Bio 107 Lab 5 Enzymes

The objective of this lab is to investigate the influence of temperature, pH and an inhibitor on enzyme activity. The enzyme we will be using in this activity is catechol oxidase derived from a potato. This is an enzyme found naturally in plants that oxidizes the organic molecule catechol (also found in plants) forming a dark brown compound that slows bacterial growth preventing “rot” in damaged plant tissues. The reactants for this enzymatic reaction are a light brown color when initially mixed together. The products of the enzymatic reaction are very dark. Therefore, the darker the solution, the more enzyme activity has occurred. Using what you already know about enzymes from lecture, form hypotheses about the function of this enzyme at different temperatures (eg. Enzymatic activity increases with decreasing temperatures), different pH, and in the presence of an inhibitor. Make predictions that will allow you to test your hypotheses (eg. If enzymatic activity increases with decreasing temperatures, then the tube at 4^oC will be the darkest brown, and the tube at 90^oC will be the lightest.)

Procedures

Temperature

Place 1ml of catechol in 4 different test tubes.

Place 1ml of catechol oxidase in 4 different test tubes.

Place 1 tube of catechol and 1 tube of catechol oxidase at each of the following temperatures and label the tubes: 4^oC, 20^oC, 40^oC, 90^oC.

Let the tubes stand for 5 minutes.

Pour each catechol tube into the catechol-oxidase tube at the same temperature.

Let the mixture stand for 15 minutes and record the color of each mixture.

pH

Place 1ml of catechol oxidase in 3 different test tubes and label them acid, water, base.

To the appropriate tube of catechol oxidase, add 1ml of acid, water, or base.

Add 1 ml of catechol to each of the tubes.

Let the tubes stand for 15 minutes and record the color of each mixture.

Inhibitor

In a single test tube, place 1ml of catechol, 1ml of Fruit Fresh (an inhibitor), and 1ml of catechol oxidase.

Let stand at 20^oC for 15 minutes and record the color of the mixture.

Again, you will be writing a lab report in the following format.

Introduction:

What are the objectives of your study?

What hypothesis (or hypotheses) do you propose and are testing?

What predictions did you make that allowed you to test the hypothesis?

Methods:

Exactly how did you test your hypothesis (or hypotheses)?

Be specific enough so that someone can read your report and repeat your experiments?

Results:

Report and summarize your data. Briefly note trends and report raw data in table.

DO NOT MAKE CONCLUSIONS IN THIS SECTION OF THE PAPER! Conclusions are for the “Discussion” section.

Discussion:

Restate your hypotheses and discuss whether or not the tests you perform supported or refuted them. What modifications would you make to your original hypotheses following these tests? Were there any variables that could have influenced the outcome of the tests that were not controlled for or considered beforehand?