH 2 buffer from the Materials shelf to the first test tube. Double-click on this test tube to rename it “pH 2 buffer”.
7. Add 5 mL of pH 6 buffer from the Materials shelf to the second test tube. Double-click on this test tube to rename it “pH 6 buffer”.
8. Add 5 mL of pH 10 buffer from the Materials shelf to the third test tube. Double-click on this test tube to rename it “pH 10 buffer”.

1. Wait until the temperature stabilizes and the tubes reach the same temperature as the baths.
2. Take three gas syringes from the Instruments shelf and place one in each test tube.
3. Click the gas syringe in the pH 2 buffer test tube and note the initial volume of gas in the syringe.
4. Add 1 mL of catalase solution from the Materials shelf to the pH 2 buffer test tube and immediately note the time on the lab clock, particularly the seconds.
5. After 15 seconds elapse, click the gas syringe in the pH 2 buffer test tube again to determine the volume of gas released from the reaction. Record the two volumes (initial and after 15 seconds) to reference later.
6. Repeat steps 11 – 13 with the pH 6 buffer and pH 10 buffer test tubes. Record the optimum pH for catalase activity.
7. Clear the bench of all materials, containers, and instruments, then return to your course page to complete any assignment for this lab.