AP Bio Lab # 13Enzyme Activity

Pre-Lab

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)

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.

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)

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.

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.

Show lab video

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

The General Procedure

• H2SO4 stops the reaction! Measure using Potassium Permanganate Drops

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

Part C: Demo Overnight

• Control Group: Do NOT add any catalase enzyme, and measure rate of Hydrogen Peroxide Spontaneous Decomposition

Part D: Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis

1. After you get your baseline2. Add Hydrogen Peroxide (H2O2) and Catalase

solution, swirl for 10 seconds3. Add Sulfuric Acid (H2SO4) to stop reaction

4. Titrate with Potassium Permanganate (KMnO4)

5. Repeat for 30, 60, 120, & 180 seconds***Do in reverse order to improve your accuracy, start with 180, 120, 60, 30, 10***

1. Start Adding one drop at a time

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.

Data

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

Conclusion Analysis Questions