Enzyme kinetics of Turnip Peroxidase
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Enzyme Kinetics with Spectrovis CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 1 Contents 1 Enzyme kinetics of Turnip Peroxidase 2 Set-up and Calibrate SpectroVis Plus 3 Effect of pH on Peroxidase Activity 4 Effect of Temperature on Peroxidase Activity 5 Effect of Substrate Concentration on Peroxidase Activity 6 Effect of Enzyme Concentration on Peroxidase Activity Enzyme kinetics of Turnip Peroxidase Hydrogen peroxide (H 2 O 2 ) 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 H 2 O by oxidizing an organic compound (AH 2 -> 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. Enzymatic activity can then be traced using a spectrophotometer to measure the amount of brown being formed. Set-up and Calibrate SpectroVis Plus Plug SpectroVis to computer via USB cable 1. Launch Vernier Spectral Analysis (https://www.vernier.com/product/spectral-analysis/ ) 2. Begin a new experiment by choosing “Absorbance vs. Time (Kinetics)” 3. Wait for Lamp to warm up if necessary 4. Place a blank cuvette in the unit and press “Finish Calibration” 5. Enter a wavelength of 500nm and press “done” 6. Proceed to the individual exercises 7.
Transcript of Enzyme kinetics of Turnip Peroxidase
Enzyme Kinetics with Spectrovis
CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 1
Contents
1 Enzyme kinetics of Turnip Peroxidase2 Set-up and Calibrate SpectroVis Plus3 Effect of pH on Peroxidase Activity4 Effect of Temperature on Peroxidase Activity5 Effect of Substrate Concentration on Peroxidase Activity6 Effect of Enzyme Concentration on Peroxidase Activity
Enzyme kinetics of Turnip PeroxidaseHydrogen peroxide (H2O2) is a strong oxidizing agent that can damage cells and is formed asa by-product of oxygen consumption. Fortunately, aerobic cells contain peroxidases thatbreak down peroxide into water and oxygen. This enzyme reduces hydrogen peroxide intoH2O by oxidizing an organic compound (AH2 -> A).
This exercise uses turnip extract as a source of peroxidase. This turnip extract requires asource of electrons (a reducing agent) in order for the reaction to occur. In this case, acolorless organic compound called guaiacol is used. Guaiacol is oxidized in the process ofconverting the peroxide and becomes brown. Enzymatic activity can then be traced using aspectrophotometer to measure the amount of brown being formed.
Set-up and Calibrate SpectroVis PlusPlug SpectroVis to computer via USB cable1.Launch Vernier Spectral Analysis (https://www.vernier.com/product/spectral-analysis/)2.Begin a new experiment by choosing “Absorbance vs. Time (Kinetics)”3.Wait for Lamp to warm up if necessary4.Place a blank cuvette in the unit and press “Finish Calibration”5.Enter a wavelength of 500nm and press “done”6.Proceed to the individual exercises7.
Enzyme Kinetics with Spectrovis
CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 2
Effect of pH on Peroxidase ActivitySet-up cuvette for pH 31.
Add 10 drops 0.02% hydrogen peroxide1.Add 5 drops 0.2% guaiacol2.Add 20 drops of pH 3 buffer3.Add 10 drops turnip extraction last4.Quickly invert the cuvette to mix and place the upright cuvette into the5.Spectrovis PlusPress “Collect” to begin recording data6.
Stop data collection after 200s1.“Export Data” as a “.csv” file to computer or a USB flash drive2.
Ensure that it has saved as a “.csv” filethis will open in a text editor or spreadsheet
Sequentially repeat the experiment exchanging the pH buffer with pH 5, 7, 102.
Effect of Temperature on Peroxidase ActivitySet-up cuvette for 0ºC1.
Add 10 drops 0.02% hydrogen peroxide (incubated at 0ºC for 10 minutes)1.Add 5 drops 0.2% guaiacol2.Add 20 drops of extraction buffer at 0ºC3.
Enzyme Kinetics with Spectrovis
CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 3
Add 10 drops turnip extraction (incubated at 0ºC for 10 minutes) last4.Quickly invert the cuvette to mix and place the upright cuvette into the5.Spectrovis PlusPress “Collect” to begin recording data6.
Stop data collection after 200s1.“Export Data” as a “.csv” file to computer or a USB flash drive2.
Ensure that it has saved as a “.csv” filethis will open in a text editor or spreadsheet
Sequentially repeat the experiment exchanging the buffers stored at 20ºC, 40ºC, 60ºC2.
Effect of Substrate Concentration on PeroxidaseActivity
Set-up cuvette for 1X substrate1.Add 10 drops 0.02% hydrogen peroxide1.Add 5 drops 0.2% guaiacol2.Add 20 drops of extraction buffer3.Add 10 drops turnip extraction last4.Quickly invert the cuvette to mix and place the upright cuvette into the5.Spectrovis PlusPress “Collect” to begin recording data6.
Stop data collection after 200s1.“Export Data” as a “.csv” file to computer or a USB flash drive2.
Ensure that it has saved as a “.csv” filethis will open in a text editor or spreadsheet
Sequentially repeat the experiment with differing amounts of buffer and peroxide:2.Repeat the experiment with 0.2X substrate and export data to USB drive1.Repeat the experiment with 2X substrate and export data to USB drive2.Repeat the experiment with 3X substrate and export data to USB drive3.
Enzyme Kinetics with Spectrovis
CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 4
Effect of Enzyme Concentration on Peroxidase ActivitySet-up cuvette for 1X enzyme1.
Add 10 drops 0.02% hydrogen peroxide1.Add 5 drops 0.2% guaiacol2.Add 20 drops of extraction buffer3.Add 10 drops turnip extraction last4.Quickly invert the cuvette to mix and place the upright cuvette into the5.Spectrovis PlusPress “Collect” to begin recording data6.
Stop data collection after 200s1.“Export Data” as a “.csv” file to computer or a USB flash drive2.
Ensure that it has saved as a “.csv” filethis will open in a text editor or spreadsheet
Sequentially repeat the experiment with differing amounts of buffer and extract:2.Perform the experiment on 1X enzyme and export data to USB drive1.Repeat the experiment with 0.2X enzyme and export data to USB drive2.Repeat the experiment with 2X enzyme and export data to USB drive3.Repeat the experiment with 3X enzyme and export data to USB drive4.
Enzyme Kinetics with Spectrovis
CC-BY-NC-SA | Jeremy Seto | New York City College of Technology | 5
Tags: quantitative reasoning
