Materials Included In Kit

Additional Materials Required

Prelab Preparation

pH 7 extraction buffer: Prepare 750 mL of pH 7 phosphate buffer by mixing equal volumes, 375 mL each, of 0.1 M sodium phosphate monobasic (NaH₂PO₄) and sodium phosphate dibasic (Na₂HPO₄) solutions. Verify pH using pH meter; it should be between 6.8 and 7.2.

pH 5 reaction buffer: Dissolve a pH 5 buffer envelope or buffer capsule in distilled or deionized water according to package instructions.

Hydrogen peroxide solution, 0.006%: Dilute 3 mL of 3% hydrogen peroxide to a final volume of 500 mL using distilled or deionized water. Store in a cool, dark area protected from heat and light. Prepare fresh before use.

Turnip peroxidase enzyme extract: Peel and cut a turnip root into small cubes, about 1 cm on each side. Measure approximately 2 g (2 pieces) in a weighing dish and add to 500 mL of pH 7 phosphate extraction buffer in a blender. Pulse the turnip root in 1−3 minute bursts three times, with 2 minutes rest between pulses, to homogenize and extract the enzymes. Filter the turnip enzyme extract through filter paper and store the extract over ice or in the refrigerator. Prepare within one week of use.

Safety Precautions

Guaiacol is toxic by ingestion. It has an aromatic, creosote-like odor and may be irritating to the nose and throat. Isopropyl rubbing alcohol (70%) is a flammable liquid. Keep away from heat, flames and other sources of ignition. Dilute hydrogen peroxide solution may be irritating to the eyes and skin. Wear chemical splash goggles, chemicalresistant gloves and a lab coat or chemical-resistant apron. Avoid contact of all chemicals with eyes and skin, and remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Buffers and leftover isopropyl alcohol solutions may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Prior to lab, check the enzyme activity of the turnip peroxidase extract using the amounts shown in trial A-1. It is important to pretest the extract so that the absorbance (color) change occurs at a convenient rate that can be accurately measured, that is, neither too fast nor too slow. Absorbance values greater than one correspond to very low transmittance and are less accurate. Enzyme activity will depend on the source of the vegetable and its freshness. Adjust the amount of turnip used, as well as the volume of buffer solution, to obtain a convenient rate (an increase in absorbance of 0.3−0.8 units over a 3-minute period).
• A variety of plants may be used as sources of peroxidase. The options are abundant (e.g., turnip, horseradish, radish, lettuce, tomatoes, spinach, legumes). Fish peroxidases have also been widely studied.
• Peroxidases from different sources will likely have different pH and temperature profiles. In addition, peroxidase extracts from a single source normally contain a mixture of enzymatic forms, called isoenzymes or isozymes, which will also have different optimum values for pH and temperature stability.
• Initial rates are generally used to compare reaction rates for different concentrations of enzyme or substrate, and for determining optimum pH and temperature values. The initial rate is calculated from the slope or linear portion of a graph of product concentration (absorbance) versus time, corresponding to approximately 5–10% of reaction completion. This is done because a graph of product concentration versus time for a chemical reaction begins to curve or level off as the reaction proceeds.
• The procedure calls for pouring the contents of the enzyme tube into the substrate tube, and then back into the enzyme tube, prior to beginning absorbance measurements. This is done to ensure adequate mixing of the contents and to improve accuracy and precision in the measured reaction rates. Students should begin timing with the first pour, however, as soon as the enzyme and substrate are combined.
• The effect of substrate concentration on reaction rate gives rise to a characteristic hyperbolic or “saturation” kinetics curve. Understanding the shape of this curve provides insight into the single most important take-home lesson relating the structure and function of enzymes, namely, formation of the enzyme−substrate complex. At low substrate concentrations, the rate of the reaction increases almost linearly as the concentration increases. At higher concentrations of hydrogen peroxide, the rate of the reaction behaves differently than a typical chemical reaction. The rate increase becomes more gradual and eventually levels off and reaches a maximum saturation velocity. Saturation kinetics is observed because once all of the enzyme in solution (or in a cell) is bound with substrate at its active site, adding further substrate does not increase the rate of reaction.
• The concept of saturation kinetics discussed above may make it challenging to identify suitable substrate concentrations that will illustrate the “desired” results. If the substrate concentration in trial B-1 is already near the saturation level for the amount of enzyme (which depends on the activity of the extract and other factors), increasing or decreasing the substrate amount by a factor of two will not change the rate much at all. It may be necessary to modify the amounts of hydrogen peroxide in Part B to find concentrations that will work under the specific laboratory conditions.
• If spectrophotometers or colorimeters are not available, the concentration of colored product after a specific time interval can be estimated by color comparison with a series of standard solutions. Prepare a concentrated stock solution of the orange product by using a large concentration of hydrogen peroxide and guaiacol. Call the concentration of the stock solution x and then dilute it to obtain solutions that have concentrations equal to 0.2x, 0.4x, 0.6x and 0.8x. When comparing the color of a test solution versus the standards, it is essential that the path length for viewing the solution colors be identical. This means that all the solutions must be in the same size test tube, filled to the same depth or volume, and the color must be viewed in the same direction, either vertically down the length of the test tube or horizontally across the tube. As an example of this technique, imagine that in trial A-1 the color of the mixed enzyme–substrate solution after three minutes most closely matches the 0.4x color standard. When the enzyme concentration is doubled in trial A-2, the color of the solution after the same length of time (three minutes) might be expected to match the 0.8x color standard. This technique requires considerable trial-and-error experimentation to prepare a color series in the same general concentration range as the rate trials.
• Enzyme kinetics is the basis for important medical tests called enzyme assays to determine the concentration of an enzyme in blood, cells or tissue. Enzyme assays are useful in clinical diagnosis of disease. In an enzyme assay, the amount of product formed in a specific time period is measured to determine the reaction rate. This rate is then compared against a reference or calibration curve of enzyme activity versus enzyme concentration to determine the actual enzyme concentration in blood.
• Various naming systems are currently in use for identifying enzymes. Many enzymes, such as the digestive enzymes pepsin and trypsin, may have medical, consumer or commercial applications and are known by their common names. Recommended scientific names for enzymes generally consist of two words, the name of the substrate followed by the type of reaction that is catalyzed. The enzyme alcohol dehydrogenase, for example, catalyzes the oxidation—also known as dehydrogenation, because two hydrogen atoms are removed—of alcohol. In 1972 a systematic way of naming enzymes was adopted to prevent confusion and to show the relationships among different enzymes. According to this system, enzymes are classified into six groups based on the general type of chemical reaction that is catalyzed. Classes of enzymes and the reactions they catalyze include:
  • Oxidoreductases—oxidation/reduction reactions
  • Transferases—transfer of a functional group from one substrate to another
  • Hydrolases—hydrolysis reactions (reaction of water with substrates
  • Lyases—elimination reactions of substrate molecules to generate products containing a double bond
  • Isomerases—isomerization reactions
  • Ligases—reactions that form bonds between molecules

Answers to Prelab Questions

1. Define each of the following terms related to the properties of enzymes.
   1. Kinetics

      Kinetics is defined as the study of the rates of chemical reactions.

   2. Activation energy

      Activation energy is the minimum or threshold energy that reactants must have in order for a chemical reaction to take place. The activation energy depends on the reaction pathway or mechanism of a reaction. An enzyme changes the reaction pathway and decreases the activation energy.

   3. Substrate

      The substrate is the reactant in an enzyme-catalyzed reaction.

   4. Active site

      The active site is a specific region in the three-dimensional structure of a protein (enzyme) where the substrate binds to the enzyme prior to undergoing a chemical reaction.

2. Most enzymes have an optimum pH at which they are most active. Explain in terms of the properties of proteins.

   Most enzymes are proteins, which are composed of amino acids with acidic and basic side chains. The charge or ionization of these side chains depends on pH. Changing the pH may alter the total charge of a protein and disrupt the noncovalent interactions that keep the protein in its native, three-dimensional structure. Altering the shape of an enzyme may compromise the binding characteristics and the catalytic properties of the active site. The optimum pH reflects the optimum three-dimensional structure for enzyme–substrate binding and chemical reaction.

3. The most common method of increasing the rate of a chemical reaction in the laboratory is to increase the temperature. What are the limits or drawbacks of this method for studying enzymatic reactions?

   Enzymes are usually most active at the physiological temperature of an organism. Increasing the temperature beyond a certain point may lead to reversible or irreversible protein denaturation, which alters the enzyme structure and may destroy its function.

4. Compare the volumes of H₂O₂ (substrate) and peroxidase (enzyme) used in Parts A and B.
   1. Let x = concentration of enzyme in Trial A-1. Express the enzyme concentrations [E] in Trials A-2 and A-3 as multiples of x.

      Trial 1, [E] = x. Trial 2, [E] = 2x. Trial 3, [E] = 0.5x.

   2. Let y = concentration of H₂O₂ in trial B-1. Express the substrate [S] concentrations in trials B-2, B-3 and B-4 as fractions of y.
      {14005_PreLabAnswers_Table_1}

Sample Data

Laboratory Report

Part A. Effect of Enzyme Concentration

Part B. Effect of Substrate Concentration

Answers to Questions

Laboratory Report

1. Analyze the data for Part A: Using different colors and/or shapes for the data points in each trial, graph absorbance versus time on the following graph. Draw a best-fit straight line through the data points for each separate trial. Add a legend to identify the data corresponding to each trial.
   {14005_Answers_Figure_1}
2. Determine the slope (ΔAbsorbance/ΔTime) for each best-fit straight line in the above graph. This is the rate of reaction for each trial. Plot the rate of reaction versus the amount (volume) of enzyme.
   {14005_Answers_Figure_2}
3. Explain how the above graph for the effect of enzyme concentration on the reaction rate supports a dynamic theory for biological reactions.

   The rate of the enzyme-catalyzed reaction is directly proportional to the enzyme concentration. The rate increases in a linear manner. Biological reactions are dynamic events requiring collisions or interactions between enzyme and substrate molecules. Increasing the number of enzyme molecules increases the collision frequency and thus the rate.

4. Analyze the data for Part B: Using different colors and/or shapes for the data points in each trial, graph absorbance versus time on the following graph. Draw a best-fit straight line through the data points for each separate trial. Add a legend to identify the data corresponding to each trial.
   {14005_Answers_Figure_3}
5. Determine the slope (ΔAbsorbance/ΔTime) for each best-fit straight line in the above graph. This is the rate of reaction for each trial. Plot the rate of reaction versus the amount (volume) of substrate.
   {14005_Answers_Figure_4}
6. Explain the shape of the curve for the effect of substrate concentration on reaction rate in terms of enzyme–substrate binding.

   The effect of substrate concentration on reaction rate gives rise to a characteristic hyperbolic or “saturation” kinetics curve. At low substrate concentrations, the rate of the reaction is proportional to the concentration. As the substrate concentration increases further, however, the rate increase becomes more gradual and levels off or reaches a plateau. This happens because once all of the available enzyme is bound with substrate at its active site, additional substrate does not increase the rate.

Introduction

Studying enzyme kinetics provides the central basis for understanding how enzymes function. Among the thousands of enzymes in a single cell, peroxidases are among the most active and the most widely distributed. Peroxidases protect plants and animals against cell damage by catalyzing the breakdown of hydrogen peroxide, a natural but toxic byproduct of aerobic respiration. Investigate the activity of peroxidase by measuring its rate of reaction with hydrogen peroxide and a natural reducing agent called guaiacol.

Concepts

• Enzyme structure and function
• Kinetics and rate laws
• Oxidation−reduction
• Enzyme−substrate binding
• Active site
• Spectroscopy

Background

Enzymes are the catalysts of biological systems. Enzymes and catalysts increase the rates of biological or chemical reactions by decreasing the activation energy required for a reaction and providing a lower energy pathway from reactants to products. Like all catalysts, enzymes are also not “consumed” during a typical reaction. Most enzymes are globular proteins that are able to bind reactant molecules, called substrates, at their active sites. Although it enters into the reaction pathway, the free enzyme is restored at the end of the reaction when the product is released from the enzyme.

Three unique features differentiate enzymes from other catalysts. First, enzymes are the most efficient catalysts known, giving rise to rate enhancements 10³ to 10⁷ faster than uncatalyzed reactions. Enzymes are also highly specific with respect to both the reactions they catalyze (reaction specificity) and the choice of reactants (substrate specificity). Finally, the activity of enzymes can be regulated—activated or inhibited—by interactions with various small molecules and other proteins, as well as by environmental factors. Enzyme regulation allows an organism to control its metabolism and respond to external stimuli and changes in the environment.

Peroxidase enzymes occur in all plants and animals, including bacteria, to protect cells against oxidative stress and cell damage due to hydrogen peroxide. Peroxidases are easily extracted from turnips and other root vegetables and provide a model enzyme for studying enzyme activity—how the rate of an enzyme-catalyzed reaction depends on biotic and abiotic factors, such as enzyme and substrate concentration, pH, temperature and the presence of inhibitors and activators.

The term peroxidase refers to both a class of oxidation−reduction enzymes and to specific enzymes within that class. As a general class of enzymes, peroxidases catalyze the decomposition reaction of hydrogen peroxide. There are two general types of peroxidases—catalase and peroxidase. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen gas (Equation 1).

In this reaction, hydrogen peroxide substrate molecules act as both the oxidizing agent (electron acceptor) and reducing agent (electron donor). Peroxidase acts in the presence of other naturally occurring organic reducing agents, such as ascorbic acid (Equation 2). The organic reducing agent, abbreviated AH₂, transfers hydrogen atoms and electrons to hydrogen peroxide, resulting in the formation of water and an oxidized organic compound, represented by A₂ in Equation 2. The differences in these two equations provide a basis for studying the enzyme activity of turnip peroxidase. Guaiacol, a colorless compound having the formula C₇H₈O₂, is a common reducing agent (AH₂) for Equation 2. Oxidation of guaiacol converts it to a dark orange compound called tetraguaiacol. The rate of the reaction may be followed by measuring the color intensity or absorbance of this orange product versus time. Enzyme activity studies reflect the structure and function of enzymes and provide a foundation for understanding the mechanism of enzyme action. The mechanism of a reaction is essentially a step-by-step description of the course of the reaction at a molecular level.

Enzyme-catalyzed reaction mechanisms are generally determined through a deductive process by analyzing enzyme kinetics. The rate of an enzymatic reaction is studied to learn how different factors affect the rate, giving rise to a general model of enzyme activity. The model should be consistent with the kinetic evidence and should explain the unique properties of enzymes, including their immense catalytic power, substrate specificity, and regulatory control.

Experiment Overview

The purpose of this activity is to investigate the effects of enzyme concentration and substrate concentration on the rate of the peroxidase-catalyzed decomposition of hydrogen peroxide. The rate of a reaction can be determined by measuring the concentration of product(s) as a function of time. Formation of an orange-colored product from guaiacol provides a simple visual clue to follow how fast the reaction occurs. The absorbance or color intensity of the combined enzyme−substrate solution will be measured at specific time intervals for different concentrations of enzyme (Part A) and substrate (Part B) using a spectrophotometer or colorimeter. Since absorbance is directly proportional to the concentration of the product, a graph of absorbance versus time will have the same characteristics as a graph of concentration versus time. The rate of the reaction for each trial will be obtained from the slope of the linear portion of each graph. Further graphical analysis of the rate of reaction versus enzyme or substrate concentration in Parts A and B, respectively, will be correlated with a model of enzyme activity.

Materials

Prelab Questions

1. Define each of the following terms related to the properties of enzymes.
   1. Kinetics
   2. Activation energy
   3. Substrate
   4. Active site
2. Most enzymes have an optimum pH at which they are most active. Explain in terms of the properties of proteins.
3. The most common method of increasing the rate of a chemical reaction in the laboratory is to increase the temperature. What are the limits or drawbacks of this method for studying enzymatic reactions?
4. Compare the volumes of H₂O₂ (substrate) and peroxidase (enzyme) used in Parts A and B.
   1. Let x = concentration of enzyme in Trial A-1. Express the enzyme concentrations [E] in Trials A-2 and A-3 as multiples of x.
   2. Let y = concentration of H₂O₂ in Trial B-1. Express the substrate [S] concentrations in Trials B-2, B-3 and B-4 as fractions of y.

Safety Precautions

Guaiacol is toxic by ingestion. The guaiacol solution is prepared in isopropyl alcohol and has an aromatic, creosote-like odor that may be irritating to the nose and throat. Isopropyl rubbing alcohol (70%) is a flammable liquid. Keep away from heat, flames and other sources of ignition. Dilute hydrogen peroxide solution may be irritating to the eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Avoid contact of all chemicals with eyes and skin, and wash hands thoroughly with soap and water before leaving the laboratory. Please follow all normal laboratory safety guidelines.

Procedure

General Procedure
We recommend that students work in groups of four to complete this activity, with one pair of students performing Part A and the second pair working on Part B. Data and graphs for Parts A and B should be shared by all members of the group.

Read the entire procedure (Parts A and B) before beginning. Precise volume measurements and accurate timing are crucial for rate studies. Pay special attention to the requirements for mixing the contents of the substrate (S) and enzyme (E) tubes and timing the reaction. Do NOT mix the S and E tubes for each trial until ready to begin timing. Use separate pipets for the solutions needed for the S versus E tubes.

Part A. Effect of Enzyme Concentration

1. Turn on the spectrophotometer, adjust the wavelength setting to 500 nm, and allow the instrument to warm up for 15−20 minutes.
2. Prepare a “blank” by combining 4 mL pH 5 buffer, 2 mL hydrogen peroxide, 1 mL guaiacol and 2 mL pH 7 buffer in a 13 x 100 mm test tube.
3. Zero the spectrophotometer (zero absorbance, 100% transmittance) at 500 nm using the blank solution.
4. Using separate 2- and 5-mL serological pipets for the solutions in the S and E tubes, respectively, prepare three series of labeled 13 x 100 mm test tubes containing substrates (S tubes) and enzyme (E tubes). Use the amounts shown in the following table. Note that the enzyme concentration is varied in trials 1−3 whereas the concentration of substrate is held constant. The pH 5 buffer provides pH control, while the presence of pH 7 phosphate extraction buffer in the E tubes makes it possible to vary the enzyme amount while maintaining the overall buffer composition constant.
   {14005_Procedure_Table_1}
5. When ready to begin trial A-1, carefully pour the contents of tube S into tube E and immediately start timing. Pour the combined contents back into tube S, wipe the outside of the tube with lens tissue, and place the test tube in the spectrophotometer cell holder.
6. Measure and record the absorbance at 500 nm as a function of time every 20 seconds for 240−300 seconds. Since initial rate data is more accurate than longer term rate data, it is crucial to begin measurements as soon as possible. The elapsed time between mixing the tubes and recording the first absorbance measurement should be no greater than 40 seconds!
7. Repeat steps 5 and 6 for the remaining two trials A-2 and A-3. These have different concentrations of the enzyme.
8. Dispose of the test tube contents as directed by the instructor and rinse well with distilled water.

Part B. Effect of Substrate Concentration

1. Repeat steps 1−3 from Part A, if necessary, to warm up the spectrophotometer and zero the instrument. These steps do not need to be repeated if Parts A and B are carried out consecutively.
2. Using separate 5- and 2-mL serological pipets for the solutions in the S and E tubes, respectively, prepare four series of separately labeled 13 x 100 mm test tubes containing substrates (S tubes) and enzyme (E tubes). Use the amounts shown in the table below. The concentration of substrate is varied in trials 1−4. Changing the volume of pH 5 buffer in the S tubes makes it possible to maintain a constant total volume for each trial, thus keeping the enzyme concentration the same while varying the substrate concentration.
   {14005_Procedure_Table_2}
3. When ready to begin trial B-1, carefully pour the contents of tube S into tube E and immediately start timing. Pour the combined contents back into tube S, wipe the outside of the tube with lens tissue, and place the test tube in the spectrophotometer cell holder.
4. Measure and record the absorbance at 500 nm as a function of time every 20 seconds for 240−300 seconds. It is important to obtain accurate measurements as early as possible. The elapsed time between mixing the tubes and recording the first absorbance measurement should be no greater than 40 seconds!
5. Repeat steps 3 and 4 for the remaining three trials with different concentrations of hydrogen peroxide (the substrate).
6. Dispose of the test tube contents as directed by the instructor and rinse well with distilled water.

Student Worksheet PDF

14005_Student1.pdf