Edvotek kit # 282

Why?

For Biology II or AP biology Follow 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?

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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  H 2 O 2 -- catalase -  H 2 O + ½ O 2  How can we measure substrate used or product made?

We will measure the amount of H 2 O 2 remaining after catalysis by catalase by coupling to a secondary reaction with KI as follows: 2 I - + H + + H 2 O 2  2H 2 O + I 2 (colorless)(reddish brown)

How So? You will catalyze H 2 O 2 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 H 2 O 2 is broken down  Contains the acidic KI that can react with the remaining H 2 O 2

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 H 2 O 2 concentration  Therefore, as the catalase reaction proceeds, more H 2 O 2 is consumed, so overtime, Absorbance readings should decrease.

Divide into four groups Materials for each groupShared materials - 6mL reaction cocktail (buffered H 2 O 2 )  25mL Assay solution (contains acidic K I & 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 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 5 o C?  How would the graph be different if the reaction was done at 37 o C?  How would the graph be different at ph 4?