BIOLOGY

Lab5-PropertiesofEnzymes.pdf

Laboratory 5 The Properties of Enzymes

Learning Objectives

• Perform a quantitative assay of enzymatic activity from a tissue extra using a spectrophotometer. • Understand the principles of a spectrophotometer including the absorption and transmission of light

by matter. • Organize and summarize data in concise tables and graphs including the rate of chemical reactions,

and incorporate these into a laboratory report on enzymatic activity. • Test the following hypotheses:

o The amount of enzyme does not affect the rate of the chemical reaction. o The temperature of the chemical reaction does not affect enzyme activity. o The pH of the solution does not affect enzyme activity. o Molecularly similar compounds do not affect the enzyme activity. o Boiling an enzyme prior to conducting a chemical reaction does not affect enzyme activity.

Introduction Thousands of chemical reactions take place in your body every minute of every day, day or night. Without enzymes, most of these reactions do not take place. Enzymes are unique biological protein catalysts, or molecules that at speed up chemical reactions generally by several orders of magnitude. As with all catalysts, enzymes lower the activation energy of a chemical reaction, or the amount of energy (i.e., spark) needed to initiate a reaction. All protein enzymes have unique 3D shapes that are determined by unique amino acid sequence. The protein sequence is determined, or coded, by specific genes. Within the enzyme lies a cavity, or the active site, which binds only those molecules that can fit in it. For instance, glucose and ribose are saccharides, but some enzymes will bind one and not the other because the selectivity of the active site. (Remember that glucose is a six-carbon sugar and ribose is a five-carbon sugar.) The active site is also where the chemical reaction occurs. Molecules that bind to an enzyme and are transformed by are called substrates. Once the substrate binds an enzyme, it causes the enzyme to change its shape, a process known as the induced-fit. At this point the enzyme is ready to perform its chemical reaction. Active sites often contain metallic ions such as Fe+3, Mg+2, Ca+2 or Mn+2, referred to as cofactors, which help bind the substrate and/or contribute to the catalytic activity of the enzyme. Vitamins or other small molecules also aid in the binding of the substrate to the active site, and these are called coenzymes. The enzyme binds the substrate with weak, non-covalent chemical bonds that forms an enzyme-substrate complex lasting a few milliseconds. In the induced-fit state of the enzyme-substrate complex, the covalent bonds of the substrate either come under stress or are oriented in such a manner that they more easily attacked by other molecules, for example, water such as in a hydrolysis reaction. The resulting chemical reaction creates a new molecule or the product of the reaction, and is immediately released by the enzyme. The enzyme is unchanged by the chemical reaction and will be ready to transform more substrates as soon as the active site is empty. Modified from Biology Investigations: Form, Function, Diversity & Process, 8th Edition, ed. By Warren D. Dolphin (2008) McGraw-Hill Companies.

Individual enzyme molecules can process several thousand substrates per second. Thus, a small amount of enzyme can convert large quantities of the substrate into product. Enzymes eventually wear out, break apart or lose their catalytic activity. Cellular proteinases degrade inactive enzymes into individual amino acids, which are recycled by the cell to make other structural and functional proteins. In cells, enzyme levels are determined by a balance between the processes that degrade enzymes and those that synthesize them. The shape of an enzyme is critical to its function. Several factors affect the shape of an enzyme including pH, salt concentration, and temperature. All of these factors can modify the shape of the enzyme and when the shape of the active site is modified then this has dire consequences for the activity of that specific enzyme. Temperature, for instance, has multiple effects. It can affect the frequency with which the enzyme collides with the substrate and there for it affects binding of the substrate; temperature extremes may affect intramolecular bonds within the enzyme and change its shape. Any factor that influences binding of the substrate to the active site, such as inhibitors, will obviously affect the rate of the enzyme catalyzed reaction. We will investigate several of these factors in this laboratory.

Peroxidase We will study an enzyme called peroxidase in this lab. It is a large protein containing several hundred amino acids and it has an iron ion located in its active site. Peroxidase is ideal for study because it is readily available, easy to prepare and assayed. Turnips, horseradish roots, and potatoes are rich in peroxidase. Peroxidase normally converts toxic hydrogen peroxide (H202), which is produced in certain metabolic reactions, into harmless water H20 and oxygen 02:

2 H202 2 H20 + 2 0:

The activity of the peroxidase enzyme is measured by determining the amount of water or radical oxygen generated. In this experiment, the amount of radical oxygen will be measured, but indirectly. An indicator dye that changes color when it reacts with the radical oxygen will track the activity of peroxidase. Guaiacol, which is normally colorless, reacts with radical oxygen and turns to a brown color that we can easily see and measure. A key feature of this approach is that guaiacol does not affect peroxidase activity itself. Measuring the amount for brown color in the enzyme solution will be an indication of peroxidase activity. By tracking the appearance of a brown color in the chemical reaction, we can gain key insights into peroxidase’s activity including the speed of the reaction.

Spectrophotometry There are many ways to quantify the amount of product generated by an enzyme or any chemical reaction. In this experiment, we will evaluate the amount of brown reaction created in an instrument called a spectrophotometer. A spectrophotometer measures the amount of light molecules absorb and transmit. Molecules and all matter exposed to light or electromagnetic radiation will absorb and/or transmit it. For instance, a red colored shirt appears red because the most of the visible light shining on it is absorb except the red colored wavelengths, which is transmitted and seen by our eyes. (In the dark, the shirt has no color due to the absence of light and for it absorb or transmit.) The ability to absorb visible light or any other type of electromagnetic magnetic radiation depends on the chemical structural composition of each molecule. A spectrophotometer works by shinning a specific wavelength of light on a sample and measuring the amount of light absorbed and transmitted by the sample. The intensity of light that penetrates the sample is compared to the input intensity. This allows us to determine how much light pass through the sample as well as how much light was absorbed by the sample. Spectrophotometry measurements or readings are reported as either as absorbance (in arbitrary units) or transmission (as % transmission); note that these are inversely proportional.

Laboratory Set-Up In this exercise, you will determine the effects of several factors on the enzymatic activity of peroxidase in a tissue extract. There are five parts to the lab and you must complete each. Before beginning, familiarize yourself with key three aspects of the lab.

• Spectrophotometric Quantitation

To quantify the amount of brown color generated by the peroxidase enzyme in tissue extract, the substrate (H202), tissue extract and dye indicator are mixed, transferred to a cuvette and immediately placed into the spectrophotometer to measure the change in solution’s color. As the color accumulates, the sample’s absorbance of light at 500 nm increases. You will record absorbance measurements at regular intervals for all of the chemical reactions performed. Prior to testing the tissue extract, the instrument settings must be adjusted to deliver “absorbance” measurements, instead of “transmission.” Second, background absorbance by factors other than the brown reaction product such as the plastic of the cuvette, water, salts and buffers are subtracted from the readings by “blanking” the instrument. Place a cuvette into the spectrophotometer with a solution that represents a “blank,” and select the blanking or zeroing option on the instrument. The instructor will guide you through this process.

• Pipetting

The transfer of specific amounts of liquids in experiments is performed with devices that provide accuracy and consistency. Pipettes are ideal measuring and transferring 1-ml – 20-ml volumes. A pipette is a cylindrical device with two openings -- one for picking-up and delivering the solution and the other where pressure is applied for moving the liquid. A pipetting bulb provides the pressure for picking-up and dispensing liquids, and is attached to the latter end. Pipetting or moving liquids with a pipette is an art! Practice transferring small volumes of water such a 1-ml, 1.5-ml, 2-ml and 4-ml from one test tube to another before you begin working on this lab. Get comfortable with this process because the accuracy and thus success of your experiments will depend on it. Note, use a 1ml or 5-ml pipette instead of a 10-ml pipette to measure a 1ml volume for greater accuracy and consistency in these experiments. Use one pipette per solution otherwise you will cross-contaminate your solutions. Label the sleeve or wrapping of each pipette with its solution being pipetted. Keep the sleeve to reinsert a used pipette for later use.

• Assay of a Peroxidase Activity – a Fast Chemical Reaction

The enzymatic reaction proceeds quickly. To control for this factor, the reaction’s reagents are divided into two test tubes. Once you are ready to perform the reaction, the tubes are mixed together quickly poured into a cuvette that is then placed into the spectrophotometer for immediate analysis. You will record the absorbance at 500 nm every 20 seconds for two minutes. At the end of each assay, you should have 6 readings or measurements of the absorbance. Work in groups of three (3) to streamline this process. Use the following scheme to run the assay. Student 1 will mix the two tubes (e.g., Tubes 2 & 3) twice, pour the mixture into the cuvette and place it into the spectrophotometer. This must be done quickly and within 20 seconds. Student 2 will start a watch as soon as the two tubes are mixed. They will call out time every 20 seconds for 2 minutes. Student 3 will observe the absorbance readings on the spectrophotometer and record them every 20 seconds after the reaction starts.

Preparation of the Peroxidase Tissue Extract The peroxidase tissue extract will be prepared before class by your instructor as follows in order to save time:

1. Weigh 1g - 10 g of peeled turnip, horseradish root, or potato on a double or triple beam balance. 2. Add 100 ml of ice cold (4°C) 0.1 M phosphate buffer at pH 7 to the tissue and mix to homogeneity in

an ice-cold grinding blender. Keep the extract on ice, or in the refrigerator at 4°C. 3. Each group will receive aliquots or portions of the tissue extract for the experiment.

Part I: The Effect of Tissue Extract Concentration on Enzymatic Activity The tissue extract contains hundreds of different types of enzymes, including peroxidase. The activity of the peroxidase enzyme will vary based on the size and age of the turnip, horseradish root, or potato; the extent of tissue homogenization; and the age of the extract. Only peroxidase, however, will react with H202. Before starting each experiment, develop a null hypothesis Ho and an alternative Ha hypothesis. For Part I, these should relate the rate of the reaction to the amount of tissue extract or enzyme amount to the rate of the reaction. An example will be given here, but you can make your own hypothesis:

Ho: The amount of enzyme added to the reaction will have no effect on the rate of the reaction. Ha: The amount of enzyme added to the reaction changes the rate of the reaction.

Materials and Methods 1. Label one 50 ml tube for each of the following solutions as follows:

• Tissue extract (keep on ice) • Buffer, pH 5 • 10 mM H202 • 20 mM guaiacol Stock chemical solutions are made by your instructor. Fill each 50 ml tube about 1/3 full with the appropriate solution. Never pour a solution back into its stock container due to the possible contamination.

2. Label seven glass test tubes from 1 to 7. Each tube will contain the following:

Tube 1: Control (no tissue extract) Tube 2: Substrate and indicator dye Tube 3: Tissue extract (0.5 ml) Tube 4: Substrate and indicator dye (same as Tube 2) Tube 5: Tissue extract (1.0 ml) Tube 6: Substrate and indicator dye (same as Tube 2) Tube 7: Tissue extract (2.0 ml)

Table 1. Mixing Table for the Effect of Tissue Extract on Enzyme Activity

Solution Test Tube #

1 – control 2 3 4 5 6 7 Buffer (pH 5) 5.0 ml

4.5 ml

4.0 ml

3.0 ml

H2O2 2.0 ml 2.0 ml

2.0 ml

Tissue Extract

0.5 ml

1.0 ml

2.0 ml Guaiacol (dye) 1.0 ml 1.0 ml

1.0 ml

Total Volume 8.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml

3. Fill each of the seven test tubes with the appropriate solution specified in the mixing table using a pipette. 4. Use the control Tube 1 to blank the spectrophotometer. First, mix its contents by pouring the solution back- and-forth with another empty test tube. Once the instrument is blanked, return the solution back to its tube. 5. Mix Tubes 2 & 3 by pouring the solutions back-and-forth between the two tubes. Start the timer once you begin mixing the solution. Fill a cuvette 2/3rd of its volume with the mixed solution and place the cuvette into the spectrophotometer immediately. Record six (6) absorbance measurements at 500 nm in Table 2. Dump the reaction mixture in the waste receptacle and rinse the cuvette clean with distilled water. 6. Perform Step 5 with Tubes 4 & 5. 7. Perform Step 5 with Tubes 6 & 7.

Table 2. Absorbance Measurement at 500 nm for the Effect of

Tissue Extract on Enzyme Activity Time (sec.) Tubes 2 & 3

0.5 ml Extract Tubes 4 & 5

1.0 ml Extract Tubes 6 & 7

2.0 ml Extract 20 40 60 80 100 120

Data Analysis Perform these analyses after class. However, answer question 4 below with your raw data before moving forward to the next assay. 1. Plot the values on Table 2 on one graph. The abscissa is the independent variable (time) and the ordinate is the dependent value (absorbance). The abscissa is usually plotted on the x-axis and the ordinate on the y-axis. Explain why the ordinate is considered the dependent variable. 2. In mathematical terms, what is the characteristic shape of the graphed line for each reaction run? What does this say about the rate of chemical reaction? 3. What are the units of activity of this experiment? 4. Did any of the enzyme reactions produce a linear change in absorbance from 0 to 1 units over the 2 minutes that you measured the reaction? Use this tissue concentration for all subsequent assays. 5. Why is it important to examine enzyme activity over a linear concentration range? 6. Do your results support or reject your Ho and Ha hypothesis? Explain why in each case.

Part II: The Effect of Temperature on Enzymatic Activity In this assay, you will determine the effect of temperature on enzymatic activity. Peroxidase activity will be measured at four different temperatures including 4˚C, 23˚C, 32˚C and 48˚C. Develop a null hypothesis Ho and an alternative Ha hypothesis for the effect of temperature on peroxidase activity.

Ho: Ha:

Materials and Methods 1. Label one 50 ml tube for each of the following solutions as follows:

• Tissue extract (keep on ice) • Buffer, pH 5 • 10 mM H202 • 20 mM guaiacol • Ice bucket • Heated water baths at 32˚C and 48˚C

2. Label nine glass test tubes from 1 to 9. Fill each of the nine test tubes with the appropriate solution specified in the mixing Table 3.

* This volume may differ based on the results from Part I.

3. Place each set of test tubes in their corresponding temperature for 10-15 minutes prior to running the reaction. 4. Use the control Tube 1 to blank the spectrophotometer. Once the instrument is blanked, return the solution back to its tube. 5. Mix Tubes 2 & 3 by pouring the solutions back-and-forth between the two tubes. Start the timer once you begin mixing the solution. Fill a cuvette 2/3rd of its volume with the mixed solution and place the cuvette into the spectrophotometer immediately. Record six (6) absorbance measurements at 500 nm in Table 4. Dump the reaction mixture in the waste receptacle and rinse the cuvette clean with distilled water. 6. Perform Step 5 with Tubes 4 & 5. 7. Perform Step 5 with Tubes 6 & 7. 8. Perform Step 5 with Tubes 8 & 9.

Table 3. Mixing Table for the Effect of Temperature on Enzymatic Activity

Solution Test Tube #

1 2 3 4 5 6 7 8 9 control 4˚C 23˚C 32˚C 48˚C

Buffer (pH 5) 5.0 ml

4.0 ml

4.0 ml 4.0 ml H2O2 2.0 ml 2.0 ml

2.0 ml

Tissue Extract

1.0 ml *

1.0 ml * 1.0 ml * Guaiacol (dye) 1.0 ml 1.0 ml

1.0 ml

Total Volume 8.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml

Table 4. Absorbance Measurement at 500 nm for the Effect of Temperature on Enzymatic Activity

Time (sec.) Tubes 2 & 3 4˚C

Tubes 4 & 5 23˚C

Tubes 6 & 7 32˚C

Tubes 8 & 9 48˚C

20 40 60 80 100 120

Data Analysis Perform these analyses after class. 1. Plot the values on Table 4 on one graph. The abscissa is the independent variable (time) and the ordinate is the dependent value (absorbance). 2. Does peroxidase activity depend on temperature, and if so, how? 3. Calculate the slope in the linear portion of each reaction. 4. Plot the slope calculated at each temperature in Step 4 against its temperature. What are the independent and the dependent variable?

Temp. (˚C)

Enzyme Activity (△A/min)

4˚ 23˚ 32˚ 48˚

5. What is the shape of the curve plotted in Step 4? What does this plot tell us? 6. What is the optimum temperature for peroxidase activity and why? 7. Do your results support or reject your Ho and Ha hypothesis? Explain why in each case.

Part III: The Effect of pH on Enzymatic Activity In this assay, you will determine the effect of pH on enzymatic activity. Peroxidase activity will be measured at four different pHs including pH 3, pH 5, pH 7 and pH 9. Develop a null hypothesis Ho and an alternative Ha hypothesis for the effect of pH on peroxidase activity.

Ho: Ha:

Materials and Methods 1. Label one 50 ml tube for each of the following solutions as follows:

• Tissue extract (keep on ice) • Buffer, pH 3 • Buffer, pH 5 • Buffer, pH 7 • Buffer, pH 9 • 10 mM H202 • 20 mM guaiacol

2. Label nine glass test tubes from 1 to 9. Fill each of the nine test tubes with the appropriate solution specified in the mixing Table 5.

* this volume may differ based on the results from Part I.

3. Use the control Tube 1 to blank the spectrophotometer. Once the instrument is blanked, return the solution back to its tube. 4. Mix Tubes 2 & 3 by pouring the solutions back-and-forth between the two tubes. Start the timer once you begin mixing the solution. Fill a cuvette 2/3rd of its volume with the mixed solution and place the cuvette into the spectrophotometer immediately. Record six (6) absorbance measurements at 500 nm in Table 6. Dump the reaction mixture in the waste receptacle and rinse the cuvette clean with distilled water. 5. Perform Step 5 with Tubes 4 & 5. 6. Perform Step 5 with Tubes 6 & 7. 7. Perform Step 5 with Tubes 8 & 9.

Table 5. Mixing Table for the Effect of pH on Enzymatic Activity

Solution Test Tube #

1 2 3 4 5 6 7 8 9 control pH 3 pH 5 pH 7 pH 9

Buffer 5.0 ml (pH 5)

4.0 ml (pH 3)

4.0 ml (pH 5)

4.0 ml (pH 7)

4.0 ml (pH 9)

H2O2 2.0 ml 2.0 ml

2.0 ml

2.0 ml Tissue Extract

1.0 ml *

1.0 ml * 1.0 ml *

Guaiacol (dye) 1.0 ml 1.0 ml

1.0 ml

1.0 ml Total Volume 8.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml 3.0 ml 5.0 ml

Table 6. Absorbance Measurement at 500 nm for the Effect of pH on Enzymatic Activity

Time (sec.) Tubes 2 & 3 pH 3

Tubes 4 & 5 pH 5

Tubes 6 & 7 pH 7

Tubes 8 & 9 pH 9

20 40 60 80 100 120

Data Analysis Perform these analyses after class. 1. Plot the values on Table 6 on one graph. The abscissa is the independent variable (time) and the ordinate is the dependent value (absorbance). 2. Does peroxidase activity depend on pH, and if so, how? 3. Calculate the slope in the linear portion of each reaction. 4. Plot the slope calculated at each temperature in Step 3 against its pH. What are the independent and the dependent variable?

pH Enzyme Activity (△A/min)

3 5 7 9

5. What is the shape of the curve plotted in Step 4? What does this plot tell us? 6. What is the optimum pH for peroxidase activity and why? 7. Do your results support or reject your Ho and Ha hypothesis? Explain why in each case.

Part IV: The Effect of an Inhibitor on Enzymatic Activity In this assay, you will determine the effect of an inhibitor, hydroxylamine (HONH2), on enzymatic activity. Peroxidase activity will be measured at three different hydroxylamine concentrations. Develop a null hypothesis Ho and an alternative Ha hypothesis for the effect of an inhibitor on peroxidase activity.

Ho: Ha:

Materials and Methods 1. Label one 50 ml tube for each of the following solutions as follows:

• Tissue extract (keep on ice) • Buffer, pH 5 • 5% hydroxylamine • 10 mM H202 • 20 mM guaiacol

2. Dilute the 1% hydroxylamine to make a 2.5% and 1% solution with distilled water. Make 10 ml of each. 3. Label nine glass test tubes from 1 to 9. Fill each of the nine test tubes with the appropriate solution specified in the mixing Table 7.

* this volume may differ based on the results from Part I.

4. Use the control Tube 1 to blank the spectrophotometer. Once the instrument is blanked, return the solution back to its tube. 5. Mix Tubes 2 & 3 by pouring the solutions back-and-forth between the two tubes. Start the timer once you begin mixing the solution. Fill a cuvette 2/3rd of its volume with the mixed solution and place the cuvette into the spectrophotometer immediately. Record six (6) absorbance measurements at 500 nm in Table 8. Dump the reaction mixture in the waste receptacle and rinse the cuvette clean with distilled water. 6. Perform Step 5 with Tubes 4 & 5. 7. Perform Step 5 with Tubes 6 & 7. 8. Perform Step 5 with Tubes 8 & 9.

Table 7. Mixing Table for the Effect of an Inhibitor on Enzymatic Activity

Solution Test Tube #

1 2 3 4 5 6 7 8 9 control H2O 5% inhibitor 2.5% inhibitor 1% inhibitor

Buffer 5.0 ml

4.0 ml

4.0 ml 4.0 ml H2O2 2.0 ml 2.0 ml

2.0 ml

Tissue Extract

1.0 ml *

1.0 ml * 1.0 ml * Guaiacol (dye) 1.0 ml 1.0 ml

1.0 ml

Inhibitor 1 ml H2O

1.0 ml 5% inh.

1.0 ml 2.5% inh.

1.0 ml 1% inh.

Total Volume 8.0 ml 3.0 ml 6.0 ml 3.0 ml 6.0 ml 3.0 ml 6.0 ml 3.0 ml 6.0 ml

Table 8. Absorbance Measurement at 500 nm for the Effect of an Inhibitor on Enzymatic Activity

Time (sec.) Tubes 2 & 3 Control

Tubes 4 & 5 5% inhibitor

Tubes 6 & 7 2.5% inhibitor

Tubes 8 & 9 1% inhibitor

20 40 60 80 100 120

Data Analysis Perform these analyses after class. 1. Plot the values on Table 8 on one graph. The abscissa is the independent variable (time) and the ordinate is the dependent value (absorbance). 2. Does the inhibitor affect peroxidase activity, and if so, how? 3. What about hydroxylamine’s molecular structure allows it to inhibit peroxidase activity? 4. Do your results support or reject your Ho and Ha hypothesis? Explain why in each case.

Part V: The Effect of Boiling on Enzymatic Activity Most, but not all, proteins are denatured and completely corrupted when they are heated to temperatures above 70˚C. Denaturation produces an irreversible change in a protein’s shape. In this assay, you will determine if boiling the tissue extract will affect peroxidase activity. Develop a null hypothesis Ho and an alternative Ha hypothesis for the effect of boiling on peroxidase activity.

Ho: Ha:

Materials and Methods 1. Label one 50 ml tube for each of the following solutions as follows:

• Tissue extract (keep on ice) • Buffer, pH 5 • 10 mM H202 • 20 mm guaiacol

2. Add 3 ml tissue extract to a glass test tube. Place the test tube in a boiling water bath for 10 min. Remove from the test tube from the water bath and cool to room temperature (approx. 5-10 min.). 3. Label three glass test tubes from 1 to 3. Fill each of the three test tubes with the appropriate solution specified in the mixing Table 9.

* this volume may differ based on the results from Part I.

4. Use the control Tube 1 to blank the spectrophotometer. Once the instrument is blanked, return the solution back to its tube. 5. Mix Tubes 2 & 3 by pouring the solutions back-and-forth between the two tubes. Start the timer once you begin mixing the solution. Fill a cuvette 2/3rd of its volume with the mixed solution and place the cuvette into the spectrophotometer immediately. Record six (6) absorbance measurements at 500 nm in Table 9. Dump the reaction mixture in the waste receptacle and rinse the cuvette clean with distilled water.

Table 9. Mixing Table for the Effect of Boiling on Enzymatic Activity

Solution Test Tube #

1 2 3 control

Buffer (pH 5) 5.0 ml 4.0 ml H2O2 2.0 ml 2.0 ml

Tissue Extract

1.0 ml * Guaiacol (dye) 1.0 ml 1.0 ml

Total Volume 8.0 ml 3 5

Table 10. Absorbance Measurement at 500 nm for the Effect of Boiling on

Enzymatic Activity Time (sec.) Tubes 2 & 3

20 40 60 80 100 120

Data Analysis Perform these analyses after class. 1. Plot the values on Table 8 on one graph. The abscissa is the independent variable (time) and the ordinate is the dependent value (absorbance). 2. Does the boiling the tissue extract affect peroxidase activity, and if so, how? Compare the effect of boiling with previous experiments where the tissue extract was not boiled. 3. Do your results support or reject your Ho and Ha hypothesis? Explain why in each case.

Summary Questions 1. What is meant by temperature optimum? 2. What is meant by pH optimum? 3. Provide a molecular level explanation for protein denaturation. Why is this process irreversible? 4. What evidence, if any, do you have from these experiments that peroxidase’s activity depends on its shape? Critical Thinking Questions 1. Would performing the “temperature effects” part of this lab on a very hot and humid day have any effect on the results? Explain. 2. If you used a cooked turnip to create the tissue extract for this lab, what results would you expect? 3. In the human digestive system, different enzymes have different optimal pHs. What happens when stomach enzymes, which are optimally effective in acid, enter the alkaline environment of the small intestine? 4. If you are hired to supervise a manufacturing plant that enzymatically converts complex sugars in corn into monosaccharides, how do you go about determining if the chemical processes are operating at maximum rates? For the Lab Report In preparing your Lab Report, consider the following points along with the other instructions: 1. In the Introduction, present a null and alternative hypothesis, H0 and Ha, for all five experiments conducted. 2. Clearly describe the methods for each experiment. 3. In the Results, include all graphs of raw data and derivative data. 4. In the Results, graph and describe the enzyme activity as △A/min for temperature and pH. 5. In the Discussion, describe whether the results support or reject the null and alternative hypothesis, H0 and Ha, for all five experiments conducted. 6. In the Discussion, describe how the different factors evaluated here may or may not affect enzymatic activity. Discuss how these factors modify the enzyme’s molecular structure and affect its activity.