INQUIRY LAB: ENZYMES Scientists __________________________________ Date __________ Block __________ Background Information: Enzymes are important in living organisms. They speed up chemical reactions. Without enzymes, we would not be alive. Their function is determined by their shape, so they are highly specific – they will work only with one set of reactants. If their shape gets changed (unfolds or denatures), they can’t work. Some environmental factors might change the shape of the enzymes and make them non-functional. A basic enzymatic and substrate reaction can be depicted as follows: Enzyme + Substrate → Enzyme-Substrate Complex → Enzyme + Products + ∆ G Two common types of enzymes are (1) catabolic enzymes that break things down (like lactase breaks down lactose) and (2) anabolic enzymes that build up. (You may know this second word already from stories about athletes who have been caught using illegal anabolic steroids to build muscle.) Catalytic enzymes, called proteases, break down proteins and are found in many organisms; one example is bromelain, which comes from pineapple and is an ingredient in commercial meat marinades. Papain is an enzyme that comes from papaya and is used in some teeth whiteners to break down the bacterial film on teeth. People who are lactose intolerant cannot digest milk sugar (lactose); however, they can take supplements containing lactase, the enzyme they are missing. All of these enzymes hydrolyze large, complex molecules into their simpler components; bromelain and papain break proteins down to amino acids, while lactase breaks lactose down to simpler sugars. Anabolic enzymes build larger molecules, such as combining amino acids to make proteins. In this investigation, you will focus on the enzyme peroxidase found in cells of many organisms (this peroxidase is from a turnip). Peroxidase is one of several enzymes that break down peroxide, a toxic metabolic waste product of aerobic respiration. Using peroxidase, you will develop essential skills to examine your own questions about enzyme function. You will use hydrogen peroxide (H2O2) as a substrate. A peroxidase in your cells is what makes hydrogen peroxide bubble if poured on a cut. Like all enzymes the peroxidase is not used up during the reaction. The reaction is: Peroxidase + Hydrogen Peroxide Peroxidase + Water + Oxygen 2H2O2 → 2H2O + O2 (gas) In the second part of this investigation you will have an opportunity to design and experiment to test the effect of one environmental factor on the rate of this chemical reaction. Prelab: Answer the following review questions in complete sentences. 1. What is the purpose of enzymes in a biological system? 2. How does shape of the active site affect the function of the enzyme? 3. What is the function of peroxidase in a cell? 4. How will the following affect the rate of an enzymatic reaction? (a) increase in the amount of substrate (b) increase in the amount of enzyme (c) increased concentration of products 5. What are two environmental factors that affect enzyme function? active site 6. What causes denaturation of a protein? 7. How does denaturation affect the structure and function of an enzyme? Part 1: Showing the effect of an enzyme on the rate of a chemical reaction. We will be using the reaction : 2H2O2 → 2H2O + O2 (gas). To see how fast the reaction occurs, we will measure how much oxygen (O2) is produced. This could be done by measuring the actual volume of oxygen gas released or by using an indicator. In this experiment, an indicator for oxygen will be used. The compound, guaiacol, has a high affinity for oxygen, and in solution, it binds instantly with oxygen to form tetraguaiacol, which is brownish in color. The more oxygen gas produced the darker brown the solution will become. To get a measure of the color, we’ll use a color chart ranging from 1 to 10 (available at each lab station) is sufficient to compare relative amounts of oxygen produced. Step 1 Using tape and a marker label the test tubes A and B. Step 2 Using the syringes Add solutions to test tubes A and B as indicated in the table below. tube label A B solution to add 7.5 mL of diluted H2O2 (substrate or reactant) 0.2 mL of guaiacol (color indicator) – located in the hood 7.5 mL enzyme Step 3 Pour the contents of test tube A into test tube B. Pour back and forth twice to mix, then immediately begin timing the reaction. Compare the color of the solution in the test tube to the color chart at your lab station, and record the color at time zero. Step 4 Observe the color change for the next 5 minutes and record the observed color at 0, 1, 2, 3, 4, and 5 minutes in the ‘with enzyme’ column. The reaction without enzyme will be done as a demonstration. Minutes 0 1 2 3 4 5 Color of solution (1-10) Without enzyme With enzyme GRAPH 1: compare reaction rate by graphing the color of solution (y axis) vs the time (x axis)be sure to include all items needed for a graph (DRY, MIX, TALK) Analysis questions for Part 1. 1. How does the color change indicate that the reaction is occurring? (Refer to the information at the top of this page and be specific.) 2. What is the effect of the enzyme peroxidase on the rate of reaction? Part 2: What are the effects of environmental factors on the rate of reaction? A. Model: What things affect a chemical reaction? Create a mind map or model of the things that can affect the rate of reaction. Don’t forget enzymes and the things that affect enzyme shape and function. Indicate if they will increase or decrease the rate. B. Design an investigation: Select a variable from your model above that you would like to investigate and write a Research Question. You will be using the same reaction and basic directions. RESEARCH QUESTION C. After you get approval from the teacher, write the hypotheses: Null hypothesis (factor will not change rate): Alternative hypothesis (factor will increase or decrease rate and why): Get approval again before going on. EXPERIMENTAL DESIGN INDEPENDENT VARIABLE: ___________________________________ Experimental groups: (be specific; give actual temperature or pH – include all 5 of the groups) _________________________________________________________________ Control group: (choose one of the experimental groups for the control) ____________________________ DEPENDENT VARIABLE: (what is measured, how) _________________________________________________ CONSTANTS: (refer to the model) ______________________________________________________________ PROCEDURES (Use the basic directions for Part 1. Number the steps, label first, say what and how much of each solution, record the color of the reaction at 4 minutes only.) DATA TABLES (proper data tables) Group raw data table for your lab group Class average data table pH or 0C color GRAPH 2: compare the Effect of the IV on the DV, by graphing the color indicator (y axis) vs pH or temp ( x axis). (this is a graph of your groups data, NOT the class data; include all necessary labels) RESULTS Restate the hypothesis (The hypothesis was “...”), state the class means in words and numbers, summarize the class means, and state if hypothesis was supported or not supported. TABLES AND GRAPHS On a separate sheet of paper include a typed table of the class average from Part 2 (either pH or temperature depending on your group’s variable). On a separate sheet of paper include a typed graph done in EXCEL of the class averages from Part 2 (either pH or temperature, depending on your group’s variable) *** be sure to include Title (in all CAPS), table and axis labels, proper scales, , Conclusion Restate the hypothesis (The hypothesis was “...”), summarize the class means, and state if hypothesis was supported or not supported. Add three references to the conclusions to support your findings. ERROR ANALYSIS Error analysis discusses what went wrong (NOT everything that could have possibly gone wrong!). Look at the data. Does something not make sense? What might have caused that? Was some factor not held constant? How would that have affected the data? FUTURE RESEARCH Future research looks at the ‘next question.’ What other experiment could be done to learn more about enzymes? REFERENCES At least 3 references that are cited in the conclusion section and in proper APA FORMAT This paper is needed for the PEER Review to be done in class on _________________________. Turn in this portion of the lab (with the PEER Review comments) and the Results and Conclusions formal lab. For the lab write up – you will be completing a separate results and conclusions section based on the class average data. You will need 3 references used as in paper citations in your final conclusions section. Follow the instructions and rubric for this lab.