Factors Affecting Enzyme Activity

Group Members: Lydia, Tiffany, Megan, Omid

Introduction

Enzymes are globular proteins that act as catalysts to speed up chemical reactions such as the conversion of hydrogen peroxide (H2O2) into oxygen and water. They can be very efficient and are reusable. However, enzymes operate best only under optimal conditions. An enzyme under extreme temperature conditions will not function as well as it would in its optimum temperature range. Environments that are too acidic or too basic may cause the enzyme to denature. It is critical for the the enzymes that convert hydrogen peroxide, like catalase (in animals and protists) and peroxidase (in plants), to be able to function optimally because hydrogen peroxide is toxic to most living organisms. By measuring the amount of oxygen produced in the reaction, the rate of reaction at different temperatures can be deduced.

Research Question

How does more acidic or basic pH affect the rate of enzyme activity?

Hypotheses

Explanatory: We hypothesize that extreme pH (very basic or very acidic) will cause enzyme activity (rate of reaction) to decrease.

Prediction: If pH is either too basic or too acidic, then the enzyme's reaction rate will decrease, because the enzyme will denature in those extreme conditions.

Null: The level of pH has no effect on the reaction rate of the enzyme.

Alternative: The level of pH has an effect on the reaction rate of the enzyme.

Materials

  • LabQuest
  • LabQuest app
  • Vernier Gas Pressure Sensor
  • rubber-stopped assembly
  • 10mL graduated cylinder
  • 250mL beaker of water
  • 3% H2O2 (hydrogen peroxide)
  • enzyme suspension
  • three 18 x 150mm test tubes
  • pH buffers (pH 4, 7, 10)
  • test tube rack
  • three disposable pipettes
  • Logger Pro

Variables

Independent Variable: pH of the buffer used

Dependent Variable: Pressure (rate of reaction)

Constants:

  • Temperature
  • Amount of enzyme used
  • Amount of substrate
  • Amount of buffer
  • Type of enzyme used

Methodology


  1. Obtain and wear goggles.
  2. Connect the plastic tubing to the valve on the gas pressure sensor, and the gas pressure sensor to LabQuest.
  3. On LabQuest, change the data-collection rate to 0.5 samples/second and the collection length to 180 seconds.
  4. Place the three test tubes onto the rack and label them pH 4, pH 7, and pH 10.
  5. Add 3mL of 3% H202 (hydrogen peroxide) and 3mL of each pH buffer to each according test tube.
  6. In the tube labeled pH 4, add 2 drops of the enzyme solution.
  7. Stopper the test tube, and gently swirl to mix the contents. Connect the plastic tubing to the connector in the rubber stopper as quickly as possible, and then click the "Play" button to start data collection.
  8. Monitor the pressure readings on the device to make sure that it does not exceed 130 kPa. If that occurs, disconnect the plastic tubing from the gas pressure sensor because the pressure inside the test tube may cause the stopped to pop off.
  9. When data collection is done, disconnect the tubing from the stopper, remove the stopper from the test tube, and discard the contents into the sink.
  10. Determine the rate of enzyme activity for the pressure vs. time curve. Record the slope of the Linear Fit equation (trendline).
  11. Store the data of the run by tapping the File Cabinet icon, and make sure to label the runs according to the pH of the buffer used.
  12. Repeat Steps 6 - 11 for at least two more trials with the pH 4 buffer, and then for the test tubes labeled pH 7 and pH 10. Each pH condition should be tested in at least three trials in total.

Data

Big image

Results

The graph lines of all the pH buffers begin around the same point, between 101.5 kPa to 102 kPa. At the end of the timeframe given to collect data (180 seconds, or 3 minutes), the pH 4 run ended at around 103.35 kPa of pressure, the pH 7 run ended up at around 104.9 kPa of pressure, and the pH 10 run ended at around 102.7 kPa of pressure. The pH 4 and pH 10 runs have lower slopes as shown by the linear regression lines, and a small amount of plateauing in the data collection line itself. In comparison, the run for pH 7 showed little to no plateauing. The pH 10 line especially took the longest to begin increasing in slope. Slope of the linear regression lines decreased as pH either became more basic or more acidic. All of the lines experienced a small dip in pressure at the beginning of the data collection, and smaller dips and rises throughout the rest of the lines. The slope in the line of pH 4 had an unexpected jump, possibly as a result of error.

Discussion

The data collected appears to support the prediction and the alternative hypothesis. Of the three tested pH buffers, pH 7 was the optimum for the enzyme. As pH conditions grew either more acidic or basic, the slopes of the their lines were smaller, meaning that their rate of reaction was smaller. The pH 7 condition produced the largest slope, and by observing the pressure at the end of the timeframe given for data collection, it is clear that much more oxygen was produced in that test tube. This signifies that the enzyme had a faster rate of reaction in an environment of pH 7 than those with pH 4 and pH 10, because the enzyme was able to convert more hydrogen peroxide into oxygen and water. The decrease in the rate of reaction occurred because enzymes often denature (unravel) when their environment is too acidic or basic. When enzymes are denatured, they cannot continue catalyzing the reaction because they have lost the shape that is so crucial to their function. A weakness of this experiment is that crucial seconds are lost in the time that it takes for the stopper to be inserted into test tube and for the experimenter to begin the data collection in LabQuest after the enzyme is actually added into the water and H2O2 solution. This breadth of this inaccuracy can be reduced by making sure to complete this step as quickly as possible. Another weakness is that the drops of enzyme dispensed from the pipettes are not completely accurate and are mostly likely all slightly varying in amount. Some test tubes may end up with marginally more enzyme suspension than others, which skews accuracy. To fix this limitation, drops of enzyme suspension can be measured out beforehand into a small graduated cylinder. Other questions for future study can include the effect of substrate concentration on the rate of reaction, the effect of the presence of an inhibitor, or what enzymes have the highest tolerance in environments of high acidity and basicity.

References

Lab Setup Image: http://vernier-videos.s3.amazonaws.com/training_html5/posters/16_9/Enzyme_Action_(Computer).jpg


Example poster layout created in Easel.ly


Factors Affecting Enzyme Activity Lab Handout

Lydia Guo

AP Biology, Mrs. Ferguson, Period 1