Lab 8 Activities Continued

Effect of Substrate Concentration on Peroxidase Activity

Overview

This activity will explore the activity of peroxidase when exposed to different concentrations of its substrate, hydrogen peroxide. The substrate concentrations that will be tested are 0.2X, 1X (standard amount), 2X, and 3X.

Additional Materials

In addition to the materials listed on Page 1, each lab group will need the following:

• extraction buffer

Method

Note: When adding the materials to the cuvette, always add the turnip extract LAST and place the cuvette in the spectrophotometer as quickly as possible. The reaction begins as soon as the extract comes in contact with hydrogen peroxide.

1. Set-up a cuvette for 1X substrate by adding each material in the order listed below:
   • 10 drops 0.02% hydrogen peroxide
   • 5 drops 0.2% guaiacol
   • 20 drops of extraction buffer
   • 10 drops turnip extraction last
2. Secure the lid and quickly invert the cuvette to mix. Place the upright cuvette into the SpectroVis Plus.
3. Press “Collect” to begin recording data.
4. Stop data collection after 200s.
5. “Export Data” as a “.csv” file to computer or a USB flash drive.
   • Ensure that it has saved as a “.csv” file.
   • This will open in a text editor or spreadsheet.
6. Empty the contents of the cuvette into the trash beaker.
7. Thoroughly rinse the cuvette using the water bottle.
8. Sequentially repeat the experiment by changing the amount of hydrogen peroxide and extraction buffer added to the cuvette, as seen in the table below.

Make sure all group members are given an opportunity to save the data to their USB drive or make arrangements to have another student email them the data.

Effect of Enzyme Concentration on Peroxidase Activity

Overview

This activity will explore the activity of peroxidase when different concentrations of the enzyme are used. The enzyme concentrations that will be tested are 0.2X, 1X (standard amount), 2X, and 3X.

Additional Materials

In addition to the materials listed on Page 1, each lab group will need the following:

• extraction buffer

Method

Note: When adding the materials to the cuvette, always add the turnip extract LAST and place the cuvette in the spectrophotometer as quickly as possible. The reaction begins as soon as the extract comes in contact with hydrogen peroxide.

1. Set-up a cuvette for 1X enzyme by adding each material in the order listed below:
   • 10 drops 0.02% hydrogen peroxide
   • 5 drops 0.2% guaiacol
   • 20 drops of extraction buffer
   • 10 drops turnip extraction last
2. Secure the lid and quickly invert the cuvette to mix. Place the upright cuvette into the SpectroVis Plus.
3. Press “Collect” to begin recording data.
4. Stop data collection after 200s.
5. “Export Data” as a “.csv” file to computer or a USB flash drive.
   • Ensure that it has saved as a “.csv” file.
   • This will open in a text editor or spreadsheet.
6. Empty the contents of the cuvette into the trash beaker.
7. Thoroughly rinse the cuvette using the water bottle.
8. Sequentially repeat the experiment by changing the amount of enzyme and extraction buffer added to the cuvette, as seen in the table below.

Make sure all group members are given an opportunity to save the data to their USB drive or make arrangements to have another student email them the data.

Presenting the Data

Use the data you saved on your USB drive to create scatterplots for each data set. You should make four scatterplots: pH, temperature, substrate concentration, and enzyme concentration. After creating the graphs, determine the optimum pH and temperature for turnip peroxidase.

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