The Biotechnology Education Company 246 EDVO-Kit # New Microtiter Plate- Based on AP Biology Lab #2 Storage: See page 3 for storage instructions. EXPERIMENT OBJECTIVES: The objective of the experiment is to demonstrate microarray technology using enzyme catalysis. Students will perform an enzyme assay in a microtiter plate and determine the rate of a biochemical reaction. All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
2 Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 4 Experiment Procedures Experiment Overview 7 Experimental Procedures 8 Enzyme Data Analysis 13 Study Questions 14 Instructor's Guidelines Notes to the Instructor 15 Pre-Lab Preparations 16 Experiment Results and Analysis 18 Study Questions and Answers 19 Material Safety Data Sheets 21
3 Experiment Components This experiment is designed for 10 groups. Store Component A in the freezer. All other components can be stored in the refrigerator. Component Storage A Catalase solution Freezer B Hydrogen peroxide, stabilized Refrigerator C Phosphate buffer, ph 7.2 (conc.) Refrigerator D Assay reagent, potassium iodide (conc.) Refrigerator E Acidification solution (conc.) Refrigerator F Color enhancer (conc.) Refrigerator G Color developer (conc.) Refrigerator Microtiter Plate All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. Requirements Automatic Micropipet and tips Spectrophotometer/microplate reader (optional) Timers or clock with second hand Test tubes (13 x 100 mm) for large assay Distilled water Ice EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. EDVOTEK - The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com 24-hour FAX: (301) 340-0582 email: edvotek@aol.com
4 Principles of Enzyme Catalysis Enzymes as Biological Catalysts Background Information A biological catalyst is used in trace amounts and accelerates the rate of a biochemical reaction without being consumed or transformed during the reaction. The equilibrium constant of reactions are not altered by catalysts. Only the rate of approach to equilibrium is changed. Reactions in cells are catalyzed by biological catalysts known as enzymes which can accelerate reactions by as much as 10 14 to 10 20 times. Enzymes function best under mild physiological conditions of neutral ph, and temperatures of 37 C. Enzymes are generally very specific for the reactions they catalyze. Certain enzymes are regulated by intracellular concentrations of key metabolites that are not directly involved with the reaction they catalyze. Enzymes that are regulated in this way are termed allosteric. Measuring Enzyme Activity The reactant molecule in an enzyme catalyzed reaction is called the substrate. The substrate (S) is transformed to product (P). Before the enzyme can transform the substrate it must first bind to it. Only a relatively small portion of the enzyme molecule is involved with substrate binding and catalysis. This region is called the active site. The active site contains the critical amino acid residues and, if applicable, the prosthetic groups required for activity. Initial binding is non-covalent and can be in rapid equilibrium. After productive binding has been achieved, the enzyme-substrate complex begins to generate product which is subsequently released. The free enzyme (E) can react with additional substrate and this reaction is repeated rapidly and effectively. The reaction is summarized using a single substrate, single product in a non-reversible reaction: E + S ES EP E + P Catalase is the enzyme used in this experiment. Hydrogen peroxide is a toxic by-product of aerobic oxidation in intermediary metabolism. All aerobic life forms are capable of enzymatic peroxide detoxification. Catalase (H 2 O 2 :H 2 O 2 oxidoreductase) catalyzes the rapid decomposition of hydrogen peroxide by the following reaction: 2 H 2 O 2 2H 2 O + O 2 (gas) Catalase uses the hydrogen peroxide as a hydrogen acceptor and donor and the catalytic reaction is referred to as catalatic. The enzyme can also utilize short chain organic molecules such as ethanol, phenols and formate to reduce hydrogen peroxide. This activity is
Background Information 5 Principles of Enzyme Catalysis called peroxidative. The catalatic activity is preferred in-vitro. Almost all the cell types in mammals contain catalase, with liver, kidney and erythrocytes being particularly rich sources. Catalase has one of the highest catalytic turn over rates known. Over 3.6 x 10 7 molecules of hydrogen peroxide are converted to product by an enzyme molecule per minute. The appearance of product (P) or the disappearance of substrate (S) can be measured as a function of time during a reaction. One can measure the amount of product formed or the decrease in substrate at regular intervals (in this experiment at 30-sec intervals). This quantity can be plotted as a graph. Typical results are shown in Figure 1 which demonstrate the rate of an enzymatic reaction. An enzymatic reaction measurement is referred to as an assay. At fixed concentration and reaction conditions, an enzyme reaction rate can increase by higher substrate concentrations. The probability of forming ES complexes increases with more substrate molecules present. Generally, the substrate concentration is thousands of times greater than the enzyme concentration for in vitro kinetic studies. At the early stages of such a reaction, the substrate concentration is in great excess and the rate is approximately linear per unit of time and is termed the initial velocity (v) or initial rate of the reaction. The characteristics of the enzyme molecule determine the initial velocity. It will always remain the same for an enzyme as long as the substrate is present in excess, the products are not inhibitory and the ph and temperature remain constant. Substrate Consumption (Absorbance units) 1 2 3 4 5 6 Time (min.) Figure 1 V = [S] 1 - [S] 2 T 1 - T 2 In the above equation [S] 1 is the molar concentration of substrate at some initial time T 1, and [S] 2 is the substrate concentration at a later time T 2. Note that the concentration of substrate decreases with time and the concentration of product increases with time. Graphically, this can be represented with the substrate concentration plotted on the y-axis and time on the x-axis. The decrease in the substrate concentration with time will generate a curve. The rate of decrease is fastest at the earliest time points of the reaction since the substrate concentration is comparatively higher. The rate of decrease diminishes at later times because the substrate concentration is lower and the reaction is slower (Figure 1). Within short time intervals there will be sections of the curve that are approximately linear and the rate of the reaction can be measured. At some substrate concentration, all the enzyme molecules are bound to substrate and are involved in some stage of the catalytic reaction. Under these conditions the enzyme is saturated with substrate and there is no increase in reaction velocity.
6 Principles of Enzyme Catalysis Background Information Product Formation (Absorbance units) 30 20 10 Figure 2 30 60 90 Time (min.) The initial reaction rate can also be expressed in terms of the appearance of product. To determine the rate of the reaction, pick any two points on the straight-line portion of the graph curve (Figure 2). The amount of product formed between two points divided by the difference in time between the two points will be the rate of the reaction. It can be expressed as µmoles product/sec. V = At time 0, there is no product formed. After 30 seconds,10 µmoles have been formed; 20 µmoles after 60 seconds; 30 µmoles after 90 seconds. For the initial period, the rate of this reaction could be stated as 20 µmoles of product formed per minute. Typically, less additional µmoles of product are formed by the second, third and fourth minute (Figure 2). For each successive minute after the initial 1.5 minutes, the amount of product formed is less than in the preceding minute. As the illustration of Figure 2: [P] 2 - [P] 1 T 2 - T 1 30µmoles - 10µmoles = 20 = 0.33 µmoles/sec 90 seconds - 30 seconds 60 In this experiment, a colorimetric assay will be used. It will measure the amount of hydrogen peroxide remaining after catalysis of H 2 O 2 by catalase in a coupled secondary reaction with iodide (KI). As shown by the equation that follows: 2 I + 2 H + + H 2 O 2 2 H 2 O + I 2 SAFETY: Potassium permanganate (KMnO4) is a strong oxidizing agent. It is often not allowed in schools and is not used in this experiment. In this two-step coupled reaction, hydrogen peroxide (substrate) is consumed and I 2 (product) from the secondary non-enzymatic reaction is produced in equimolar amounts. Catalase (enzyme) catalyzes the conversion of hydrogen peroxide to water and oxygen. To visualize the enzymatic time course reaction, equal volumes of the catalase incubation reaction mixture are transferred at various time points to an acidic solution of iodide (KI). In this acidic environment, catalase is denatured and the enzymatic reaction is terminated. In the second reaction which is the chemical non-enzymatic reaction, the remaining hydrogen peroxide converts Iodide (I ) to iodine resulting in the generation of a brownred color that is characteristic of iodine. During the enzymatic reaction, the remaining hydrogen peroxide (substrate) will decrease in the incubation reaction. As a consequence, the amount of brown color due to the generation of iodine (I 2 ) in the chemical reaction will also proportionally decrease.
Background Information 7 Experiment Overview EACH LAB GROUP REQUIRES: 0.6 ml Reaction cocktail (Solution #2B) 0.8 ml Assay solution (Solution #3) (Do not to contaminate with vessels or pipets that contained peroxide while dispensing) 50 µl Diluted catalase on ice (Solution #4) 200 µl Diluted phosphate buffer (Solution #1) 1 Permanent marker to label tubes 2 1 ml pipets (0.1 ml divisions) 1 Automatic Micropipet 1 Microtiter strip (2 rows) EXPERIMENT OBJECTIVE The objective is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction. WORKING HYPOTHESIS If the disappearance of substrate can be measured as a function of time during a reaction, then a corresponding qualitative observation and/or quantitative determination of enzyme rate can be made. IN THIS EXPERIMENT: Catalase will be added to a buffered solution of hydrogen peroxide. A time course of the reaction will be obtained by removing aliquots from the reaction well every 30 seconds. Aliquots removed during the time course reaction will be added to assay solution for the remaining hydrogen peroxide wells that contain a catalase denaturing solution. To determine the amount of substrate remaining in the time course reaction the remaining hydrogen peroxide will be measured. Hydrogen peroxide that is not catalyzed by the enzyme catalase will oxidize iodide (I ) in a coupled reaction to give the brown-red (Iodine) color. The color intensity increases with the amount of hydrogen peroxide remaining from the time course reaction. Therefore, in the enzymatic reaction as the incubation time (between substrate and enzyme) increases, there will be a corresponding decrease in the amount of hydrogen peroxide and therefore a decrease in the intensity of the red-brown color. The color intensity may be qualitatively estimated or quantitatively using a spectrophotometer. LABORATORY SAFETY Gloves and safety goggles should be worn routinely as good laboratory practice.
8 Overview of the Reaction Steps Experiment Procedures Wear Safety Goggle and Gloves. In this experiment, microtiter plates will be used to preform the assay. The first well, in lane 2, labelled Control, "C" contains 100 µl of buffered substrate (hydrogen peroxide) and 10 µl of dilute phosphate buffer. No enzyme is added. Ten µl from the "control" well (lane 2) will be added to the well labelled "0" in lane 1. This establishes the zero time point (highest color intensity of iodine conversion due to hydrogen peroxide buffer. The first well labelled "blank" in lane 1 is used to blank the spectrophotometer for background. If 30 sec. intervals are too short for students, you can increase time increments to 45 sec. or 1 min. This will mean less H 2 0 2 for the color reaction and a lighter color.
Experiment Procedures 9 Microtiter Plate Enzyme Assay 1. Place the microtiter plate on a white piece of paper and with a water resistant pen, line up and label 8 wells in the top row as follows: 1. B (blank) 2. 0 min. 3. 0.5 min 4. 1.0 min 5. 1.5 min 6. 2.0 min 7. 2.5 min 8. 3.0 min A fixed or variable (5-50µl) automatic micropipet is required for most of the steps. 2. In the second row, label the first well for the control C and the second well for the enzyme reaction Rxn". 3. Using a fresh micropipet tip, dispense 10 µl of dilute phosphate buffer (Solution #1) to the well labelled Control ( C ) and 100 µl to the well labelled blank ( B ). Microtiter Plate - Catalase Reaction Assay Blank Assay Buffer Phosphate Buffer (used to blank spectrophotometer for background) Each well (0.5 to 3.0 min.) contains ( I ) solution and HCl. Enzyme reactions are stopped upon the addition of the timed enzyme reaction in the wells that contain I and HCl (0.5 to 3.0 min.). Chemical color reaction is initiated to determine amount of substrate (H 2 O 2 ) remaining. B 0 0.5 1.0 1.5 2.0 2.5 3.0 C Rxn Control establishes Zero Timepoint. Peroxide (Substrate) / Buffer. No Catalase. Catalase (Enzyme) Reaction Assay Well Peroxide / Buffer / Catalase. Catalase converts Peroxide into Products. Timepoints: 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 min.
10 Microtiter Plate Enzyme Assay Experiment Procedures 4. Using a fresh micropipet tip, dispense 100 µl of Reaction Cocktail (Solution #2B hydrogen peroxide in phosphate buffer) in the wells labelled C for the control and Rxn for reaction well. Discard the tip. 5. Using a micropipet and fresh tip, transfer 100 µl of the Assay solution (Solution #3) to each of the wells in the top row except the blank. The wells are labelled in the top row (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0). When you have completed these additions, discard the tip. 6. Using a fresh micropipet tip, add 10 µl of the solution from the control well (lane 2) to the 0 well in (lane 1). This will be the zero time point reaction. The zero time point reaction will have the highest amount of hydrogen peroxide since no catalase was added to the 0 reaction well. This will also result in the highest amount of color due to the amount of iodide converted to iodine in the color reaction. 7. With a fresh micropipet tip, add 10 µl of diluted enzyme (catalase Solution #4) to the well labelled Rxn. At this point, the enzymatic reaction has been initiated. Immediately start your timer. Discard the pipet tip. Well Diluted Buffered Catalase Buffer Substrate H 2 O 2 (Solution #4) (Solution #1) (Solution #2B) "B" 110 µl 0 µl 0 µl "C" 10 µl 100 µl 0 µl "Rxn" 0 µl 100 µl 10 µl Table 1: Preparation of Wells "B", "Con", & "Rxn" TIMED ENZYME REACTION 8. After 30 seconds from the addition of catalase, remove 10 µl from the Rxn well and add to the well that is labelled "0.5". You may retain the tip and rinse liberally with distilled water. 9. Using a fresh pipet tip, remove an additional 10 µl from the reaction well after an additional 30 second interval and add to the well labelled 1.0 min. Use a fresh pipet tip or rinse the tip liberally with distilled water.
Experiment Procedures 11 Microtiter Plate Enzyme Assay 10. Using a fresh pipet tip, remove an additional 10 µl from the reaction well after an additional 30 second interval and add to the well labelled 1.5 min. Use a fresh pipet tip or rise the tip liberally with distilled water. Table 2: Preparation and Detection of Well 11. Using a fresh pipet tip, remove an additional 10 µl from the reaction well after an additional 30 second interval and add to the well labelled 2.0 min. Use a fresh pipet tip or rise the tip liberally with distilled water. 12. Repeat the transfer of 10 µl aliquots from the reaction well after additional 30 second intervals and add to the wells labelled 2.5 min, and 3.0 min respectively. 13. After the addition of the last reaction aliquot, incubate the reactions at room temperature for 4 minutes to allow color development. 14. A visual examination will determine the rate of the reaction. Alternatively, the reaction can be quantified using a microplate reader or a laboratory spectrophotometer that uses cuvettes which hold volumes in the range of 0.1 and 0.2 ml. TIME (min.) Enzyme Assay #3 Solution I + HCl buffer From "C" well (H 2 O 2 ) From "Rxn" well (Enzyme and Substrate) Qualitative or Quantitative results (color) Blank (See Table 1) --- --- 0 100µl --- 10µl 0.5 100µl --- 10µl 1.0 100µl --- 10µl 1.5 100µl --- 10µl 2.0 100µl --- 10µl 2.5 100µl --- 10µl 3.0 100µl --- 10µl * Requires the use of a spectrophotometer with small curvettes or a microplate reader.
12 Microtiter Plate Enzyme Assay Experiment Procedures QUANTITATION OF THE ENZYME REACTION (OPTIONAL - Experiment needs to be adapted for use with spectrophotometer) Note: This quantitation procedure requires a spectrophotometer that utilyzes cuvettes that accomodate 0.2-0.4 ml samples. 1. To obtain quantitative spectral results, transfer each of the reactions to a cuvette. 2. Zero the instrument with solution labelled B (Blank). Be sure the instrument is set at 500 nm wavelength. The instrument should read 0 absorbance with the blank solution (no color). Read the samples and record your data. 3. In the case of a single beam spectrophotometer, after setting the absorbance to zero replace the blank solution in cuvette with each of the samples to obtain the reading. In between samples wash the cuvette with distilled water and adjust the absorbance to zero using the blank. 4. For a dual beam spectrophotometer, leave the blank cuvette in the blank position and place the second cuvette in the holder to obtain the readings. Again check the blank between sample readings. 5. Using a clean pipet tip for each sample, transfer the sample sequentially from each well to the corresponding cuvette and mix. 6. Read the absorbance at A 500 and record the absorbancy values for each solution in wells labelled 0.5 to 3.0.
Experiment Procedures 13 Enzyme Data Analysis The reaction rate can be obtained by graphing the absorbancy data versus time. However, the rate can also be expressed in terms of substrate consumed. 1. To express your data in terms of molar concentration of peroxide: Absorbance x 2.0 = Molarity of hydrogen peroxide in well. ε ε, the extinction coefficient for this assay system, is 5 x 10 3. The molar concentration of either a decrease in substrate or increase in the product can be obtained by dividing the absorbance at A 500 by the molar extinction coefficient. Multiplication by 2.0 (dilution factor) gives the peroxide concentration in the reaction tube. Scientific notation will make the calculations more convenient. 2. Graph the peroxide concentration on the y-axis versus time on the x- axis provided. 3. Draw the best straight line through the data points. You may notice some curvature to the data points. This is normal, especially between 0 and the first time point, and between later time points. You are making a linear approximation. 4. Determine the rate of change in the molarity of hydrogen peroxide with time. The rate is equivalent to the slope of the line. Pick a time, go vertically up to the line, then horizontally to the y-axis. Determine the concentration in this way for the next time point. V = [peroxide 1 - peroxide 2 ] [ time 1 - time 2 ] In this experiment, catalase, the enzyme, catalyzes the conversion of hydrogen peroxide to water and oxygen. In this coupled reaction, the molar concentrations of peroxide (substrate) consumed and I 2, the product from the non enzymatic reaction are equimolar. To visualize the enzymatic reaction, equal samples of the catalase incubation reaction are transferred at various time points to an acidic solution of potassium iodide (KI). In the acidic environment catalase is denatured and the enzymatic reaction stops. In the second reaction, which is a non-enzymatic chemical reaction, the remaining hydrogen peroxide is used at each of the time points to convert the Iodide (I - ) to iodine (I 2 ). Over the time course of the enzymatic reaction, the amount of the substrate (hydrogen peroxide) will decrease and consequently the brown-red color in the chemical reaction will also decrease corresponding to the amount of iodine (I 2 ) generated.
14 Study Questions 1. What is the enzyme in this reaction? Experiment Procedures 2. What is the substrate in this reaction? 3. What are the products in this reaction? 4. What is in the assay solution? 5. What is the function of the Potassium Iodine Solution? 6. Explain inhibiting effect of HCl in relation to enzyme structure and function. 7. Explain why the color intensity of the peroxide assays decreased with time? 8. What makes the rate of a reaction of an enzymatic reaction decrease? 9. Assuming optimal reaction conditions (ph, temperature, etc.) how could you increase the rate of the reaction other than increasing the substrate concentration? 10. The velocity of a catalase reaction was found to increase with increasing hydrogen peroxide concentrations as expected. However, at high peroxide concentrations, the reaction rate decreased and eventually went to zero. What could explain this observation?
15 Notes to the Instructor Online Ordering now available www.edvotek.com Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology and biology education. OVERVIEW OF LABORATORY INVESTIGATIONS The "hands-on" laboratory experience is a very important component of the science courses. Laboratory experiment activities allow students to identify assumptions, use critical and logical thinking, and consider alternative explanations, as well as help apply themes and concepts to biological processes. EDVOTEK experiments have been designed to provide students the opportunity to learn very important concepts and techniques used by scientists in laboratories conducting biotechnology research. Some of the experimental procedures may have been modified or adapted to minimize equipment requirements and to emphasize safety in the classroom, but do not compromise the educational experience for the student. The experiments have been tested repeatedly to maximize a successful transition from the laboratory to the classroom setting. Furthermore, the experiments allow teachers and students the flexibility to further modify and adapt procedures for laboratory extensions or alternative inquirybased investigations. ORGANIZING AND IMPLEMENTING THE EXPERIMENT Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. Technical Service Department If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. Instructor's Guide M o n EDVO-TECH 1-800-EDVOTEK (1-800-338-6835) - Fri 9 SERVICE am - 6 E T pm Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) 340-0582 web: www.edvotek.com email: edvotek@aol.com Please have the following information: The experiment number and title Kit Lot number on box or tube The literature version number (in lower right corner) Approximate purchase date www. edvotek.com In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).
16 Pre-Lab Preparations Instructor's Guide Note: All glassware needs to be clean and dry for preparation of enzyme solutions. Wear gloves and safety glasses. ON THE DAY OF THE LAB, PREPARE THE FOLLOWING: Solution #1 - Dilute Phosphate Buffer ph 7.2 (1x) 1. Pour 19 ml of distilled water into a clean small beaker. 2. Transfer 1 ml of phosphate buffer concentrate (Component C) to the beaker containing the water. Mix. Label the vessel Dilute Phosphate Buffer. Store on ice or in the refrigerator. Each group requires 200 µl. WITHIN 30 MINUTES OF THE LAB, PREPARE THE FOLLOWING: Solution #2A - 0.12% Hydrogen Peroxide Substrate 1. Transfer 2.7 ml diluted phosphate buffer (Solution #1) to clean 10 ml beaker or test tube. 2. Using a 1 ml pipet, add exactly 0.3 ml of 1.2% peroxide (Component B) to the 2.7 ml of buffer. 3. Mix. 4. Label the vessel 0.12% Peroxide. 5. Let the diluted peroxide cool on ice or in the refrigerator in the dark. Useful Hint! If the assay solution is prepared too far in advance it will begin to yellow due to oxidation of the iodide. The yellowing will create higher blank values for the spectrophotometer. Solution #2B - Enzyme Reaction Cocktail 1. Transfer 6 ml of diluted phosphate buffer (Solution #1) to a clean 15 ml plastic tube. 2. Using a washed or a fresh pipet tip, add 0.3 ml of 0.12% peroxide (Solution #2A) to the buffer. 3. Mix. 4. Label the vessel Reaction Cocktail. 5. Each group will need 0.6 ml. Solution #3 - Assay Solution (for Colorimetric Assay) 1. Add 15 ml of distilled water to a clean small beaker. 2. Add 2.5 ml of potassium iodide solution (Component D). 3. Add 2.5 ml of the 0.1 M HCl solution (Component E ). 4. Add 2.5 ml of the enhancer solution (Component F). 5. Add 2.5 ml of the developer solution (Component G). 6. Mix. 7. Label the vessel Assay Solution. Store in the dark, on ice or in the refrigerator. Each group requires 0.8 ml.
17 Pre-Lab Preparations WITHIN 20 MINUTES OF THE LAB, PREPARE THE FOLLOWING: Solution #4 - Catalase Enzyme 1. With a pipet or graduate cylinder, transfer 2.6 ml of ice cold, diluted phosphate buffer (Solution #1) to the 15 ml plastic tube provided. 2. Label Enzyme. Keep the tube on ice. 3. Carefully add 40 µl of catalase stock (Component A) to the buffer. The enzyme solution is viscous. Rinse the pipet in the buffer to remove residual enzyme. 4. Cap the tube and gently mix by inverting so that no enzyme solution remains on the bottom. 5. Use within 20 to 30 minutes. 6. Each group requires 50 µl. Instructor's Guide Spectrophotometer A well maintained and calibrated spectrophotometer or microplate reader will give good results for your lab activities. 1. Allow the spectrophotometer to warm up one half hour before the lab. 2. Set the wavelength at 500 nm. Absorbance will be measured. Color is stable for approximately 1 hour. EACH LAB GROUP REQUIRES: 0.6 ml Reaction cocktail (Solution #2B) 0.8 ml Assay solution (Solution #3) (Do not to contaminate with vessels or pipets that contained peroxide while dispensing) 50 µl Diluted catalase on ice (Solution #4) 200 µl Diluted phosphate buffer (Solution #1) 1 Permanent marker to label tubes 2 1 ml pipets (0.1 ml divisions) 1 Automatic Micropipet 1 Microtiter strip (2 rows)
18 Experiment Results and Analysis Instructor's Guide EXAMPLE OF EXPECTED RESULTS: Absorbancy reading should decrease with time. The pipeting accuracy will affect observed reaction velocities. Representative experiment results are presented below: MINUTES ABSORBANCE MOLARITY* 0 1.087 1.86 x 10-3 - (determined experimentally) 0.5 0.915 1.57 x 10-3 1.0 0.811 1.39 x 10-3 1.5 0.693 1.19 x 10-3 2.0 0.594 1.02 x 10-3 * This represents the calculated molarity multiplied by 2. Rate between 0.5 and 1 min is 3.6 x 10-4 Molar/min. INSTRUCTOR NOTES: Catalase, the enzyme catalyzes the conversion of hydrogen peroxide to water and oxygen. In this coupled reaction, the molar concentrations of peroxide (substrate) consumed and I 2, the product from the non enzymatic reaction are equimolar. To visualize the enzymatic reaction, equal samples of the catalase incubation reaction are transferred at various time points to an acidic solution of potassium iodide (KI). In the acidic environment catalase is denatured and the enzymatic reaction stops. In the second reaction, which is a non-enzymatic chemical reaction, the remaining hydrogen peroxide is used at each of the time points to convert the Iodide (I - ) to iodine (I 2 ). Over the time course of the enzymatic reaction, the amount of the substrate (hydrogen peroxide) will decrease and consequently the brown-red color in the chemical reaction will also decrease corresponding to the amount of iodine (I 2 ) generated.
Please refer to the kit insert for the Answers to Study Questions
Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X None Incompatibility Acetic Anhydride-alc. Hydrogen Cyanide, AL, Bases, Bronze, CA HCI IDENTITY (As Used on Label and List) 1 N HCI Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) 14676 Rothgeb Drive Rockville, MD 20850 Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV 1MHCI CAS# 7647-01-0 Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) 251-5990 Telephone Number for information (301) 251-5990 Date Prepared Signature of Preparer (optional) Unusual Fire and Explosion Hazards Emits toxic fumes under fire conditions 09/11/02 Other Limits Recommended % (Optional) Section III - Physical/Chemical Characteristics Boiling Point 384 F Specific Gravity (H 0 = 1) 2 1.2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) 1.3 Evaporation Rate (Butyl Acetate = 1) Solubility in Water Water soluble Appearance and Odor Colorless Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL UEL N.D. N.D. Extinguishing Media Water spray, carbon dioxide, dry chemical powder, or appropriate foam Special Fire Fighting Procedures Wear SCBA and protective clothing to prevent contact with skin and eyes IDENTITY (As Used on Label and List) Color Developer Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) 14676 Rothgeb Drive Rockville, MD 20850 Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV CAS # 10102-40-6 No hazardous components Section III - Physical/Chemical Characteristics Boiling Point Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water soluble No Data No Data No Data Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) 251-5990 Telephone Number for information (301) 251-5990 Date Prepared 09-16-2002 Signature of Preparer (optional) Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) Other Limits Recommended % (Optional) No Data No Data No Data Hazardous Decomposition or Byproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Fumes cause irritation of throat, coughing/choking. Skin contact is corrosive. Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Skin irritation, inflammation, ulceration Medical Conditions Generally Aggravated by Exposure available Emergency First Aid Procedures Wash with large amounts of water Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method Precautions to be Taken in Handling and Storing Store away from incompatibles Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Yes Special Mechanical (General) No Other Protective Gloves Other Protective Clothing or Equipment Work/Hygienic Practices Stability Wear protective equipment Observe all Government regulations Avoid contact Chemical resistant Section V - Reactivity Data Incompatibility Unstable Stable Hazardous Decomposition or Byproducts NIOSH/MSHA approved respirator Lab coat Eye Protection Avoid contact - wash thoroughly after handling Conditions to Avoid None Safety goggles Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X none Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Yes Yes Ingestion? Yes Health Hazards (Acute and Chronic) May cause irritation Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Irritation of eye, skin, mucous membranes and upper respiratory tract Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Eye/skin contact: flush with water Inhalation: remove to fresh air Ingestion: wash out mouth with water. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Mop up with absorbent material Waste Disposal Method Carefully acidify suspension of material to ph with Sulfuric acid. Add a 50%excess of aqueous sodium bisulfite (heat liberated). Observe federal, state and local regulations for disposal Precautions to be Taken in Handling and Storing Avoid incompatibles X Strong oxidizing agents Appearance and Odor colorless solution, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL Extinguishing Media Use extinquishing media for surrounding fire Special Fire Fighting Procedures Wear SCBA and protective clothing to prevent contact with skin and eyes Unusual Fire and Explosion Hazards none UEL Other Precautions Avoid contact Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Ventilation Local Exhaust No Special None Mechanical (General) Yes Other None Protective Gloves Rubber Eye Protection Safety goggles Other Protective Clothing or Equipment Rubber boots Work/Hygienic Practices Avoid contact
IDENTITY (As Used on Label and List) Hydrogen Peroxide, Stabilized Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) 14676 Rothgeb Drive Rockville, MD 20850 Special Fire Fighting Procedures Unusual Fire and Explosion Hazards Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) 251-5990 Telephone Number for information (301) 251-5990 Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) Hydrogen peroxide, H2O2 1.2% CAS # 7722-84-1 Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) at 30 C 22.3 Melting Point Vapor Density (AIR = 1) 1 Evaporation Rate (Butyl Acetate = 1) Solubility in Water Soluble Appearance and Odor Colorless liquid, no odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL N.D. Extinguishing Media Waterspray Wear SCBA and protective clothing to prevent contact with skin and eyes. 09-17-2002 1.110 Strong oxidizer, contact with other material may cause fire. Container explosion may occur under fire conditions. UEL N.D. Section V - Reactivity Data Stability Unstable Stable Conditions to Avoid Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X None Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Acute: Irritates mucous membranes upperrespiratory tract, eyes, skin Chronic: May have mutagenic affect Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Inhalation: burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea. Irritation. Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Ingestion: Wash mouth out with water. Contact physician Eyes: Flush with water Inhalation: Move to fresh air Skin: Flush with water Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method Cautiously acidify to ph2 with Sulfuric acid. Add a 50% excess of aqueous sodium bisulfate with stirring (heat generated). If no heat is evident, cautiously add until heat is liberated. Precautions to be Taken in Handling and Storing Observe federal, state, and local laws Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Excessive heat Incompatibility Strong acide, aluminum, steel Hazardous Decomposition or Byproducts Toxic oxides of phosphorous Mop up with absorbent material and dispose of properly Store away from incompatibilities Ventilation Local Exhaust No Special Mechanical (General) No Other Protective Gloves Rubber Eye Protection X NIOSH/MSHA approved No Chemical fume hood Safety goggles Other Protective Clothing or Equipment Rubber boots Work/Hygienic Practices Avoid inhalation. Keep away from incompatibilities and combustible material. IDENTITY (As Used on Label and List) Assay Reagent, Potassium Iodide Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) 14676 Rothgeb Drive Rockville, MD 20850 Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Potassium Iodide CAS# 7681-11-0 Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) 251-5990 Telephone Number for information (301) 251-5990 Date Prepared 09-19-2002 Signature of Preparer (optional) Other Limits Recommended Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water soluble Appearance and Odor Clear solution, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL Extinguishing Media Use media appropriate for surrounding fire % (Optional) Special Fire Fighting Procedures Wear SCBA and protective clothing to prevent contact with skin and eyes UEL Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X light, air Incompatibility Strong reducing agents, strong acids, steel, aluminum, alkali metals, brass, magnesium,, Zinc, cadmium, copper, tin, nickel Hazardous Decomposition or Byproducts Hydrogen Iodide Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Ingestion? Yes Yes Yes Health Hazards (Acute and Chronic) Acute: irritation to mucous membranes, upper respiratory tract, eyes, and skin Chronic: may cause reproductive disorders. Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Irritation Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Eye/skin contact: flush with water Inhalation: remove to fresh air Ingestion: wash out mouth with water. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Mop up with absorbent material and dispose of properly Waste Disposal Method Observe federal, state and local regulations Precautions to be Taken in Handling and Storing Avoid contact and do not store with incompatibles Other Precautions None Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Ventilation Local Exhaust No Special None Mechanical (General) No Other Chemical safety goggles Protective Gloves Rubber or PVC gloves Eye Protection Safety goggles Unusual Fire and Explosion Hazards Emits toxic fumes under fire conditions. Other Protective Clothing or Equipment Work/Hygienic Practices Rubber boots Avoid contact