Transcript AP Bio Lab # 13 Enzyme Activity

AP Bio Lab # 13
Enzyme 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 baseline
2. Add Hydrogen Peroxide (H2O2) and Catalase
solution, swirl for 10 seconds
3. 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***
2. Swirl, if
solution turns
back clear,
continue
adding one
drop at a time
1. Start 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