1. AP Bio Lab # 13 Enzyme Activity Pre-Lab

2. Before doing this lab, you should understand:
The general functions and activities of enzymes
The relationship between the structure and function of enzymes
The concept of initial reaction rates of enzymes
How the concept of free energy relates to enzyme activity (view graph)
That changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions (view graph)

3. Learning Objectives
Students will use the catalase reaction to demonstrate different variables’ effect on enzyme activity:
such as temperature and the presence of catalase.
Students will observe the reaction of hydrogen peroxide and catalase:
establish a baseline determining the amount of hydrogen peroxide in a 1.5% solution,
Determine the rate of spontaneous conversion of hydrogen peroxide to water and oxygen
determine the rate of hydrogen peroxide decomposition by enzyme catalysis.

4. 2 H2O2  2 H2O + O2 (gas) Background Info
In this experiment, we will be using the enzyme catalase.
found inside peroxisomes in almost all organisms.
catalyses the breakdown of hydrogen peroxide to water and oxygen.
Without this enzyme, hydrogen peroxide would destroy our cells because it is a toxic metabolic waste product of aerobic respiration.
2 H2O2  2 H2O + O2 (gas)

5. Background Info Cont:
Reaction is spontaneous (will occur naturally), but very slowly.
If catalase is boiled, it will become denatured and will no longer function to break down hydrogen peroxide.
The rate of this reaction will decrease as the concentration of hydrogen peroxide (the substrate) decreases.

6. Safety Read all instructions before beginning lab.
Wear personal protective eyewear (PPE) at all times, included during clean-up.
Wear gloves and aprons, will stain clothes!
Wash hands before leaving lab.

7. Show lab video

8. Part A: Demo (3 parts) Mix Hydrogen Peroxide (H2O2) + Catalase

9. The General Procedure H2SO4 stops the reaction!
Measure using Potassium Permanganate Drops
H2SO4 stops the reaction!

10. Part B: Establishing a Baseline – Determining the Amount of Hydrogen Peroxide (H2O2) in a 1.5% solution
Add Hydrogen Peroxide (H2O2)
Add Water (control)
Add Sulfuric Acid (H2SO4)
Titrate with Potassium Permanganate (KMnO4)
Amount of (KMnO4) used to titrate is proportional to amount of (H2O2) present in the solution

11. Part C: Demo Overnight
Control Group: Do NOT add any catalase enzyme, and measure rate of Hydrogen Peroxide Spontaneous Decomposition

12. Part D: Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis
After you get your baseline
Add Hydrogen Peroxide (H2O2) and Catalase solution, swirl for 10 seconds
Add Sulfuric Acid (H2SO4) to stop reaction
Titrate with Potassium Permanganate (KMnO4)
Repeat for 30, 60, 120, & 180 seconds
***Do in reverse order to improve your accuracy, start with 180, 120, 60, 30, 10***

15. 2. Swirl, if solution turns back clear, continue adding one drop at a time
3. Stop adding drops when solution remains a pinkish – yellowish color, measure & record amount of KMnO4 used.
1. Start Adding one drop at a time

16. Data
Record your data in the appropriate data table as you complete the lab.

17. Conclusion Analysis Questions