Peroxidase Enzyme Activity
Inquiry Laboratory Kit for AP® Biology
Materials Included In Kit
Buffer envelopes, pH 3−6, and 8, 1 each
Guaiacol, C7H8O2, 0.2% solution in isopropyl alcohol, 100 mL
Hydrogen peroxide, H2O2, 3%, 100 mL
Sodium phosphate, dibasic, solution, Na2HPO4, 0.2 M, 300 mL
Sodium phosphate, monobasic, solution, NaH2PO4, 0.2 M, 300 mL
Pipets, serological, 2-mL, 5
Additional Materials Required
Water, distilled or deionized water*
Computer with LoggerPro or LabQuest (optional)*
Erlenmeyer flask, 500-mL†
Erlenmeyer flask, 1-L†
Dark bottle with cap, 500-mL, 2†
Filter paper and funnel†
Graduated cylinder, 10-mL†
Spectrophotometer or colorimeter*
Test tubes, 13 x 100 mm, 6, and rack (optional)*
Thermometer (for inquiry investigations)*
*for each lab group
†for Prelab Preparation
Phosphate Extraction Buffer: Prepare one liter of pH 7 phosphate buffer by mixing equal volumes, 250 mL each, of 0.2 M sodium phosphate monobasic and sodium phosphate dibasic solutions. Dilute to one liter with distilled or deionized water.
Reaction Buffers: Dissolve one each pH 3−8 buffer envelopes in 500 mL distilled or deionized water according to packet instructions. Prepare separate buffer solution for each desired pH.
Dilute Hydrogen Peroxide, 0.02%: Dilute 3 mL of 3% hydrogen peroxide to a final volume of 500 mL using distilled or deionized water. Store in a dark bottle with cap in a cool, dark area protected from heat and light.
Enzyme Extraction: Peel and cut a turnip root into small pieces. Measure approximately 4 g in a weighing dish. Place 500 mL of pH 7 phosphate extraction buffer and the turnip root pieces in a blender. Blend the turnip root for six minutes using three, 1-minute bursts with 2 minutes rest between pulses, to homogenize and extract the enzymes. Filter the turnip enzyme extract through filter paper and store the extract in a dark bottle with cap in the refrigerator. Use within two weeks.
Check the enzyme activity of the turnip peroxidase extract before doing the Baseline Activity. 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 are less accurate due to the limitations of most spectrophotometers. The concentration of the stock is likely much higher than necessary, so start by diluting 1 mL of stock enzyme extract in 4 mL of pH 7 extraction buffer and then follow the instruction in the baseline activity. If, after five minutes the absorbance is greater than one, dilute further until the desired concentration is reached. If after ten minutes the absorbance is less than 0.2, use a higher concentration of enzyme.
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 (3%) may be irritating to the eyes and skin. Exercise care when using a knife to peel and cut the turnip. Wear chemical splash goggles, chemical-resistant gloves and a 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.
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.
- Enough materials are provided in this kit for eight groups of students to complete the baseline activity as written. Buffers and substrate solutions are also provided for individual groups of students to each perform additional guided-inquiry investigations testing one variable, such as substrate concentration, pH, or temperature, on the rate of reaction of enzyme peroxidase. See the Sample Data section for possible inquiry investigations.
- If the extraction will be performed by students, this step can be incorporated into the Opportunities for Inquiry. 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 (increase in absorbance of 0.3−0.6 units over a 3-minute period for Trial 1).
- The enzyme extract should be stored in the refrigerator and used within two weeks of preparation. Students should run a new baseline for each series of inquiry experiments.
- A variety of plants may be used as sources of peroxidase and may be incorporated into the Opportunities for Inquiry. Students may research suitable examples. The options are abundant—turnip, horseradish, radish, lettuce, tomatoes, spinach, legumes, etc. 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.
- The College Board has stipulated knowledge and laboratory skill prerequsities for AP Biology in both math and chemistry content areas. Chemistry prerequisities that are relevant to this laboratory investigation include knowing how to measure volume and temperature, determine the rate of a chemical reaction, and calculate solute concentrations. Graphing skills are also listed as a relevant math prerequisite. We recommend that teachers provide a worksheet or activity for students to review the concepts of kinetics and reaction rates with students before beginning this laboratory investigation. A short primer is provided in the Teaching Tips section.
- 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. Rate is proportional to reactant concentration, so as the reactants are depleted the rate decreases.
- The Procedure for the Baseline Activity calls for pouring the contents of the substrate tube into the enzyme tube, and then back into the substrate tube, prior to beginning absorbance measurements. This is done to ensure adequate mixing of the contents and to obtain accurate reaction rates. Students should begin timing with the first pour, however, as soon as the enzyme and substrate are combined.
- Students must remember to keep the total volume of the enzyme and substrate solutions (test tubes) constant when they design their own inquiry experiments in Part B. This is done by varying the amount of enzyme or hydrogen peroxide solution and then adjusting the volume of buffer to accommodate the difference. See the baseline activity for an example.
- The effect of substrate concentration on reaction rate gives rise to a characteristic hyperbolic or “saturation” kinetics curve (see graphs in the Sample Data section). 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 or 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 cannot and does not increase the rate of reaction.
- The concept of saturation kinetics discussed above may make it challenging for students to identify suitable substrate concentrations that will illustrate the “desired” results. If the substrate concentration in the baseline activity 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. See the graphs in the Additional Sample Data section for concentrations that worked in our independent lab testing. Students must indeed experiment to find concentrations that will work under their laboratory conditions. Remind students that they should change only the volume of substrate, and adjust the volume of buffer to keep the total volume of the contents of the “S” tube constant.
- If a spectrophotometer or colorimeter is 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 mixing 15 mL of pH 5 buffer, 10 mL of 0.02% H2O2, 5 mL of 0.2% guaiacol, and 10 mL enzyme. Call the concentration of the stock solution x and then dilute it to obtain solutions that have concentrations equal to 0.1x, 0.2x, 0.4x, 0.5x, 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 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 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 will require considerable trial-and-error experimentation to prepare a color series in the same general concentration range as the rate trials will produce.
- Kinetics is the study of the rate of chemical reactions, which describes how fast a reaction occurs. The greater the rate of a reaction, the less time that is needed for reactants to be converted to products. Reaction rates are therefore inversely proportional to time. In general the rate of a reaction increases as the concentration of reactants increases in accord with the dynamic or collision theory of a chemical reaction. In order for a reaction to occur, reacting molecules must first collide. Any factor that changes the total number of collisions, the average energy or favorable orientation of the colliding molecules will affect the reaction rate.
- As reactants are transformed into products in a chemical reaction, the amount of reactants will decrease and the amount of products will increase. The rate of the reaction is determined by measuring the concentration of reactants or products as a function of time. In the case of the peroxidase-catalyzed decomposition of hydrogen peroxide, the formation of an orangecolored product from guaiacol provides a simple visual clue to determine the reaction rate based on the time it takes for the color to appear.
- The purpose of this technology-based experiment is to use spectrophotometry or colorimetry and graphical analysis to determine the reaction rate. A spectrophotometer measures the absorbance or transmittance of light. The absorbance of the solution is measured at specific time intervals. Since the absorbance is directly proportional to the concentration of the colored product, a graph of absorbance versus time has the same characteristics as a graph of concentration versus time. The rate of the reaction is obtained from the slope of the linear portion of the graph.
- 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 the sample.
Alignment with Concepts and Curriculum Framework for AP® Biology
Big Idea 2: Biological systems utilize free energy and molecular building blocks to grow, to reproduce, and to maintain dynamic homeostasis.
Enduring Understandings and Essential Knowledge
2D1: All biological systems from cells and organisms to populations, communities, and ecosystems are affected by complex biotic and abiotic interactions involving exchange of matter and free energy.
Big Idea 4: Biological systems interact, and these systems and their interactions possess complex properties.
Enduring Understandings and Essential Knowledge
4A1: The subcomponents of biological molecules and their sequence determine the properties of that molecule.
4B1: Interactions between molecules affect their structure and function.
- The student is able to design a plan for collecting data to show that all biological systems are affected by complex biotic and abiotic interactions (2D1 and SP 4.2, 7.2).
- The student is able to use models to predict and justify that changes in the subcomponents of a biological polymer affect the functionality of the molecule (4A1 and SP 6.1, 6.4).
- The student is able to analyze data to identify how molecular interactions affect structure and function (4B1 and SP 5.1).
4.2 The student can design a plan for collecting data to answer a particular scientific question.
5.1 The student can analyze data to identify patterns or relationships.
6.1 The student can justify claims with evidence.
6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
7.2 The student can connect concepts in and across domains to generalize or extrapolate in and/or across enduring understandings and/or big ideas.
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering Practices
Asking questions and defining problems
Developing and using models
Planning and carrying out investigations
Analyzing and interpreting data
Using mathematics and computational thinking
Constructing explanations and designing solutions
Engaging in argument from evidence
Disciplinary Core Ideas
HS-PS1.A: Structure and Properties of Matter
HS-PS1.B: Chemical Reactions
HS-LS1.A: Structure and Function
Cause and effect
Scale, proportion, and quantity
Systems and system models
Energy and matter
Structure and function
Stability and change
HS-PS1-1. Use the periodic table as a model to predict the relative properties of elements based on the patterns of electrons in the outermost energy level of atoms.
HS-PS1-2. Construct and revise an explanation for the outcome of a simple chemical reaction based on the outermost electron states of atoms, trends in the periodic table, and knowledge of the patterns of chemical properties.
HS-PS1-5. Apply scientific principles and evidence to provide an explanation about the effects of changing the temperature or concentration of the reacting particles on the rate at which a reaction occurs.
HS-LS1-1. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins, which carry out the essential functions of life through systems of specialized cells.
HS-LS1-3. Plan and conduct an investigation to provide evidence that feedback mechanisms maintain homeostasis.
Additional Sample Results
AP® Biology Investigative Labs: An Inquiry-Based Approach. College Entrance Examination Board: New York, 2012.
||FlinnPREP™ Inquiry Labs for AP® Biology: Peroxidase
||Flinn Scientific Spectrophotometer
||Cuvets, ½" Square Disposable, Set of 100 Cuvets and 20 Lids
||Test Tubes without Rims, Borosilicate Glass, 13 x 100 mm, 9.0 mL
||Test Tube Rack, Polypropylene, Submersible, 13 mm Tubes, 90 Place
||Timer, Count Up/Count Down, with Clock, Large Display
||Bulb, Rubber, 15-mL