Edvotek kit # 282
PRINCIPLES OF ENZYME
CATALYSIS
Why?
For Biology II or AP biology
Follow up to:
Introduction to
 Protein structure &
 How do enzymes work/
function
 Properties of enzymes
 Factors that effect proteins
 How is enzyme activity
measured?
 What is an enzyme assay
and why are they
important?
What enzymes do
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Measuring Enzyme Activity
 Corn shucking analogy – what is measured?
 We are the enzymes (my friend)
 Corn = substrate
 Shucked corn = product
 How do we measure how fast we the
enzymes work ?
 How fast does our substrate/ corn disappear or…
 How fast does our shucked corn/product appear
What are factors that effect
enzyme activity?
 What if room is filled with corn?
 What if the room is too cold? Too hot?
 What if your fingers were broken?
 What if had to wear mittens?
Enzyme Assay
 Measuring activity at different temperatures
 Measuring activity at different pHs
 Measuring activity at different substrate
concentrations
This lab
 Enzyme being measured – catalase
 Reaction being catalyzed
 H2O2 --catalase- H2O + ½ O2
 How can we measure substrate used or
product made?
We will measure the amount of H2O2
remaining after catalysis by
catalase by coupling to a secondary
reaction with KI as follows:
2 I- + H+ + H2O2 2H2O + I2
(colorless)
(reddish brown)
How So?
You will catalyze H2O2 with
catalase in a reaction tube.
 Then you will take
aliquots out of the
reaction tube at 6
different time intervals
and mix with an assay
solution.
 The assay solution
does two things
 Stops the action of
catalase, so no more
H2O2 is broken down
 Contains the acidic KI
that can react with the
remaining H2O2
Color change can be measured by a
spectrophotometer.
 The more hydrogen peroxide present in the
aliquot removed, the more iodine is produced
in the secondary reaction
 The more iodine produced in the secondary
reaction, the more reddish brown color
produced.
 The more reddish brown color produced, the
greater the Absorbance reading (A) on the
spectrophotometer.
Thus,
 Absorbance corresponds to H2O2
concentration
 Therefore, as the catalase reaction proceeds,
more H2O2 is consumed, so overtime,
Absorbance readings should decrease.
Divide into four groups
Materials for each group
- 6mL reaction cocktail
(buffered H2O2)
 25mL Assay solution
(contains acidic KI &
chemicals to inactivate
catalase)
 1 mL catalase on ice
 1 mL phosphate buffer
 10 15mL tubes
 1000uL micropipet & tips
 2 5mL disposable pipets &
bulbs
Shared materials
 Tray for
spectrophotometer
 spectrophotometer
Procedure
Prepare tubes as directed
p. 10-12 labeled as follows
For spectrophotometer
readings
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 Load 300uL from each of
Con (control)
RXN (reaction)
B (Blank for spec)
0
0.5
1.0
1.5
2.0
2.5
3.0
your ten tubes into the
proper labeled well of the
spectrophotometer tray.
 Each group’s wells will be
read at the same time
 Record your Absorbance
readings in chart on p. 12
 Plot absorbance vs time on
graph on page 16
How should the graph look?
 How would the graph be different if the
reaction was done at 5oC?
 How would the graph be different if the
reaction was done at 37oC?
 How would the graph be different at ph 4?