The Effects of Temperature and pH on Enzymatic Activity

Activity 2: Pre-Assessment

1. Which environmental conditions could alter the rate at which an enzymatic reaction takes place? Why would this occur?
2. Which environmental conditions could affect an enzyme’s active site? Why would this occur?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 2: The Effects of Temperature and pH on Enzymatic Activity

What types of environmental factors may affect enzymatic activity? Why? Several factors known to affect enzymatic activity are temperature, pH, and substrate concentration. In a typical chemical reaction, increasing temperature causes the substrates to become more energetic and hence more likely to bump into each other in solution. However, changes in temperature can cause an enzyme to denature, which changes the three-dimensional structure of the enzyme molecule. In addition, cellular enzymes each work within a certain pH range because the side chains within their active sites are optimized for efficient catalysis and are thus quite sensitive to changes in pH. Different enzymes may have different pH ranges and pH optima, conditions under which they work maximally; while many enzymes work best around a neutral pH, some are adapted to an acidic pH, while others are adapted to a basic pH.

Safety Precautions

• Goggles should be worn at all times while in laboratory.
• No open-toe shoes worn in laboratory.
• Measure fluids carefully using graduated cylinders to avoid breakage and spillage.
• Be careful not to touch solutions of concentrated acids and bases directly.
• Take precautions when using a hot plate and touching hot glassware.
• Inform your teacher immediately of any broken glassware as it could cause injuries.
• Clean up any spilled fluids to prevent other people from slipping.

For this activity, you will need the following:

• Graduated cylinder
• Beaker
• Water
• Buffer solutions of pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, and pH 9.0
• Milk
• Lactase (obtained from laboratory supply company)
• Test tubes
• Stirring rod
• Labeling pencil
• Hot plate
• Ice bath
• Refrigerator set to approximately 4 °C
• Incubator set to 37 °C
• Thermometers
• Glucose test strips
• Timer
• Graph paper

For this activity, you will work in pairs.

Structured Inquiry: Temperature

Step 1: Prepare a large beaker of boiling tap water on a hot plate. Prepare five identical test tubes, each containing 2 mL of milk. Label five test tubes accordingly with each of the following temperatures: 0°C (ice bath), 4°C (refrigerator), room temperature, 37°C, and 100°C (boiling temperature). Place one tube of milk at each of the five temperatures. Create a data table to enter your results for each of these test tubes over time. Measure the room temperature using a thermometer.

Step 2: Hypothesize/Predict: Based upon your knowledge of enzymes and the effects of temperature on their activity, rank the tubes from fastest (1) to slowest (5) glucose production predicted over time after the addition of lactase. Add your predictions to the data table you created in step 1.

Step 3: Student-led Planning: Discuss with your partner how you could use the data you collect to calculate a rate of lactase activity for each temperature.

Step 4: Make your lactase enzyme solution per your teacher’s instructions. Add 1 mL of the lactase enzyme solution to each of the five tubes listed above and immediately start timing. Immediately after adding the lactase enzyme solution, determine the glucose concentration in each tube using glucose test strips and the color chart that came with the test strips. Record this in your data table.

Monitor the temperatures of each of these locations, both before and after the experiment using thermometers.

Step 5: Every 3 minutes for 15 minutes, record the concentrations of glucose in each tube using the color chart that came with the test strips and record in your data table.

Step 6: Critical Analysis: Calculate the rate of enzymatic activity for lactase at each temperature using the method you devised in step 3. Using graph paper, graph your data of rates of lactase activity versus temperature. Which is the independent variable? Which is the dependent variable? Are the predictions you made in step 2 supported by your data? Explain how you know in your notebook.

Guided Inquiry: Temperature

Step 1: Hypothesize/Predict: Based on the data already collected, predict a temperature range that includes the optimal temperature for lactase activity. How do you think you could more finely pinpoint the optimal temperature for lactase activity? Write your ideas in your notebook.

Step 2: Student-led Planning: Determine how you could change the set-up of your test tubes to determine the optimal temperature for lactase activity. Once your teacher approves, create a table to record your data, prepare your test tubes per your design, and record data on glucose production every 3 minutes for 15 minutes. Determine the rates of lactase enzyme activity under each of your chosen conditions as you did in the Structured Inquiry. Graph the rates of lactase enzyme activity versus temperature and estimate the optimal temperature.

Step 3: Critical Analysis: Are the predictions you made in step 1 supported by your data? Is there any way you can improve your experiment? Discuss your answer with your lab partner and write it in your notebook.

Structured Inquiry: pH

Step 1: Prepare three test tubes, each containing 2 mL of milk, and label the three tubes as follows: 4.0 (acidic), 7.0 (neutral), and 9.0 (basic). To the first test tube, add 1 mL of pH 4.0 buffer. To the second test tube, add 1 mL of pH 7.0 buffer. To the third test tube, add 1 mL of pH 9.0 buffer. Create a data table to enter your results for each of these test tubes over time.

Step 2: Hypothesize/Predict: Based upon your knowledge of enzymes and the effects of pH on their activity, order the tubes from highest (1) to lowest (3) glucose production predicted over time. Add your predictions to the data table you created in step 1.

Step 3: Student-led Planning: Discuss with your partner how to calculate a rate of lactase activity for each pH.

Step 4: Make your lactase enzyme solution per your teacher’s instructions. Add 1 mL of the lactase enzyme solution to each of the three tubes listed above and immediately start timing. Immediately after adding the lactase enzyme solution, determine the glucose concentration in each tube using glucose test strips and the color chart that came with the test strips. Record this in your data table.

Step 5: Every 3 minutes for 15 minutes, record the concentrations of glucose in each tube using the color chart that came with the test strips and record in your data table.

Step 6: Critical Analysis: Calculate the rate of enzymatic activity for lactase at each pH. Using graph paper, graph your data of rates of lactase activity versus pH. Which is the independent variable? Which is the dependent variable? Are the predictions you made in step 2 supported by your data? Explain how you know in your notebook.

Guided Inquiry: pH

Step 1: Hypothesize/Predict: Based on the data already collected, predict a pH range that includes the optimal pH for lactase activity. How do you think you could more finely pinpoint the optimal pH for lactase activity? Write your ideas in your notebook.

Step 2: Student-led Planning: Determine how you would change the set-up of your test tubes to determine the optimal pH for lactase activity. Once your teacher approves, create a table to record your data, prepare your test tubes per your design, and record data on glucose production every 3 minutes for 15 minutes. Determine the rates of lactase enzyme activity under each of your chosen conditions. Graph the rates of lactase enzyme activity versus pH and estimate the optimal pH.

Step 3: Critical Analysis: Are the predictions you made in step 1 supported by your data? Is there any way you could improve your experiment? Discuss your answer with your lab partner and write it in your notebook.

Assessments

1. If an enzyme has an optimal activity at 25°C, what do you think will happen to the enzyme’s activity if the temperature is raised to 37°C? Why?
2. If an enzyme has a largely acidic active site, what do you think will happen to the enzyme’s activity if the pH is made basic? Why?