Hanover College



Environmental effects on enzymesBiology 185, Winter 2020Objective of laboratory:Analyze activity of an enzyme under different environmental conditions, including temperature, and pHOverview: Enzymes work most effectively under specific environmental conditions. Depending on the physiological system an enzyme is working in, its activity can be negatively affected by pH, temperature, and other factors (such as enzyme inhibitors). For example, enzymes in the human stomach work under relatively acidic conditions, and those same enzymes could be affected negatively if the stomach was to undergo a rapid change to a more basic environment. This week, we will work with the same enzyme, potato tyrosinase, and examine the activity of this enzyme under changing environmental conditions. As one might expect, tyrosinase works optimally under certain environmental conditions that, when altered, can adversely affect the ability for the enzyme to bind substrate (catechol) and produce measurable product (ortho-quinone). Effect of Temperature on Enzyme ActivityEnzymes work only if the active site has the proper shape and charge. While raising and lowering temperatures have little effect on the charge of the functional groups in the active site, altering temperatures dramatically affects the bonds that shape it. Hydrogen bonds and van der Waals forces are weak and may break with even moderate increases in temperature, thus disrupting the active site. On the other hand, reducing temperature makes it more difficult to move bonds that need to be flexible during the course of the reaction. In general, the “right temperature is that at which the organism most commonly lives, and fluctuations account for changes in activity and viability of cells and organisms. Any wonder why our bodies work so hard to keep their internal temperatures steady?In this experiment, five different temperatures will be measured: 0°C (in ice), 23°C (room temperature), 37°C (human body temperature and where most of our enzymes perform optimally), 50°C and 70°C. The last three temperatures will be altered using water baths. Effect of pH on Enzyme ActivityWhile temperatures disturb the active site (the area on the enzyme where a substrate is bound) by breaking (high temperature) or stiffening (low temperature) weak bonds critical to its function, changes in pH affect ionic and hydrogen bonds in a different but analogous way. To form either bond, a pair of atoms must be held together by an attraction between partial (hydrogen) or full (ionic) opposite charges. Reducing the pH increases the number of H+ ions in solution and they, at characteristic points, bind either to -O-, =O, -OH, -NH2 functional groups causing them to become -OH, =OH+, -OH2+, or -NH3+, respectively. Similarly, increasing the pH and reducing the concentration of H+ (or increasing the OH- concentration) changes -OH to -O- and -NH3+ to -NH2. In all cases, critical pairings are disrupted, bonds fall apart and the active site is altered, resulting in the inability to properly bind a substrate.In order to determine the effect of varying concentrations of hydrogen ions on tenability of the tyrosinase to catalyze the production of ortho-quinone, four different pH conditions will be examined with buffers adjusted to pH 2, 5, 7, 9, and 12. Review your notes to determine which of these measures are more acidic, neutral, or more basic. Obtain the following supplies from the back benches for each group:1 rack spec tubes4 serological pipettes20 Erlenmeyer flasks50 test tubes200mL flask/beaker diH2O6mM Catechol (on ice)0.1M Tris, pH 7Tyrosinase enzyme (on ice)If doing pH: 0.1M Tris: pH 2, 5, 9, 12Initial Setup:Collect 50 test tubes and arrange into 5 sets of 5-paired test tubes (the 5 tubes are for the five time increments, 0, 5, 10, 15, and 30 minutes). One set of 5 tubes will serve to collect a test sample (T) and the other set of 5 tubes to collect the negative control (NC) or blank for each condition of the experiment. Due to time constraints, each group will be given one experimental condition to examine (either pH, or temperature). Each of the conditions has five variations to test (five pH conditions or five temperature conditions). To not get overwhelmed with the different conditions, divide the time into two periods by doing only the first three experimental conditions initially. The additional two will be done after successful completion of the first part. For example, if examining the effect of pH on enzyme activity, test the effect of pH 2, 5, and 7. After completion of the first three pH conditions, start over and test the remaining conditions (pH 9 and pH 12). If examining the effect of temperature, test the effect of 0?C (freezing), 23?C (room temperature), and 37?C. After completion of the first three temperature conditions, start over and test the remaining conditions (50?C and 70?C). Label one row of tubes NC and the other T for each set. To keep reactions in order, label tubes by time increments (i.e. NC0, NC5, NC10, NC15 and NC30, plus T0, T5, T10, T15, and T30) for each condition. Collect 20 125 ml Erlenmeyer flasks, 4 flasks for each of the five experimental conditions. Note: Each reaction condition initially begins with two flasks, a catechol and an enzyme, so that the reaction does not begin before you are ready. Later, these two flasks will be combined, at which point the reaction will begin.The reactions are set up identically to the 1X test condition from last week. Various temperatures are achieved with water baths and effects of pH determined by altering buffer pH.Setup for pH and temperature conditions (with standard concentration [1X] of enzyme and substrate): *Note: To alter pH, for each condition a buffer of different pH should be used1X Negative Control (Blank)1X Test Condition (all pH and ?C)NC Catechol Flask:T Catechol Flask:0mL Substrate (6 mM Catechol)5mL Substrate (6 mM Catechol)15mL Water10mL WaterNC Enzyme Flask:T Enzyme Flask:5mL Buffer (Tris)5mL Buffer (Tris)2mL Enzyme2mL Enzyme8mL Water8mL WaterProcedure: After setting up the flasks, an initial zero must be taken for each test condition and for the negative control:Take 2mL of solution from the first condition (e.g. 0°C or pH 2) NC catechol flask and add it to the NC0 test tube for that condition. Take 2ml of solution from the first condition enzyme flask and combine it with the NC0 test tube. This test tube will now have 4 ml of solution. Take 2mL of solution from the first condition (e.g. 0°C or pH 2) T catechol flask and add it to the T0 test tube for that condition. Take 2mL of solution from the first condition enzyme flask and combine it with the T0 test tube. This test tube will now have 4 mL of solution. Repeat steps 1a and 1b for each condition being sure to collect time 0 for both the NC and T.After taking time point 0 begin the experiment:Combine the remaining solutions of the first condition NC catechol flask with the NC enzyme flask (so, combine, for example, the 0°C or pH 2 catechol and enzyme flasks). The two flasks will now be one and have a total volume of 26mL. This is the 0°C or pH 2 NC flask. As soon as flasks are combined the reaction begins (enzyme and substrate will bind and begin to form product). Repeat the same procedure for the remaining flasks. Once finished you will have 6 flasks: a negative control (NC) and a test (T) for three different experimental conditions. As flasks are combined start a timer and provide the reaction with oxygen by swirling flasks constantly. As the reaction occurs a distinct color develops.At the following intervals, 5, 10, 15, and 30 min, remove 4mL from each flask and transfer to the collection test tubes. Pour the freshly collected 4mL NC 0 sample into a spec tube and use this sample to blank the spectrophotometer at an absorbance of 540nm. After blanking the spectrophotometer, spec the T0 sample. Record the optical density of all Test samples (Table 1). Repeat the steps in 5 above for each reaction condition (NC and T) and time (5, 10, 15, and 30min), and record all Test absorbance readings (Table 1). Note: The spectrophotometer must be blanked with the corresponding NC sample prior to measuring Test absorbance.Repeat the entire process with the final two experimental conditions. For example, if working with temperature repeat the process with the 50°C and 70°C conditions.Download the Excel spreadsheet titled “Enzyme II: Rate ” from Moodle. Follow the instructions in the spreadsheet. Note: There are two tabs at the bottom of the spreadsheet; one is titled “Temperature” another is titled “pH.” Click on the correct tab to enter the corresponding absorbance values measured. Enter the 5 calculated initial reaction rate into the Data collection: Effect of environment on enzyme spreadsheet posted on Moodle (Table 1).5 Initial velocity (Vi) or more simply the initial reaction rate (the change in absorbance at 540nm over time) for each of the reactions performed. Maximum product does not need to be recorded at this time, other than for calculating initial velocity. After completing the experiments clean writing off test tubes; rinse flasks and test tubes with deionized water. Table 1. Optical density for the effects of changes in temperature or pH on enzyme functionTime (min.)0?C or pH 2 or 23?C or pH 5 or 37?C or pH 7 or 50?C or pH 9 or 70?C or pH 12 05101530GlossaryActive site: area of a substrate where the enzyme bindsCatalyze: Speed upEnzyme: Protein that functions as a catalyst; increases the rate at which reactions occurProduct: Molecule resulting from an enzymatic reactionSubstrate: Molecule bound by an enzyme in a reactionin vivo: in the cellin vitro: in the test tubeLab write-upWhile you may have done only one of the conditions, your report must include and address both. Additionally, the data from this lab should be added to the data from week 1 of enzymes to generate a single write-up.The full scientific write-up will include: Goal: What was the reason the experiment(s) was/were performed (what was discovered/studied/identified)?Checklist:Is it brief? (Typically one sentence per unique experiment)Is it descriptive? (Does the goal mention the tested variables?)Results: What were the major findings of the experiments performed (include means, SD, and t-test values when appropriate)? What information do the figures present (figures must be cited in the text)? If t-tests were performed, what is indicated, does a significant difference exist between the treatments?ChecklistFindings in paragraph form, includingDescriptive statistics (Mean, SD) for each treatment are included?If a t-test was performed, do results include whether the means are significantly different or not?Do the results avoid interpretation or analysis?Does the results cite the figure (s) (at the end of sentences)?Pertinent figures from the Excel spreadsheet, with captionsDiscussion: How do the results help meet the goal(s)? How should the results be interpreted? Are trends present? If t-test was performed, what do the significance and non-significance mean?The questions below should be addressed in the Discussion section:?When examining the initial reaction rate for the experiment performed by your group:Is there a significant increase in the enzyme (tyrosinase) activity under different temperature/pH? What does this indicate about the interactions between the enzyme (tyrosinase) and the substrate (catechol)? Write a summary paragraph for each experiment not perform in lab by your group, (you will have one). Address each with the following questions: What were the general findings (examine the figures)? How did the enzyme activity change with different environmental treatments in temperature/pH? Which condition(s) appear to be the most optimal for an enzyme to work properly? Relate this last question to the structure or integrity of the enzyme and its ability to bind a substrate. Final Tips and Suggestions:Read over the report at least twice before submitting to ensure major points are addressed following correct grammatical rules. Use proper units throughout (mL, μL, g, nm, etc.).Do not do the report at the last minute; give yourself a few days to come up with an outline. Come see me if you need assistance. ................
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