The Effect of Temperature on an Enzyme Catalyzed Reaction - Student Handout

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Jan 9, 2024

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General Biology, BIO 123 Investigating the action of Horseradish Peroxidase – Week #2 During week #2, we will be investigating the effect of varying temperatures on the rate of an enzyme-catalyzed reaction. Investigating the Effect of Enzyme Concentration on an Enzyme-Catalyzed Reaction Experimental protocol Materials list Enzyme extract prepared during session #1 10 mM H 2 O 2 hydrogen peroxide solution 25 mM Guaiacol solution Citrate Phosphate buffer, pH 5 (2) 100 ml beakers Test tube rack (9) clean test tubes Green pipette pump (for use with 5 - 10 mL pipettes) 5 mL pipettes Blue pipette pump (for use with 1 mL pipettes) 1 mL pipettes Spectrophotometer (5) clean cuvettes for spectrophotometer (1) cuvette rack (1) box of kimwipes to clean outside of cuvettes 100 mL graduated cylinder Masking tape (use to label test tubes) Marker Metric ruler Timer Step 1: Determining the Dilution of Enzyme to be Used For this experiment, we will be testing the effects of different temperatures on the rate of an enzyme catalyzed reaction. As such, during this experiment you need to chose one enzyme dilution to study. To do this, you will review your graph generated by the data you gathered during the first lab session. You should select the enzyme extract dilution from the previous experiment that resulted in an increase in absorbance over the entire 120- second time period. You want to select the line that illustrates a large increase, but does not ‘max out’ to infinity early in the time period. Once you have determined which dilution you want to use, please review your choice with your instructor for confirmation. Once your choice is confirmed, fill in the volume of enzyme extract and buffer that you will be using for test tubes # 3, 5, 7 and 9 in Table #3. Step 2: Setting up the Experiment The exposure temperatures to be used in this study will be that of a cooler of ice at approximately 5 o C, room temperature at approximately 20 – 24 o C, as well as 34 o C and a 60 o C water baths. Since the temperature of the ice and room temperature may vary, you must confirm and fill in the actual temperatures in Table #3. Protocol: 1. Using masking tape, label your other 100 mL beaker “citrate phosphate buffer pH 5 ” 2. Using a graduated cylinder, add 25 mL room temperature citrate-phosphate buffer pH 5 to the beaker 3. Obtain (9) clean test tubes. 4. Using masking tape, label the test tubes 1 – 9. 5. Before you begin filling the test tubes, review Table #3. Note the following information: a. What is going in to test tube #1? Note the color of the solution.

b. What is going in to test tubes 2,4,6, and 8? Note the color of the solution c. What is going in to test tubes 3,5,7, and 9? Note the color of the solution d. What is the total volume of test tube #1? e. What is the total volume of test tubes 2,4,6, and 8? f. What is the total volume of test tubes 3,5,7, and 9? 6. When you are setting up the test tubes for the experiment, it is very important that you do not mix or cross contaminate the contents of the test tubes. 7. The solutions containing guaiacol and H 2 O 2 will be stored in separate bottles that have dispensers (called dispensettes) on them. You will use the dispensettes to deliver the specified volumes to specific test tubes as indicated in table #3. a. The bottle containing guaiacol will be set to deliver 1.0 mL. b. The bottle containing H 2 O 2 will be set to deliver 2.0 mL. 8. You will use pi-pumps and disposable pipettes to deliver the specified volumes of citrate phosphate buffer pH 5 to the specific test tubes as indicated in table #3. 9. Once you have set up all of your test tubes, show them to your instructor. You are now ready to move on to the next part of the experiment.

Table #3 The Effect of Various Temperatures on an Enzyme-Catalyzed Reaction: Test tube contents Test tube number Incubation Temperature Citrate- phosphate buffer, pH 5 (mL) 10 mM H 2 O 2 (mL) Enzyme extract (mL) 25 mM Guaiacol (mL) 1 Blank 5.0 2.0 0 1.0 2 Ice _____ o C 0 2.0 0 1.0 3 Ice _____ o C 0 0 4 Room temp _____ o C 0 2.0 0 1.0 5 Room temp _____ o C 0 0 6 34 o C 0 2.0 0 1.0 7 34 o C 0 0 8 60 o C 0 2.0 0 1.0 9 60 o C 0 0 Couplet #4 Test tubes # 8 & 9 Couplet #3 Test tubes # 6 & 7 Couplet #2 Test tubes # 4 & 5 Couplet #1 Test tubes # 2 & 3

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Step 3: Incubating Samples in Varying Temperatures 1. Place the two tubes prepared for a specific temperature at that temperature for 10 minutes. DO NOT MIX THE TUBES YET. a. Important Note: After the incubation period, do not take all of your samples out of the various temperatures at the same time because by the time you take the readings for one set, all of the other samples will not be at the correct temperature anymore. Due to this, do only one couplet at a time . 2. During the incubation period, use Tube # 1 to calibrate your spectrophotometer. 3. When the incubation period (at least 10 minutes) has elapsed for each couplet, quickly bring them over to your spectrophotometer and begin taking readings. Mix the contents of the two test tubes together (time zero) and transfer them quickly to a cuvette. Read and record the absorbance at 500 nm wavelength in Table #4. 4. Continue to record absorbance at 20 sec intervals for a total of 120 secs. Table #4 Absorbance readings: The effect of varying temperature on an enzyme catalyzed reaction Incubation Temperature 0 sec 20 sec 40 sec 60 sec 80 sec 100 sec 120 sec Ice _____ o C Room temp _____ o C 34 o C 60 o C

Step 4: Generating the data and Experimental Design For this experiment, you will be using a spectrophotometer to accurately measure the final color of the enzyme-catalyzed reaction. Remember, you will start out with colorless solutions. For the experimental portion, you will be mixing substrates with varying concentrations of the HRP enzyme. To measure enzyme activity, the spectrophotometer will allow us to accurately detect the absorbance of each solution. The spectrophotometer will expose each sample to specific wavelengths of visible light and will detect and measure how much of the wavelength is absorbed by the solution.

Zeroing out the spectrophotometer protocol Spectrophotometers need to warm up before being used. Turn on the spectrophotometer. Let it warm up for 15 mins. Your instructor will setup the spectrophotometer. Once this is complete, you will do the following: 1) Set the wavelength of light to 500 nm. 2) Before you take each experimental reading, you must “zero” the spectrophotometer using the contents of test tube #1. This is your “blank”. The purpose of the blank is to “teach” the spectrophotometer what 0 absorbance is for your solution. Your blank should be completely colorless. Using this tube gives the spectrophotometer a baseline for your particular solution. a. To zero out the spectrophotometer, carefully transfer the contents of test tube #1 into a clean cuvette. b. Wipe the outside of the cuvette with a kimwipe. Be careful not to get your fingerprints all over the cuvette. Remember, anything on the cuvette could effect your experimental results. c. Place the cuvette into the chamber of the spectrophotometer. d. Turn the absorbance control knob until the spectrophotometer reads as close to 0.00 as you can get. Note: you might have to turn the knob a few times before you will see a change. Experimental protocol Once you have the spectrophotometer zeroed, you are ready to run your experimental samples. For this part of the experiment, you want to work in groups of three. One person from your group will be the time keeper, another person will be responsible for recording the data, and the third person will be zeroing out the spectrophotometer and running the experimental samples in the spectrophotometer. There are four different readings to obtain, so make sure that you are taking turns for each “job”. By the end of the experiment, everyone in your group should be able to use the spectrophotometer. 1) After the spectrophotometer has been zeroed, remove the cuvette and place it in your cuvette rack. 2) The first reading that you will take will be for couplet #1. 3) Mix the contents of test tube # 2 (contains only substrate) with test tube # 3 (contains enzyme) carefully and quickly. To do so, carefully pour the contents of test tube # 2 into a cuvette and then carefully pour the contents of test tube # 3 into that same cuvette. 4) Quickly wipe the cuvette with a kimwipes and insert it into the chamber. You have to do all of this within 10 - 15 seconds!! 5) At exactly 20 seconds, the person recording the data will record the absorbance reading of the solution into table #4. 6) The cuvette remains in the chamber and you will continue to record the absorbance data at 20 second intervals for 120 seconds. All data is to be entered into table #4. After the 120 second reading, you are done with that particular couplet. a. Note: The highest reading (called the infinity point) on these spectrophotometers is 1.999. if your sample reaches the infinity point, do not stop. Continue taking readings for the full 120 seconds. b. Remove the sample from the chamber. c. You are now ready to go on to the next couplet. 7) Swap responsibilities with your lab partners. 8) Use the “blank” test to re-zero the spectrophotometer 9) Using couplet # 2 with test tubes 4 and 5, repeat steps 3 – 6. 10) Using couplet # 3 with test tubes 6 and 7, repeat steps 3 – 6. 11) Using couplet # 4 with test tubes 8 and 9, repeat steps 3 – 6.

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12) As soon as you have completed the experimental steps, discard the dilutions in the appropriate waste container and clean out the cuvettes. Do not let the solutions sit in the cuvettes because they will stain them. a. Important Note: NEVER use a scrub brush, paper towel, or any other abrasive to clean the cuvettes. Scratches on the cuvettes will cause light to refract and will cause inaccuracies in your experimental data. Clean the cuvettes with liquid test tube soap and water only. Clean up instructions 1. All used pipettes are to be disposed of in the “pipette discard” canister located next to the sink. 2. Remove all tape and markings from test tubes and beakers. 3. As soon as you have completed absorbance readings for each couplet of enzyme + substrate make sure that you dispose of the solution in the hazardous waste container and wash the cuvette with soap and water only. 4. Allow clean test tubes and cuvettes to drain upside down in the tube racks. a. Important: Do not dry the inside of the test tubes or cuvettes with paper towels. Clean with test tube soap and water. Invert and allow to drain in rack.

Name 1. BEFORE PERFORMING THE EXPERIMENT: What is your formally worded hypothesis regarding the effect of temperature on the rate of enzyme activity? Once you have formed your hypothesis , FULLY explain the information you used to develop this hypothesis. What I want to know is what background information did you base your hypothesis on and why is this information relevant to your hypothesis? 2. What is the independent variable in this experiment? 3. What is the dependent variable?

4. What variables must be controlled? Name at least 3 a. b. c. 5. What is the control (control treatment) for this experiment? Upon completion of the experiment: 6. Prepare the following for the experiment that was performed The Effect of Temperature on the Rate of an Enzyme Catalyzed Reaction . This portion of your post-lab should be typed and handed in stapled to this sheet. 1. A title page (1 page) a. Pay attention to the format of the title page. b. Your title should be original, specific and describe the objectives of the experiment. “Enzyme lab” as a title is NOT sufficient. 2. Introduction (1 – 2 pages) i. Must include all relevant background information regarding the organism(s) or object(s) under study. i.e. basic information regarding enzymes such as the type of macromolecule it is, a complete description of the common molecular levels of protein structure found in enzymes, what effects differing temperatures will have on enzyme structures, any bonds that maybe disrupted due to experimentation, the substrates for the specific enzyme you tested, etc. ii. Must include your experimental hypothesis and prediction(s). Do NOT write the words “hypothesis” or “prediction” before your written hypothesis and prediction. The way you word each of these will tell the reader what they are.

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iii. Must include a brief description of the experiment and its outcome. iv. DO NOT write your lab report in the first person. Lab reports are written in the third person, impersonal form. v. Since you will have already completed the lab when you write the report, remember to write in the PAST tense. vi. Make sure to cite all information you include that has originated from sources other than yourself. b. Data tables and graphs i. Include the data table 1. The table must be titled and include the appropriate labels and units. ii. You must hand in the hand drawn graph you did in class as well as the same graph recreated in excel. 1. All graphs should always be properly titled, the x & y axis must be labeled and the graph must have a legend. 2. Don’t forget the units! iii. Create a second excel graph investigating the effect of temperature on the rate of reaction. 1. Make sure to include an additional data table that includes your rate of reaction calculations. Make sure the data table has a title. 2. Calculate the trend line for your excel graph. iv. All tables and graphs must have an appropriate title (absorbance versus time is NOT an appropriate title). The title must be descriptive so the reader knows exactly what is being tested. v. All graphs must be fully labeled (X and Y axis) and must have a legend. Don’t forget the units! c. References (1 page) i. Follow MLA or APA guidelines for in-text citations ii. Make sure to include a references page for all work cited.

SCIENTIFIC WRITING: WHAT NOT TO DO! (Courtesy of Dr. Erin Christensen) 1. NO DIRECT QUOTES! In a scientific paper, citations are limited to specific research only and not for general knowledge. Quotations of any type should not be used. Example: Osmolarity refers to the amount of solute in a solution. “Three terms describing osmolarity are hypertonic, hypotonic, and isotonic (Smith, 2005).” Quotes are NOT appropriate here because osmolarity terms are established knowledge and not something that Smith is credited with coining. The information should be paraphrased and Smith’s book listed as a reference at the end of the paper along with any other works consulted. If Smith did a particular experiment relevant to your research, then a citation is appropriate: Experiments have demonstrated that 0.5M aqueous sucrose is isotonic for potato cells (Smith, 2005). Here, Smith is cited in the report and in the Reference section, because specific data are reported. Note that the information is still paraphrased and not directly quoted from Smith’s paper. 2. NO DRAMA! You are not writing a novel. Pronouns, creative language, slang, etc., should not be used. Combine sentences so that your writing is succinct but complete, without the repetitive use of words or phrases. Do assume that your reader has knowledge of basic laboratory procedures. Consider: I put potatoes on a paper towel and me and my partners made cylinders by pressing it really hard through the potato. I put potatoes on the table and measured each one with a ruler very carefully. After we measured each one, we cut them into pieces that were 7 cm. long. This paragraph has many grammatical errors (“me and my partners”), casual language (“really hard”), switching tenses (“remove…measured”), repetitive phrases (“I put potatoes”), unidentified nouns (“pressing it”…what?), and a number rule violation (numbers less than ten are spelled out and hyphenated with a unit, period omitted). In contrast: Potato cylinders, prepared from peeled potatoes using a cork borer, were cut with a razor blade into 7-cm lengths and blotted with paper towels. It is assumed that you and your partner did the work, that you used a ruler, were careful, etc. Remember to use past tense and formal, grammatically correct English consistently throughout the paper. Proofread! 3. NO SLOPPY TABLES! Your data should be clear and easy to understand. Tables and graphs are numbered consecutively, titled, and appropriately labeled. Do not use pencil, markers, etc., and do not split tables across pages. Adjust the fonts to be consistent with the size of your tables. In the report, refer to a table as Table 1, etc., and a graph as Figure 1, etc. Examples: Table 1: Change in weight of potato tissues following sucrose solution incubation. Figure 1: Percent change in weight of potato tissue as compared to sucrose concentration. 4. NO USE OF MR., MRS., MS., or MISS! An instructor’s title is either Prof. or Dr., or, at the very least, a first initial and last name – correctly spelled, of course. 5. NO PLASTIC COVERS! Staple your paper in the upper left corner.

Student Name Grade Bio 123 Enzyme Lab #2 Lab Report Rubric Please fill in your name and staple this at the END of your lab report. Title of Report Properly describes experiment (5) Vague / ambiguous title (2) Missing (0) Introduction Fully explains object under study. Contains properly worded hypotheses / predictions (35) Fully explains object under study. Hypotheses and/or predictions worded improperly or missing (25) Insufficient explanation of object under study. Contains properly worded hypotheses / predictions (15) Insufficient explanation of object under study. Hypotheses and/or predictions worded improperly or missing (10) Missing (0) Data Table Data is accurately depicted. Properly titled. (10) Data is accurately depicted. Missing or improperly titled. (7) Missing (0) Hand Drawn Graph (Experimenta l data) Data is accurately depicted. All axes labeled. Legend complete. Properly titled. (10) Data is accurately depicted. Legend complete. Improperly titled (7) Data is accurately depicted. One or more labels is missing and legend is incomplete or graph titled improperly (4) Data is inaccurately depicted or graph is missing. (0) Excel Graph # 1 (Experimenta l Data) Data is accurately depicted. All axes labeled. Legend complete. Properly titled. (10) Data is accurately depicted. Legend complete. Improperly titled (7) Data is accurately depicted. One or more labels is missing and legend is incomplete or graph titled improperly (4) Data is inaccurately depicted or graph is missing. (0) Excel Graph Data is accurately Data is accurately Data is accurately Data is

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# 2 (Rate of Rxn) depicted. All axes labeled. Legend complete. Properly titled. (10) depicted. Legend complete. Improperly titled (7) depicted. One or more labels is missing and legend is incomplete or graph titled improperly (4) inaccurately depicted or graph is missing. (0) References Complete and followed format (10) Submitted reference page but did not use in-text citations. (5) Missing (0)

The Effect of Temperature on an Enzyme Catalyzed Reaction - Student Handout

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