Enzyme Kinetics of Turnip Peroxidase

Overview

In this activity, you will explore the effects of four different factors on the rate of action of the enzyme turnip peroxidase. The four factors that will be studied are pH, temperature, substrate concentration, and enzyme concentration.

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).

{H_2O_2} + {AH_2} \xrightarrow{\text{peroxidase}} {2H_2O} + {A}

This activity 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.

Before You Begin

Discuss the following questions with your lab group:

  1. What effect does pH have on the rate of enzyme activity? Explain this with respect to the nature of enzymes.
  2. Predict the optimal pH for turnip peroxidase activity.
  3. What influence do you think a cold temperature (ice bucket or 0-4ºC) would have on the rate of the turnip peroxidase activity?
  4. What influence do you think a warm temperature (around human body temperature or fever 37-40ºC) would have on the rate of turnip peroxidase activity?
  5. What outcome can you predict of the rates when changing the concentration of enzyme or substrate?

Materials Needed for Entire Lab Session

Each lab group will need the following materials for the entire lab session. Additional materials will be listed under the appropriate activity.

  • SpectroVis Plus spectrophotometer
  • laptop
  • 0.02% hydrogen peroxide
  • 0.2% guaiacol
  • turnip extract (which contains the turnip peroxidase enzyme)
  • cuvettes with lids
  • beaker
  • water bottle with nozzle

In addition, each student should bring their own USB flash drive to download the data.

Calibrating the SpectroVis Plus

  1. Plug SpectroVis to computer via USB cable.
  2. Launch Vernier Spectral Analysis (https://www.vernier.com/product/spectral-analysis/).
  3. Begin a new experiment by choosing “Absorbance vs. Time (Kinetics)”.
  4. Wait for lamp to warm up if necessary.
  5. Place a blank cuvette in the unit and press “Finish Calibration”.
  6. Enter a wavelength of 500 nm and press “Done”.
  7. Proceed to the individual activities.
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