BIOL 1406 Properties of Enzymes: Peroxidase, A Case Study

Transcription

1 BIOL 1406 Properties of Enzymes: Peroxidase, A Case Study Objectives Name the class of macromolecules to which peroxidase belongs and the monomers that make it up. Name the substrates and products of the peroxidase catalyzed reaction. Explain the role of guaiacol in this experiment. Define enzyme, activation energy, active site, ph, and denaturation. Distinguish between oxidation/reduction, activation energy/catalysis, substrate/product, and hydrogen peroxide/peroxidase. Describe how temperature, ph, enzyme concentration, and substrate concentration affect the reaction rate. Explain why peroxidase is a necessary enzyme for all aerobic or oxygen-using cells. Background Information The thousands of chemical reactions that occur in a cell at any given time do not occur randomly, but are highly under the control of biological catalysts called enzymes. A catalyst is a substance that speeds up a chemical reaction without being consumed by the reaction. Enzymes speed up reactions by lowering the activation energy of a reaction, which is that initial amount of energy necessary to bring reactants together with the proper amount of energy and in the proper orientation so that the products can be formed. Most enzymes are proteins with particular primary structures dictated by genes. As proteins, upon their synthesis, enzymes assume particular shapes. This shape, especially in its active site, determines its catalytic effects. The active site of each enzyme binds to specific molecules for example, the enzyme sucrase binds to sucrose but not to lactose, even though both are disaccharides. The reactant molecule that binds with to the active site of an enzyme and undergoes chemical modification is called the substrate of that enzyme. Some enzymes bind to two substrates to form products. Certain enzymes have metallic ions (such as Cu 2+, Fe 2+, Mn 2+ ) as part of their active site These metal ions are called cofactors. Sometimes a cofactor is organic in nature and not an ion (e.g. some vitamins); in that case the cofactor is properly called a coenzyme. The binding between enzyme and substrate(s) consists of weak, non-covalent chemical bonds, forming and enzyme-substrate complex that exists for only a few milliseconds. During this instant, the covalent bonds of the substrate(s) either come under stress or are oriented in such a way that facilitates the formation of a different molecule or molecules. This results in the formation of a product. The product leaves the enzyme s active site and is used by the cell. The enzyme is unchanged by the reaction and may enter the catalytic cycle again if more substrate molecules are available. Enzyme + Substrate Enzyme-Substrate Enzyme + Products Complex Fig. 7-1 A pictorial representation of an enzyme-catalyzed reaction. 1

2 An individual enzyme may convert about a thousand substrate molecules in one second. Their fast rates combined with the fact that they are not used up during the reaction means that enzymes are needed in small amounts. Eventually enzymes wear out, break apart and lose their catalytic capacity. Cellular enzymes called proteases degrade inactive enzymes to amino acids, which are used by the cell to make other enzymes or other proteins. The amount of a particular enzyme found in a cell is determined by the balance between the processes that degrade the enzyme and those that synthesize it. When no enzyme is present, the chemical reaction catalyzed by the enzyme does not occur at an appreciable rate. Generally, if the concentration of the enzyme increases, the rate of the reaction will also increase. This is true up to a certain point. At some point the amount of substrate also plays a role in the rate of the reaction: adding more enzyme will not speed of a reaction if there is not sufficient substrate. The ph (which is a measure of the concentration of H + in an aqueous solution) and high salt concentrations affect the shape of enzyme molecules by interfering with ionic bonds that are necessary to maintain their tertiary structure. This, in turn, affects the substrate-binding efficiency of the enzyme. Temperature, within the physiological limits of 0 to 40 o C, affects the frequency with which the enzyme and its substrates collide and, hence, also affects binding. Extremely high temperatures typically cause enzyme denaturation, rendering the enzyme completely inactive. All factors that influence binding obviously affect the rate of enzyme-catalyzed reactions. Some of these factors will be investigated during this laboratory. During this experiment you will study a type of enzyme called peroxidase, which is a large protein containing an iron ion in its active site, acting as a cofactor. Peroxidase is a good experimental enzyme because it is easily prepared and assayed. Turnips, horseradish roots, and potatoes contain a large amount of this enzyme. For this experiment, we extracted the peroxidase from turnips. The normal function of peroxidases is to rid the cell of toxic hydrogen peroxide (H 2 O 2 ), which is produced as by-product in certain metabolic reactions. It is critical to remove this H 2 O 2 before the cells become damaged by it. We will carry out the peroxidase reaction in vitro, and will monitor its activity by observing the production of an oxidized product. The dye guaiacol (extracted from the guaiacum tree of Central, South America and the Caribbean) binds to peroxidase and becomes oxidized as the hydrogen peroxide is reduced to water. The complete reaction is as follows: peroxidase 4 H guaiacol (reduced) 8 H 2 O + tetraguaiacol (oxidized) COLORLESS BROWN To quantitatively measure the amount of brown color in the final product, the enzyme and substrates can be mixed in a tube and immediately placed in a spectophotometer. As color accumulates, the absorbance at 500 nm will increase. [The procedure for using a spectrophotometer was explained in the previous lab and you should take a few minutes to review those instructions before proceeding.] For each particular run, plots of absorbance versus time can be constructed. The data points of each run are connected by drawing a best-fit line that goes through (0, 0). The slope of each plot is directly related to the enzyme reaction rate for that run. 2

3 Factors Affecting Enzyme Activity Your laboratory instructor may decide to divide your class up into teams; each team will tackle one or more of the following experimental variables (A E). The results will then be shared at the end of the lab period and should be included in your lab report. To perform each of the tests on the experimental variables, you will be following the same basic procedure. Please read over the instructions for A, Effect of Enzyme Concentration, whether or not this is the variable that your group is experimenting with and make sure you understand the instructions before you continue on with the experiment. This is the same basic procedure that you used in the Enzyme Lab Practice (Lab. 6) For each of the following experimental variables (A E), use the Mixing Table for each variable to set up the tubes. After you have all of the tubes set up, with the appropriate volumes of solutions, you are ready to begin the experiment. In each test, tube 1 is always the control and is used to blank the spectrophotometer. You will always be mixing tubes 2 & 3 together, tubes 4 &5 together, tubes 6 & 7 together, and tubes 8 & 9 together (when applicable). Remember to mix the solutions in the test tubes and then pour the contents of the tube into the cuvette. The peroxidase extract has been prepared for you. The extract contains hundreds of different types of enzymes, including peroxidase. The Effect of Enzyme Concentration: Changing the amount of turnip extract changes the amount of peroxidase! 1. Obtain a beaker or cup of each of the following solutions: Turnip Extract, ph 5 buffer, 10mM H 2 O 2, and 25 mm guaiacol. You ONLY need to pour about 20-mL of each solution. Make sure the beakers (or cups) are properly labeled. 2. Obtain two 1-mL pipets and two 5- or 10-mL pipets. Use of the wrong pipet will cross contaminate your reagents and introduce errors into your subsequent experiments in this exercise. DO NOT CROSS CONTAMINATE! 3. Using the china marker on your trays, label seven test tubes from 1 to 7. Prepare these tubes according to Table 7-1. Amounts given are in ml. These tubes will be used for the following purposes: Tube 1: Control without hydrogen peroxide; to be used in calibrating the spectrophotometer Tube 2: Substrates. Tube 3: Low concentration of peroxidase. Tube 4: Substrates (same as tube 2) Tube 5: Medium concentration of peroxidase. Tube 6: Substrates (same as tube 2) Tube 7: High concentration of peroxidase. 3

4 Table 7-1 Mixing table for effect of enzyme concentration test. (All volumes in milliters) Tube Buffer (ph5) H 2 O 2 Extract Guaiacol Total Volume 1(Blank) Refer to the handout from the previous lab for the instructions on how to use the spectrophotometer. Adjust the wavelength to 500 nm for this experiment. 5. Pour the contents of Tube 1 into a cuvette. This tube is used to blank the spectrophotometer, so that any color caused by contaminants in the reagents will not influence subsequent measurements. 6. In your lab groups, one person should be the Timer, another the Spectophotometer Reader, and another the Data Recorder. Note the time to the nearest second and mix the contents of tubes 2 and 3 by pouring the contents of tube 3 into tube 2 two or three times. Mixing should be completed within ten seconds. The contents of this tube will now be referred to as the reaction mixture. 7. Immediately after mixing, add the reaction mixture to a cuvette, wipe the outside of the cuvette with a Kimwipe, and place the cuvette in the spectrophotometer. Read the absorbance at 20 sec intervals from the start of mixing. Record your measurements in Table 7-2. After two minutes (six readings) remove the tube from the spectrophotometer and visually note the color change. Discard the solution. 8. Mix the contents of tubes 4 and 5, pour into a cuvette, and repeat your measurements for two minutes at 20-second intervals. Record the results in Table Mix the contents of tubes 6 and 7, pour into a cuvette, and record the absorbance measurements in Table 7-2. Table 7-2 Results from effects of enzyme concentration (entries should be given as absorbance units at 500nm). Time (sec) Tubes 2 & ml extract Tubes 4 & ml extract Tubes 6 & ml extract

5 Activity 7-2 Temperature Effects on Peroxidase Activity: To determine the optimum temperature for peroxidase activity, perform the following experiment. 1. Set up four water baths: 1 In an ice bath at approximately 0 o C 2. At room temperature at about 23 o C 3. At 40 o C 4. At 60 o C 5. At 100 o C (boiled extract) 2. Number nine test tubes in sequence 1 through 11. Refer to Table 7-3 for the volumes of reagents to be added to each tube. Table 7-3 Mixing table for temperature experiment (all values in milliliters) Temperature Tube Buffer H 2 O 2 Extract Guaiacol Total Volume (ph5) 1(blank) C C C C C C C C o C o C ** **For the 100 o C sample, take 2 ml of the turnip extract and place it in a boiling water bath. After five minutes, remove the tube, let it cool and add 1 ml of this boiled extract in tube Pre-incubate the sample solutions at 0 o C, 40 o C and 60 o C for at least 15 minutes before running the enzyme reaction. 5

6 5. After reaching temperature equilibrium and calibrating the spectrophotometer with the contents of test tube 1, mix tubes 4 and 5 (room temperature), and measure changes in absorbance every 40 seconds for two minutes. Record the changes in absorbance in Table Repeat Step 5 for tubes 10, 11 (boiled extract). 7. For more accurate results run the experiments at 0 o C, 40 o C and 60 o C in the following way: After incubating in the proper temperature for at least 15 minutes, mix tube pairs (one pair at a time, e.g. tubes 2 & 3), pour the mix in a cuvette, and place the cuvette back in its appropriate temperature. You will be measuring absorbance every 40 seconds, so leave the cuvette in its bath until 10 seconds before it is time to read the spectrophotometer. After reading the absorbance, return the cuvette to the temperature bath and leave it there until 10 seconds before the next reading. Using this procedure, the temperature will remain more or less constant during the experiment. Table 7-4 Results from temperature effects on peroxidase activity (entries should be given as absorbance units at 500nm). Time (sec) Tubes 2 & 3 0 C Tubes 4 & 5 23 C Tubes 6 & 7 40 C Tubes 8 & 9 60 C Tubes 10 & o C Activity 7-3 ph Effects on Peroxidase Activity To determine the optimum ph for peroxidase, perform the following experiment. 1. Number nine test tubes 1 through Obtain two 1-mL pipets and two 5- or 10 ml pipets. 6

7 3. Set up the test tubes by adding the reagents to these tubes as described in Table 7-5. Amounts given are in ml. The ph buffers have been provided for you in the laboratory. 4. After calibrating the spectrophotometer using the contents of Tube 1 (in a cuvette), mix pairs of tubes one pair at a time (2 and 3, 4 and 5, 6 and 7, 8 and 9) and measure absorbance changes at 20-second intervals for two minutes for each mixed pair. Record the results in Table 7-5. Table 7-5 Mixing table for ph experiment (all values in milliliters). Amount Buffer Tube of H 2 O 2 Extract Guaiacol Total Volume Buffer ph 5 1 (blank) ph ph ph ph ph ph ph ph Table 7-6 Results from effects of ph on peroxidase activity (entries should be given as absorbance units at 500nm). Time (sec) Tubes 2 & 3 ph 3 Tubes 4 & 5 ph 5 Tubes 6 & 7 ph 7 Tubes 8 & 9 ph

8 The Effect of an Inhibitor on Peroxidase Hydroxylamine (HONH 2 ) has a structure similar to hydrogen peroxide (HOOH). This molecule binds with the iron atom at the active site of peroxidase and prevents hydrogen peroxide from entering the site. What will be the effect on enzyme activity? To test this hypothesis follow this procedure: 1. Mix five drops of 1% hydroxylamine and 2 ml of enzyme extract in a test tube, letting the mixture stand for at least five minutes. 2. Prepare 5 test tubes as indicated in Table 7-7. Table 7-7 Mixing table for inhibitor experiment (all volumes in milliliters). Tube Buffer (ph5) H 2 O 2 Extract Guaiacol Inhibitor and Extract Total Volume 1 (Blank) After calibrating the spectrophotometer with the contents of Tube 1, mix pairs one at a time (2 and 3; 4 and 5,) and measure the changes at 20-second intervals for two minutes. Record your measurements in Table 7-8. Table 7-8 Results for the enzyme inhibition experiment (entries should be given as absorbance units at 500nm) Time (sec) Tubes 2 & 3 Normal extract Tubes 4 & 5 Inhibitor-treated extract

9 Data Analysis NOTE: Appendix 1 has instructions for plotting graphs in the computer using Microsoft Excel. QUESTIONS 1.The data of absorbances and time obtained for each experiment should be graphed as part of the lab report. The x-axis should be the independent variable (time in seconds) and the y-axis should be the dependent variable (absorbance units). Explain why absorbance is considered the dependent variable. 2. The slopes of the linear portions of the curves are a measure of enzyme reaction rate. What are the units? 3. Explain why the slopes are different. 4. Does the activity of peroxidase vary with temperature? What is the optimum temperature? How do you know this? 5. Does the activity of peroxidase vary with ph? What is the optimum ph? 6. How did boiling affect the activity of peroxidase? 7. Was hydroxylamine a strong, moderate or weak inhibitor? Give the basis for your answer. References: 1. Dolphin, W.D., Exercise 4 (Properties of Enzymes) in: Biology Laboratory Manual, 4 th edition, 1997, WCB Publishers 2. Eberhard,C., General Biology Lab Manual, 1996, Harcourt 3. MadScientist network: 9

10 Appendix 1 Plotting Enzyme Activity Data Using Microsoft Excel Purpose: This document provides information for the use of Microsoft Excel to plot best-fit lines with the enzyme activity data obtained in the lab, and to calculate the slope of each best-fit. It also provides information for plotting bar graphs that represent differential plots of enzyme reaction rates under various conditions. This document will show you how to manipulate the data of the Effect of Enzyme Concentration experiment ONLY. After you learn this, you can follow the same basic procedure to plot your data on the effect of ph, inhibitor and Temperature on peroxidase (see addendum at the end of this handout). Laboratory 8 Plotting Best-Fit Lines of Absorbance vs. Time data 1. Double click on the Microsoft Excel icon to open the program. You will see a grid. Each box in the grid is called a cell. Each cell has an address made up of a letter indicating the column the cell is in and a number indicating the row the cell is in, e.g. the upper left cell is A1. Note: a. To save paper, I will show only the whole screen view on the first picture; future diagrams will be simplified, showing only the data area. b. Unless specified otherwise, when you are asked to click, use the left mouse button (LMB). 2. Double click on cell A1 to start entering information. Using the diagram on the next page as a reference, enter the data of time (in seconds) and absorbance readings of YOUR enzyme concentration experiment (numerical data will be on cells A3 through D9). 3. Click on cell A3 with the left mouse button (LMB) and drag the mouse to highlight the area containing all the data points (go down to cell D9). The screen should look like this: 10

11 Time Absorbance 1/2 x 1x 2x Once the data is highlighted, look on the upper toolbar and click on Insert, then on Chart. A box with graph options will appear. Select XY(Scatter) and click on Next. Your data will appear already plotted on an x-y axis system. Now you will learn how to set-up the axes write the title of the graph and write the legend. You will also command the computer to draw best-fit lines that go through (0,0), and to calculate the slope of each plot. You can also choose to make some cosmetic touches (changing the size of fonts, colors of the plot etc. ) on your graph before printing. 5. Find Series towards the top of the graph and click on it. Series1 should appear highlighted. To the right of this there is an empty space for you to write the name of this plot: click on this empty box and type 1/2x [enzyme]. Now highlight Series2 by clicking on it with the LMB. Just as you did for series1, name this series: 1x [enzyme]. Repeat the procedure for Series3 (name it 2x [enzyme]). 6. Click on Next. Another box will appear with options to modify titles on the graph. a. Click on the blank space below Chart Title and name your graph (e.g. The effect of Enzyme Concentration on Peroxidase Activity ). b. Click on space below Value(X)Axis and type: Time (seconds) c. Click on space below Value(Y)Axis and type: Absorbance d. Click on Axes (upper left of graph) and make sure that Value(X)Axis and Value(Y)Axis have a checkmark. e. Click on Gridlines and alter the appearance of these as you choose. (I chose to show only Major Gridlines on the y-axis.) f. Click on Legend and choose the location of the legend (I chose bottom). 7. Click on Next and then on Finish. 11

12 Absorbance Absorban ce Your graph will appear on the screen as shown below. You can alter its size by clicking the LMB on the black squares around the graph and dragging the mouse with the LMB pressed down. Time Absorbance 1/2 x 1x 2x The Effect of Enzyme Concentration on Peroxidase Activity Time (sec) 1/2x [enzyme] 1x [enzyme] 2x [enzyme] You can move the whole graph by clicking the LMB (and holding it down) on the white of area of the graph until the cross symbol appears. With the LMB down, drag the graph below the area containing your data points. The graph should look something like this: Time Absorbance 1/2 x 1x 2x The Effect of Enzyme Concentration on Peroxidase Activity Time (sec) 1/2x [enzyme] 1x [enzyme] 2x [enzyme] 12

13 Absorbance 8. Now you are going to add the line that goes through the data points (trendline). Click on the graph to make sure that the black squares around the graph are there. a. Move the pointer to any of the data points of a particular series (any Δ, for example). As you do this, information about this data point will appear on the screen. Press the RIGHT mouse button. A box with choices appears. Select and click on Add Trendline. Another box will appear. Select Linear (should already be highlighted). Before clicking OK, go to Options on the top of the box. Click on the box Set intercept = 0 ( should appear) and on Display equation on chart. Click on OK. The graph will show a best-fit line that goes through 0,0 for the data points of that series. Also on the graph you should see an equation (e.g. y = x) which gives you the value of the slope for that series). b. Repeat the steps outlined in 8a to add the trendline through the two other Series. The graph should look something like this: Time Absorbance 1/2 x 1x 2x The Effect of Enzyme Concentration on Peroxidase Activity y = x y = x y = x Time (sec) 1/2x [enzyme] 1x [enzyme] 2x [enzyme] Linear (2x [enzyme]) Linear (1x [enzyme]) Linear (1/2x [enzyme]) Cosmetic changes for graphs: 13

14 After cosmetic changes, your graph may look like the one shown on the next page. a. Changing the size of the font used for the equation: Click on the equation to be changed. Click the RIGHT mouse button. Select and click on Format Data Labels. Click on Font and reduce the font size (I chose 8). Click on OK. b. Changing the position of the equation: Click on the equation and drag that box to the desired position. c. Changing the size of the axis label: Click on the desired label to change (e.g. the x axis label that says Time in seconds ). Click the RIGHT mouse button. Select and click on Format Axis Title. Click on Font and reduce the font size (I chose 10). Click on OK. Do the same for the other axis. You may also want to alter the size of the numbers on the scale. Click the RIGHT mouse button on top of the scale numbers. Click on Format Axis, then set the font to the desired size (I chose 8). Do this for the scale on the other axis also. d. To edit the Legend box: Click on the Legend box. Click on linear 2x [enzyme] until you see black squares around its box. Press the RIGHT mouse button, select and click on Clear. Do the same for the other items like these ( linear ) on the legend box. You can also alter the size of the font for the legend items. Click on top of 1/2x [enzyme](for example) with the RIGHT mouse button until black squares around it appear. Select Format Legend, and change the font size as desired (I chose 10). Do this for all the items in the legend. e. To change the color of the graph background: Press the RIGHT mouse button while the cursor is on the gray area. Select and click on Format Plot Area. You may want to choose a white background if your printer is black and white. 9. Go to the top toolbar. Click on File, Print Preview and check if the graph is ready to print. If it s not ready, try more cosmetic changes as desired, until you are satisfied with the product. When it is ready, SAVE your data: go to File, Save As and choose the proper destination. (If working at home, use your hard drive, if working at school, choose drive A: after inserting a formatted 3 ½ diskette. 10. Click on Print and OK. 14

15 Ab sorbance The Effect of Enzyme Concentration on Peroxidase Activity y = x y = x y = x Time (sec) 1/2x [enzyme] 1x [enzyme] 2x [enzyme] II. Obtaining the differential plots (bar graphs) Once you have the values of the slopes, you can build the bar graphs of enzyme reaction rate versus the various parameters tested. 15

16 Reaction Rate (absorbance units/sec) 1. Under your previous graph, use 6 cells to enter the relative enzyme concentrations used and the values for the three slopes obtained with the previous graph: Relative RxnRate [Enzyme] 1/2 x x x Highlight the data as you learned before (down LMB, dragging mouse over data points). Rel.[Enz] RxnRate 1/2 x x x Click on Insert Chart. Select Column (=bar graph). Click on Next. a. Click on Titles. 1. Name the chart (Example: Peroxidase reaction rates at different enzyme concentrations) 2. Name the x-axis: Relative concentrations of peroxidase 3. Name y-axis: Reaction rate (absorbance units/sec) b. Click on Legend to change the position of the legend box if desired. c. Click on Finish. 4. Drag the chart below your data points and enlarge it by dragging the black squares with the LMB. At this point your graph should look like the graph on the next page. Peroxidase Reaction Rates at Different Enzyme Concentrations /2 x 1x 2x Relative Concentrations of Peroxidase 16 RxnRate

17 4. You can make any other cosmetic changes as desired. [I chose a white plot area; axes and legend labels with a size 8 font, and a chart title with a size 12 font, boldface. I also chose a white background for the plot area and deleted the legend box completely] Note: For the color of the bars, I clicked with the RIGHT mouse button when the pointer was inside the bar. That activates the Format Data Series box, which allows you to change the color after clicking on it. I chose light gray.] 5. To check your graph, click on File, Print Preview. 6. Save your data (click on File, Save As. Type concentr2.xls after selecting the a drive as your destination) 7. The final graph may look like the one on next page. 17

18 Reaction Rate ( absorbance units /s ec) Peroxidase Reaction Rates at Different Enzyme Concentrations /2 x 1x 2x Relative Concentrations of Peroxidase Laboratory 7 ADDENDUM Notes for plotting Temperature, ph and Inhibitor graphs: 1. Temperature: a. Absorbance vs Time graphs 18

19 Enter your data with the following format: Time Temp. (oc) (sec) data data data data data 80 data data data data data 120 data data data data data add this row of 0 s When ready to Insert Chart, make sure to highlight ONLY the cells that contain data and the added row of 0 s: data data data data data 80 data data data data data 120 data data data data data After selecting Insert, Chart, XY (Scatter), and clicking on NEXT, make sure to select: Series in COLUMNS, NOT IN ROWS! Label each series according to which particular temperature they represent (0 o C, 23 o C, ) b. Bar graphs of temperature experiment Make sure that the data is entered with this specific format: T=0 slope T=23 slope T=40 slope T=60 slope T=100 slope The slopes were obtained from the best-fit plots Later on, make sure to label the X-axis: Temperature ( o C). 2. ph experiment Absorbance vs. Time graphs: Use same procedure as for Enzyme concentration. Don t forget to add the row of zero s! Time ph 3 ph 5 ph 7 ph data data data data 40 data data data data 60 data data data data 80 data data data data 100 data data data data 120 data data data data 19

20 Bar graphs: Make sure to follow this specific format: ph 3 ph 5 ph 7 ph 9 slope slope slope slope The slopes were obtained from the best-fit plots 3. Inhibitor experiment Absorbance vs. Time graphs: Enter data as follows. Proceed as for ph or enzyme concentration experiment: Time without with inh data data 40 data data 60 data data 80 data data 100 data data 120 data data Bar graphs: Enter data with this format: with inhib no inhib. slope slope The slopes were obtained from the best-fit plots 20

21 Appendix 2 Writing the Enzyme Lab Report [ NOT CREATIVE WRITING! ] {ALL SECTIONS, EXCEPT THE TITLE, MUST BE LABELED ON YOUR REPORT} TITLE Should be short, formal, complete and specific. (E.g. Enzymes, the Goodies of Life IS NOT an appropriate title). The title should mention peroxidase since that is the enzyme under study. Your name goes below the title. I. ABSTRACT: This is a short paragraph that summarizes the whole experiment. It is the part of a scientific paper that most likely will determine whether the reader will read the whole paper. Give the reader as MUCH information as possible (including purpose, procedure and RESULTS!!) in a very concise way. Usually the Abstract is written in boldface and with a smaller font than the rest of the paper. II. INTRODUCTION In this section you want to give background information on the experiment. Since we want to verify your knowledge on this material, assume that your readers will be people that are not acquainted with enzymes and want to know accurate information about how they operate. -Use this section to define: enzyme, substrate, active site, enzyme-substrate complex, competitive inhibitor and make connections to the particular reaction that you are performing (what enzyme? from what source? what is the substrate? inhibitor? ). You should show the specific reaction under study and its coupling to the oxidation of guaiacol. -You should explain briefly what a spectrophotometer is, and why you can use this instrument to monitor the progress of this particular reaction. -In this section you can also explain how the data is analyzed (explain how are the enzyme reaction rates determined). -After all the background information, you should state what your hypotheses were concerning the effect of enzyme concentration, ph, temperature, and inhibitor on the peroxidase reaction, taking in consideration that hypotheses are educated guesses. III. MATERIALS AND METHODS You may want to start this section by stating that we followed the procedure given in our Laboratory Manual. Then, try to summarize the procedure as much as possible (no need to say that 4.7 ml of this was mixed with 0.1 ml of that, or we mixed tubes 4 and 5, etc.). IV. RESULTS: The results should be presented in a neat and organized manner. For this experiment you will show: 1. Four graphs of ABSORBANCE vs. TIME, one for each of the experiments done: ph, temperature, enzyme concentration and presence of inhibitor. All graphs have a title and a legend for each plot. All plots should be BEST-FIT lines that go through 0,0 (because at time zero there is no product present). 21

22 On each graph you will show the slope of each plot, which represents the enzyme reaction rate for that particular run. When using MSExcel to plot the graphs, the computer will be commanded to show the slope of the best-fits, and the slopes will be in units of absorbance units/second. 2. Four bar graphs, EACH showing data on REACTION RATE vs. the particular parameter studied (whether ph s, presence/absence of inhibitor, enzyme concentration or temperatures). Recall that the reaction rates were the slopes previously determined. The bar graphs facilitate the visualization of optimum conditions for the enzyme and, therefore, make it easier to write the conclusions. V. DISCUSSION (Conclusions): Indicate your findings in terms of optimum ph and Temperature for the enzyme, and the effect of enzyme concentration and the presence of an inhibitor on enzyme activity. (Was it a strong, moderate or weak inhibition?) Overall, you may want to postulate reasons for your findings (make connections with the results and your ideas of how enzymes operate). Were your hypotheses supported? How could you improve or expand the experiment? VI. REFERENCES: List ALL the sources used for the experiment and the writing of the report. The reference should include author, year paper was published, publisher, etc. Lab manuals: 1) Perez-Ramos S., Couvillon, S., Laboratory 7: Properties of Enzymes, in: Laboratory Manual for Biology 1406: Molecular and Cellular Aspects of Life 2/e. McGraw-Hill, 2005 Textbooks: 2) Solomon, E.P., Berg, L.R., Martin, D.W., Biology, 7 th edition, Thomson Learning, 2005 Articles in journal and magazines: 3) Power, P. and P. J. Fonteyn Effects of Acidity and Temperature on Salivary Amylase, Journal of Bionatural Stuff 45: 1-4. Websites: 4) 5) Note: these are not real websites 22