At the conclusion of the lab, the student should be able to:
- define the following terms: metabolism, reactant, product, substrate, enzyme, denature
- describe what the active site of an enzyme is (be sure to include information regarding the relationship of the active site to the substrate)
- describe the specific action of the enzyme catalase, include the substrate and products of the reaction
- list what organelle catalase can be found in every plant or animal cell
- list the factors that can affect the rate of a chemical reaction and enzyme activity
- explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)
- explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity
- explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically
- decreases enzyme activity
- explain why increasing enzyme concentration promotes enzyme activity
- explain why the optimal pH of a particular enzyme promotes its activity
- if given the optimal conditions for a particular enzyme, indicate which experimental conditions using that particular enzyme would show the greatest and least enzyme activity
Hydrogen peroxide is a toxic product of many chemical reactions that occur in living things. Although it is produced in small amounts, living things must detoxify this compound and break down hydrogen peroxide into water and oxygen, two non-harmful molecules. The organelle responsible for destroying hydrogen peroxide is the peroxisome using the enzyme catalase. Both plants and animals have peroxisomes with catalase. The catalase sample for today’s lab will be from a potato.
Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products. Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.
The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction.
Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme.
Environmental factors may affect the ability of enzymes to function. You will design a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these environmental factors on the ability of catalase to convert H2O2 into H2O and O2.
The Scientific Method
As scientists, biologists apply the scientific method. Science is not simply a list of facts, but is an approach to understanding the world around us. It is use of the scientific method that differentiates science from other fields of study that attempt to improve our understanding of the world.
The scientific method is a systematic approach to problem solving. Although some argue that there is not one single scientific method, but a variety of methods; each of these approaches, whether explicit or not, tend to incorporate a few fundamental steps: observing, questioning, hypothesizing, predicting, testing, and interpreting results of the test. Sometimes the distinction between these steps is not always clear. This is particularly the case with hypotheses and predictions. But for our purposes, we will differentiate each of these steps in our applications of the scientific method.
You are already familiar with the steps of the scientific method from previous lab experiences. You will need to use your scientific method knowledge in today’s lab in creating hypotheses for each experiment, devising a protocol to test your hypothesis, and analyzing the results. Within the experimentation process it will be important to identify the independent variable, the dependent variable, and standardized variables for each experiment.
Part 1: Observe the Effects of Catalase
- Obtain two test tubes and label one as A and one as B.
- Use your ruler to measure and mark on each test tube 1 cm from the bottom.
- Fill each of two test tubes with catalase (from the potato) to the 1 cm mark
- Add 10 drops of hydrogen peroxide to the tube marked A.
- Add 10 drops of distilled water to the tube marked B.
- Wait 60 seconds and measure the height of any bubbling you observe.
- Bubbling height tube A
- Bubbling height tube B
- What happened when H2O2 was added to the potato in test tube A?
- What caused this to happen?
- What happened in test tube B?
- What was the purpose of the water in tube B?
Part 2: Effects of pH, Temperature, and Substrate Concentration
From the introduction and your reading, you have some background knowledge on enzyme structure and function. You also just observed the effects of catalase on the reaction in which hydrogen peroxide breaks down into water and oxygen.
From the objectives of this lab, our questions are as follows:
- How does temperature affect the ability of enzymes to catalyze chemical reactions?
- How does pH affect the ability of enzymes to catalyze chemical reactions?
- What is the effect of substrate concentration on the rate of enzyme catalyzed reactions?
Based on the questions above, come up with some possible hypotheses. These should be general, not specific, statements that are possible answers to your questions.
- Temperature hypothesis
- pH hypothesis
- Substrate concentration hypothesis
Test Your Hypotheses
Based on your hypotheses, design a set of experiments to test your hypotheses. Use your original experiment to shape your ideas. You have the following materials available:
- Test tubes
- Catalase (from potato)
- Hydrogen peroxide
- Distilled water
- Hot plate (for boiling water)
- Acidic pH solution
- Basic pH solution
- Ruler and wax pencil
Write your procedure to test each hypothesis. You should have three procedures, one for each hypothesis. Make sure your instructor checks your procedures before you continue.
- Procedure 1: Temperature
- Procedure 2: pH
- Procedure 3: Concentration
Record your results—you may want to draw tables. Also record any observations you make. Interpret your results to draw conclusions.
- Do your results match your hypothesis for each experiment?
- Do the results reject or fail to reject your hypothesis and why?
- What might explain your results? If your results are different from your hypothesis, why might they differ? If the results matched your predictions, hypothesize some mechanisms behind what you have observed.
Communicating Your Findings
Scientists generally communicate their research findings in written reports. Save the things that you have done above. You will be use them to write a lab report a little later in the course.
Sections of a Lab Report
- Title Page: The title describes the focus of the research. The title page should also include the student’s name, the lab instructor’s name, and the lab section.
- Introduction: The introduction provides the reader with background information about the problem and provides the rationale for conducting the research. The introduction should incorporate and cite outside sources. You should avoid using websites and encyclopedias for this background information. The introduction should start with more broad and general statements that frame the research and become more specific, clearly stating your hypotheses near the end.
- Methods: The methods section describes how the study was designed to test your hypotheses. This section should provide enough detail for someone to repeat your study. This section explains what you did. It should not be a bullet list of steps and materials used; nor should it read like a recipe that the reader is to follow. Typically this section is written in first person past tense in paragraph form since you conducted the experiment.
- Results: This section provides a written description of the data in paragraph form. What was the most reaction? The least reaction? This section should also include numbered graphs or tables with descriptive titles. The objective is to present the data, not interpret the data. Do not discuss why something occurred, just state what occurred.
- Discussion: In this section you interpret and critically evaluate your results. Generally, this section begins by reviewing your hypotheses and whether your data support your hypotheses. In describing conclusions that can be drawn from your research, it is important to include outside studies that help clarify your results. You should cite outside resources. What is most important about the research? What is the take-home message? The discussion section also includes ideas for further research and talks about potential sources of error. What could you improve if you conducted this experiment a second time?