Enzymes: Part 2

 

Quick Introduction

 

Previously, we investigated how the enzyme catalase increases the rate of hydrogen peroxide decomposition. Remember that catalase binds with hydrogen peroxide (the substrate) at a specific location called the active site. It is this binding that leads to the rapid formation of the products, water and oxygen gas.

 

This week we will investigate how differing environmental conditions will impact the ability of catalase to catalyze this reaction. The key to understanding this lab is to remember that anything that can change the shape of the active site of catalase has the potential to alter hydrogen peroxide binding and, therefore, catalase activity. The process that changes the shape of an enzyme is called denaturation.

 

We have provided the equipment/materials to create six different environments in which to run your catalase reaction. You will be able to:

 

 

What to do:

  1. Have a discussion with your lab group members and choose one of the above environments to test. Check with other groups in the lab to make sure that you are doing something different.
  2. Once you have chosen your environment, formulate an RQ.  Remember to look at the suggestions on the online lab manual for this week.
  3. Pose a hypothesis and design an experiment to test it. Before you do any work, answer the following questions:

 

Tools

Enzyme Extraction: You will need to extract enough enzyme so that you can do each control and experimental group three times. The extract may be provided for you by the instructor.

 

Catalase Assay: Refer to the online lab manual and last weeks “quick link” to refresh your memory about the catalase assay procedure. For most experiments, you will use the same amount of substrate and enzyme in each assay as you did last time. (The exception will be experiments designed to test the effect of varying substrate concentration on enzyme activity. In that type of experiment, the amount of enzyme will be kept constant.) The difference in this lab is that you will subject the enzyme (catalase preparation) to the various environmental conditions before you measure its activity in the assay.

 

Calculation of specific activity. Last week catalase activity was expressed as mmHg/sec. This is sufficient for comparisons within an experiment, but does not allow for comparisons with other lab groups that might have used different amounts of turnip in their catalase extraction. To compensate for this, specific activity is calculated by dividing the relative catalase activity expressed as mmHg/sec/ml by the turnip tissue concentration in the extraction expressed as g/ml.

  1. To determine your catalase activity divide the mmHg/sec rate by the amount (in ml) of catalase solution you used in that assay. So if your activity was 3 mmHg/sec with 2 ml of catalase solution, then the final activity was 3/2 or 1.5 mmHg/sec/ml
  2. To determine the turnip tissue concentration, divide the amount of turnip that you weighed by the final volume of extract that you recovered after grinding and filtering.  So, if you weighed out 30 g of turnip and measured a final volume of extract as 45 ml, then the concentration would be 30/45 or 0.67 g/ml
  3. To calculate specific activity, divide catalase activity by the turnip concentration:

1.5/0.67 or 2.24 mmHg/sec/g turnip tissue

For your work today, plot all of your catalase results as specific activity and not relative activity.

Assignment

1.      Prepare a final figure of the results from your investigation.

2.      Provide a brief explanation of the experimental design.

3.      Determine the trends in the data (if any).

4.      Determine if the results support the hypothesis.

5.      Explain your results in terms of the relevant biological principle involved.

6.      List ways that the experiment could be improved if it was repeated.