Experiment 2: Effect of Temperature on Enzyme Activity

Yeast cells contain catalase, an enzyme which helps convert hydrogen peroxide to water

Figure 4: Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.

and oxygen. This enzyme is very significant as hydrogen peroxide can be toxic to cells if allowed to accumulate. The effect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2 H2O2 → 2 H2O + O2).

In this lab you will examine the effects of temperature on enzyme (catalase) activity based on the amount of oxygen produced. Note, be sure to remain observant for effervescence when analyzing your results.



(2) 250 mL Beakers

3 Balloons

30 mL 3% Hydrogen Peroxide, H2O2

Measuring Spoon

Permanent Marker


20 cm String


3 Test Tubes (Glass)

Test Tube Rack


Yeast Packet

*Hot Water Bath


*You Must Provide



1.     Use a permanent marker to label test tubes 1, 2, and 3. Place them in the test tube rack.

2.     Fill each tube with 10 mL hydrogen peroxide. Then, keep one of the test tubes in the test tube rack, but transfer the two additional test tubes to two separate 250 mL beakers.

3.     Find one of the balloons, and the piece of string. Wrap the string around the uninflated balloon and measure the length of the string with the ruler. Record the measurement in Table 2.

4.     Create a hot water bath by performing the following steps:

1.     Determine if you will use a stovetop or microwave to heat the water. Use the 100 mL graduated cylinder to measure and pour approximately 200 mL of water into a small pot or microwave-safe bowl (you will have to measure this volume in two separate allocations).

2.     If using a stovetop, obtain a small pot and proceed to Step 4c. If using a microwave, obtain a microwave-safe bowl and proceed to Step 4e.

3.     If using a stove, place a small pot on the stove and turn the stove on to a medium heat setting.

4.     Carefully monitor the water in the pot until it comes to a soft boil (approximately 100 °C). Use the thermometer provided in your lab kit to verify the water temperature. Turn the stove off when the water begins to boil. Immediately proceed to Step 5.

CAUTION: Be sure to turn the stove off after creating the hot water bath. Monitor the heating water at all times, and never handle a hot pan without appropriate pot holders.

5.     If using a microwave, place the microwave-safe bowl in the microwave and heat the water in 30 second increments until the temperature of the water is approximately 100 °C. Use the thermometer provided in your lab kit to verify the water temperature. Wait approximately one minute before proceeding to Step 5.

5.     Place Tube 1 in the refrigerator. Leave Tube 2 at room temperature, and place Tube 3 in the hot water bath.

Important Note: The water should be at approximately 85 °C when you place Tube 3 in it. Verify the temperature with the thermometer to ensure the water is not too hot! Temperatures which exceed approximately 85  °C may denature the hydrogen peroxide.

6.     Record the temperatures of each condition in Table 2. Be sure to provide the thermometer with sufficient time in between each environment to avoid obscuring the temperature readings.

7.     Let the tubes sit for 15 minutes.

8.     During the 15 minutes prepare the balloons with yeast by adding ¼ tsp. of yeast each balloon. Make sure all the yeast gets settled to the bulb of the balloon and not caught in the neck. Be sure not spill yeast while handling the balloons.

9.     Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube.

10.   Attach the neck of a balloon you prepared in step 8 to the top of Tube 2 (the room temperature test tube) making sure to not let the yeast spill into the test tube yet. Once the balloon is securely attached to the test tube lift the balloon and allow the yeast to enter the test tube. Tap the bulb of the balloon to ensure all the yeast falls into the tube.

11.   As quickly and carefully as possible remove the Tube 1 (cold) from the refrigerator and repeat steps 9 – 10 with Tube 1 using a balloon you prepared in step 8.

12.   As quickly and carefully as possible remove Tube 3 (hot) from the hot water bath and repeat steps 9 – 10 with Tube 3 using a balloon you prepared in step 8.

13.   Swirl each tube to mix, and wait 30 seconds.

14.   Wrap the string around the center of each balloon to measure the circumference. Measure the length of string with a ruler. Record your measurements in Table 2.

Table 2: Balloon Circumference vs. Temperature
Tube Temperature (°C) Balloon Circumference (Uninflated; cm) Balloon Circumference (Final; cm)
1 – (Cold)      
2 – (RT)    
3 – (Hot)    



Post-Lab Questions

1.      What reaction is being catalyzed in this experiment?

2.      What is the enzyme in this experiment? What is the substrate?

3.      What is the independent variable in this experiment? What is the dependent variable?

4.      How does the temperature affect enzyme function? Use evidence from your data to support your answer.


5.      Draw a graph of balloon diameter vs. temperature. What is the correlation?


6.      Is there a negative control in this experiment? If yes, identify the control. If no, suggest how you could revise the experiment to include a negative control.


7.      In general, how would an increase in substrate alter enzyme activity? Draw a graph to illustrate this relationship.



8.      Design an experiment to determine the optimal temperature for enzyme function, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use?

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