3.7: Enzyme Kinetics with Spectrovis
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Enzyme Kinetics of Turnip Peroxidase
Hydrogen peroxide (H2O2) is a strong oxidizing agent that can damage cells and is formed as a by-product of oxygen consumption. Fortunately, aerobic cells contain peroxidases that break down peroxide into water and oxygen. This enzyme reduces hydrogen peroxide into H2O by oxidizing an organic compound (AH2 -> A).
This exercise uses turnip extract as a source of peroxidase. This turnip extract requires a source of electrons (a reducing agent) in order for the reaction to occur. In this case, a colorless organic compound called guaiacol is used. Guaiacol is oxidized in the process of converting the peroxide and becomes brown. The enzymatic activity can then be traced using a spectrophotometer to measure the amount of brown being formed.
Set-up and Calibrate LabQuest with SpectroVis Plus
- Set-up the cuvette to serve as a Blank.
- Add 10 drops 0.02% hydrogen peroxide.
- Add 5 drops 0.2% guaiacol.
- Add 20 drops of pH 7 buffer.
- Add 10 drops extraction buffered.
- Connect a LabQuest2 to a SpectroVis plus and start the data collection program.
- Calibrate the SpectroVis.
- Change Mode.
- Select “Time-Based” from the dropdown menu and press OK when done.
- Change Mode.
- Rate: 0.5 samples/s
- Interval: 2 s/sample
- Duration: 200 s
2. Press the Play button (green arrow).
1. Perform Warm-up for 90 seconds.
1. Choose "Finish Calibration".
2. Press "OK".
2. Press the "Stop" button (Red Square).
4. Select “Meter Icon” (top left most of the display).
- Press the large red area showing the current absorbance reading.
- Choose “Change Wavelength”.
- Set Wavelength to 500nm.
Effect of pH on Peroxidase Activity
- Set-up the cuvette for pH 3.
- Add 10 drops 0.02% hydrogen peroxide.
- Add 5 drops 0.2% guaiacol.
- Add 20 drops of pH 3 buffer.
- Add 10 drops turnip extraction last.
- Quickly invert the cuvette and place the cuvette into the Spectrovis Plus.
- Press play button to begin recording data (choose “discard data” if prompted).
- After 200s, remove SpectroVis from USB.
- Insert a USB flash drive and wait 1 minute.
- Press File → Export → choose USB icon and rename the file and add “.csv” to the end of the file name.
- Press OK.
- Sequentially repeat the experiment exchanging the pH buffer with pH 5, 7, 10.
Effect of Temperature on Peroxidase Activity
- Set-up the cuvette for 0ºC
- Add 10 drops 0.02% hydrogen peroxide (incubated at 0ºC for 10 minutes).
- Add 5 drops 0.2% guaiacol.
- Add 20 drops of extraction buffer at 0ºC.
- Add 10 drops turnip extraction (incubated at 0ºC for 10 minutes) last.
- Quickly invert the cuvette and place the cuvette into the Spectrovis Plus.
- Press play button to begin recording data (choose “discard data” if prompted).
- After 200s, remove SpectroVis from USB.
- Insert a USB flash drive and wait 1 minute.
- Press File → Export → choose USB icon and rename the file and add “.csv” to the end of the file name.
- Press OK.
- Sequentially repeat the experiment exchanging the buffers stored at 20ºC, 40ºC, 60ºC.
Effect of Substrate Concentration on Peroxidase Activity
- Set-up the cuvette for 1X substrate.
- Add 10 drops 0.02% hydrogen peroxide.
- Add 5 drops 0.2% guaiacol.
- Add 20 drops of extraction buffer.
- Add 10 drops turnip extraction last.
- Quickly invert the cuvette and place the cuvette into the Spectrovis Plus.
- Press play button to begin recording data (choose “discard data” if prompted).
- After 200s, remove SpectroVis from USB.
- Insert a USB flash drive and wait 1 minute.
- Press File → Export → choose USB icon and rename the file and add “.csv” to the end of the file name.
- Press OK.
- Sequentially repeat the experiment with different amounts of buffer and peroxide:
- Repeat the experiment with 0.2X substrate and export data to a USB drive.
- Repeat the experiment with 2X substrate and export data to a USB drive.
- Repeat the experiment with 3X substrate and export data to a USB drive.
Effect of Enzyme Concentration on Peroxidase Activity
- Set-up the cuvette for 1X enzyme.
- Add 10 drops 0.02% hydrogen peroxide.
- Add 5 drops 0.2% guaiacol.
- Add 20 drops of extraction buffer.
- Add 10 drops turnip extraction last.
- Quickly invert the cuvette and place the cuvette into the Spectrovis Plus.
- Press play button to begin recording data (choose “discard data” if prompted).
- After 200s, remove SpectroVis from USB.
- Insert a USB flash drive and wait 1 minute.
- Press File → Export → choose USB icon and rename the file and add “.csv” to the end of the file name.
- Press OK.
- Sequentially repeat the experiment with different amounts of buffer and extract:
- Perform the experiment on 1X enzyme and export data to a USB drive.
- Repeat the experiment with 0.2X enzyme and export data to a USB drive.
- Repeat the experiment with 2X enzyme and export data to a USB drive.
- Repeat the experiment with 3X enzyme and export data to a USB drive.