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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A different take

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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?