Lab#5 Enzymes: Working with Yeast Catalase

Lab session: 11Dec2016
– – Lab Overview:  This lab is about enzymes, specifically catalase, an enzyme in the peroxidase family.
– – We worked through the Toothpickase activity and did a yeast catalase experiment as laid out by Brad Williamson on the AP list. I took his ideas and suggestions and wrote up my own procedure and helpful information.
– – Please see below for a summary of the labs, as well as this link for background information (lots of helpful information and activities for learning about enzymes, don’t skip it!), and this link for the data analysis of your yeast catalase experiment. Please be ready with data and graphs and explanations of your experiment by next lab session.

 

1. Toothpickase Activity
This is a fun but surprisingly effective way to model enzyme activity using toothpicks.

In short, we have:

A bowl of toothpicks (100 to start) = bowl of substrate
A student’s fingers: the digestive enzyme that catalyzes the reaction of the toothpicks being broken into two pieces.

Here is a link to the original Toothpickase activity as invented by an AP Biology teacher named Kim Foglia years ago. She passed away but her generous sharing of activities and resources means her teaching methods live on. Bio_Toothpickase_Activity_Explore_Biology_Foglia I have included a quick summary below of the activity.

Experiment #1: Testing the Rate of Product Formation in an Enzyme  Facilitated Reaction.
– – Using a timer, have the student close their eyes and break as many toothpicks in 10-20 seconds, the others counting the number broken. Do not remove the broken toothpicks from the bowl. Do 10 runs to determine the standard rate (doing a few practice runs to get the fingers warmed up can result in more consistent data).

– – Graph the results.
– – Calculate the rate of enzyme action in toothpicks per second for each time interval:

Experiment #2: Effect of Substrate Concentration on Rxn Rate.
– – Use paper clips to make the “solution” of toothpicks less homogenous. What happens to the rate?

– – Calculate the rate of enzyme action in toothpicks per second. Compare the two rates (no paper clips and with paper clips).
– – Discuss your results and explain why the rates were different at different concentrations.
– – Summarize the effect of substrate concentration on enzyme action

Experiment #3: Effect of Temperature on Rxn Rate.
– – Have student place hands in ice/cold water before being timed in breaking toothpicks (remove paper clips and previously broken ones. What happens to the rate?
– – Graph the results.
– – Discuss your results and explain why the rates were different at different temperatures.
– – Summarize the effect of temperature on enzyme action.
– – – Explain what would happen to an enzyme-facilitated reaction if temperature were increased. Be sure to include the effect if temperature were increased to 100°C.
– – – What is the optimal temperature (°C) for enzymes functioning in the human body?
 
While the Toothpickase activity seems too simple to model a real-life enzymes, it really isn’t. Can you general a simple model of what it happening with the substrate and the enzyme? What has to happen before the enzyme can help the reaction to happen? What happens to the enzyme once the reaction is finished?

2. Yeast Catalase and H2O2
A simple paper based enzyme reaction that allows for analysis and learning about enzymes and catalysts.

First, I did a simple demo: I dissolved about a tablespoon of instant yeast in about a cup of warm water, letting it sit for about 30 minutes before the demo. Then, with the measuring cup of yeast solution on a baking sheet, I then poured about 1/2 cup of 3% hydrogen peroxide into the yeast. This is what happened!

How does it work?
This is an easy and affordable lab to measure/quantify enzyme action or kinetics using disks of filter paper soaked in a yeast solution as the enzyme source and a solution of hydrogen peroxide as the substrate.  Here’s a right up by Paula Donham on the technique:  http://www.kabt.org/wp-content/uploads/2009/02/catalase-enzyme-lab.pdf
 
Dip the paper disk (use a hole punch and some brown paper coffee filters) in the yeast solution.  The yeast solution provides a set amount of catalase per disk.  Drop the disk into a solution of hydrogen peroxide.  The catalase breaks down the hydrogen peroxide into water and oxygen.  The oxygen bubbles catch in the paper fibers and eventually cause the disk to rise. You can see the disk staring to rise in the lower right hand corner of the cup. You can use plastic cups to make a dilution series with the hydrogen peroxide and the 24 well plates for the testing.  The well plates allow you to put one disk per well (which might lead to better precision).
 
Here’s a short video of the procedure using the well plate:
 Dip a disk in the yeast, drop it into the hydrogen peroxide and time how long it takes to rise.

 
You will notice that I am not providing explicit instructions for running this lab. You will need to think about what is going on with the reaction and then design your own experiment to generate usable data that you can use to support your conclusions about what is happening in the experiment.
 Things to think about:
– – The original demo with yeast and H2O2 overran the measuring cup, so obviously these concentrations aren’t very useful for our disk assay. You need to figure out what yeast concentrations will work, and which H2O2 concentrations (you will have a fresh 3% bottle and an older 12% bottle).
– – Think about your procedure: timing, placing the disk, filling the wells with the same amount of solution, etc.

Keep copious notes in your notebook!

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