Experiment 2 effect of temperature on enzyme activity

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Experiment 2: Effect of Temperature on Enzyme Activity

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

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

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 experiment, 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.

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Materials

(2) 250 mL Beakers 3 Balloons 30 mL 3% Hydrogen Peroxide, H2O2 Measuring Spoon Permanent Marker Ruler 20 cm String

3 Test Tubes (Glass)

Test Tube Rack Thermometer Yeast Packet *Hot Water Bath *Stopwatch

*Sheet of Paper *You Must Provide

Procedure

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 9 mL hydrogen peroxide. Then, keep Test Tube 2 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:

a. 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).

b. 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.

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

d. 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.

e. 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 enzymes.

6. Let the tubes sit for 15 minutes in their respective condition.

7. During the 15 minutes, prepare three balloons by using a sheet of paper to create a funnel by wrapping a sheet of paper into a cone shape with an opening at the point of the cone. Place the point of the funnel into the mouth of the balloon. One at a time, funnel 1/4 tsp. of yeast into each of the three balloons. 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.

8. After 15 minutes, use the thermometer to record the temperature of the hydrogen peroxide in Test Tube 2 in Table 2. Remove the thermometer after a reading has been recorded and set it aside.

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Figure 5: The neck of the balloon is stretched around the opening of the test tube, but the yeast is still resting in the bulb of the balloon.

9. Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube. Remember, there is yeast inside of the balloons.

10. Attach the neck of a balloon you prepared in Step 7 to the top of Tube 2 (the room temperature test tube) making sure to not let the yeast spill into the test tube yet (Figure 5). Once the neck of the balloon is securely attached to the test tube lift the balloon so that is in a vertical position in line with the test tube and allow the yeast to drop from the bulb and enter the test tube. Tap or rub the sides of the balloon together to ensure all the yeast enters the test tube. The balloon should remain attached to the test tube.

Note: To ensure the balloon does not pop off of the test tube during the reaction, ensure the neck of the balloon extends a least two centimeters down the neck of the test tube.

11. As quickly and carefully as possible remove Tube 1 (cold) from the refrigerator and repeat Steps 8 - 10 with Tube 1 using a balloon you prepared in Step 7.

12. As quickly and carefully as possible remove Tube 3 (hot) from the hot water bath and repeat Steps 8 - 10 with Tube 3 using a balloon you prepared in Step 7.

13. Swirl each tube to mix the yeast and hydrogen peroxide. Observe the test tubes for at least two minutes.

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)

Uninflated Balloon Circumference (cm)

Final Balloon Circumference (cm)

Difference in Balloon Circumference (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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