Principles of Enzyme Catalysis
description
Transcript of Principles of Enzyme Catalysis
Edvotek kit # 282
Why?
For Biology II or AP biologyFollow up to: Introduction to
Protein structure & function
Properties of enzymes Factors that effect
proteins
How do enzymes work/
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 Shared materials
- 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
Tray for spectrophotometer
spectrophotometer
Procedure
Prepare tubes as directed p. 10-12 labeled as follows
For spectrophotometer readings
Con (control) RXN (reaction) B (Blank for spec) 0 0.5 1.0 1.5 2.0 2.5 3.0
Load 300uL from each of 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?