Enzyme Lab Part 2 Bio 110.pdf

Enzyme Action Part II Objective: ​Using the optimal salvia dilution from in Part I, determine the effect of NaCl, pH and temperature on enzyme activity. Negative and positive controls were completed to confirm that all reagents were working as expected and...

Enzyme Action Part II Objective: ​Using the optimal salvia dilution from in Part I, determine the effect of NaCl, pH and temperature on enzyme activity. Negative and positive controls were completed to confirm that all reagents were working as expected and the 1:16 saliva dilution (determine to be optimal in week #1) remained in the optimal range (no starch detection between 4-10 wells). Protocols and the results for negative and positive controls are shown below: Negative control: 1. Obtained a clean test tube and labeled it negative control. 2. Added 2 mL of pH 7 buffer. 3. Added 1 mL of water to the tube. Mixed. 4. Incubated at room temperature for 5 minutes. 5. Added 2 mL of 0.25% starch solution to the tube. Immediately started timer and mixed. 6. At 30 second intervals transferred 100 µl from “negative control” experimental set-up (using the micropipette) and added sequentially to Lugols-containing wells of the microtiter dish (96 well plate). Continued adding 100 µl of “negative control” in 30 second intervals until starch was not detectable (“no color change”) for 3 consecutive wells. Positive control: 1. Obtained a clean test tube and labeled it positive control. 2. Added 2 mL of pH 7 buffer. 3. Added 1 mL of 1:16 saliva dilution (optimal dilution determined in Part I) to the tube. Mixed. 4. Incubated at room temperature for 5 minutes. 5. Added 2 mL of 0.25% starch solution to the tube. ​Immediately​ started timer and mixed. 6. At 30 second intervals transferred 100 µl from “positive control” experimental set-up (using the micropipette) and added sequentially to Lugols-containing wells of the microtiter dish (96 well plate). Continued adding 100 µl of “positive control” in 30 second intervals until starch was not detectable (“no color change”) for 3 consecutive wells. What conclusions can you make from the negative and positive control results? 1 Examination of environmental influences on enzyme activity: ​The experimental protocol and results for each environmental factor is provided below. You will need to analyze and interpret all environmental conditions to answer questions at the end of the lab packet. Experimental protocol to test the effect of NaCl on enzyme activity 1. Obtained a clean test tube and labeled it 1% NaCl. 2. Added 2 mL of 1% NaCl (pH7) to the tube. 3. Added 1 mL of my 1:16 saliva dilution (optimal dilution determined in Part I) to the tube. Mixed. 4. Incubated at room temperature for 5 minutes. Allows enzyme to be in experimental environment before substrate (starch) is added. 5. Added 2 mL of 0.25% starch solution to the tube. Immediately started timer and mixed. 6. At 30 second intervals transferred 100 µl from “1% NaCl” experimental set-up (using the micropipette) and added sequentially to Lugols-containing wells of the microtiter dish (96 well plate). Continued adding 100 µl of “1% NaCl” in 30 second intervals until starch was not detectable (“no color change”) for 3 consecutive wells. Recorded the number of wells and the total time it takes to reach “no color change” in Table 1. 7. Repeated the protocol using the following experimental conditions in place of the 1% NaCl: 5% NaCl, 10% NaCl, 20% NaCl, and 30% NaCl. The results are shown below and data has been recorded in Table 1. Table 1. NaCl Results Tube # # of wells to “no color change” Time to “no color change” (min) 1% NaCl 2 1 5% NaCl 2 1 10% NaCl 3 1.5 20% NaCl 3 1.5 30% NaCl 4 2 2 If you were assigned NaCl as your environmental factor, answer the questions below. 1. Copy your hypothesis from Part I into the space below. Evaluate the results against your hypothesis. Do the experimental results support or refute your hypothesis? 2. Determine which type of graph will best present the data in Table 1 (# of wells or time, not both). Construct the graph using google sheets and paste the graph into the space below. What can you conclude from the experimental results? 3 Experimental protocol to test the effect of pH on enzyme activity 1. Obtained a clean test tube and labeled it pH 3. 2. Added 2 mL of pH 3 buffer to the tube. 3. Added 1 mL of my 1:16 saliva dilution (optimal dilution determined in Part I) to the tube. Mixed. 4. Incubated at room temperature for 5 minutes. Allows enzyme to be in experimental environment before substrate (starch) is added. 5. Added 2 mL of 0.25% starch solution to the tube. Immediately started timer and mixed. 6. At 30 second intervals transferred 100 µl from “pH 3” experimental set-up (using the micropipette) and added sequentially to Lugols-containing wells of the microtiter dish (96 well plate). Continued adding 100 µl of “pH 3” in 30 second intervals until starch was not detectable (“no color change”) for 3 consecutive wells. Recorded the number of wells and the total time it takes to reach “no color change” in Table 2. 7. Repeated the protocol using the following experimental conditions in place of the pH 3 solution: pH 5, pH 6, pH 9, pH 11. The results are shown below and data has been recorded in Table 2. Table 2. pH Results Tube # # of wells to “no color change” Time to “no color change” (min) pH 3 >12 >6 pH 5 3 1.5 pH 6 3 1.5 pH 9 >12 >6 pH 11 >12 >6 4 If you were assigned pH as your environmental factor, answer the questions below. 1. Copy your hypothesis from Part I into the space below. Evaluate the results against your hypothesis. Do the experimental results support or refute your hypothesis? Will starch be detected in Ph levels using Lugol’s reagent? The experimental results do support the hypothesis, but not all of the wells had starch in them. 2. Determine which type of graph will best present the data in Table 2 (# of wells or time, not both). Construct the graph using google sheets and paste the graph into the space below. What can you conclude from the experimental results? (I wasn’t sure if I needed to deviate from the chart and remove the < or > it seems to mess up the chart) 5 Experimental protocol to test the effect of temperature on enzyme activity 1. Obtained a clean test tube and labeled it 0°C. 2. Added 2 mL of pH 7 buffer to the tube. 3. Added 1 mL of my 1:16 saliva dilution (optimal dilution determined in Part I) to the tube. Mixed. 4. Incubated at 0°C for 5 minutes. Allows enzyme to be in experimental environment before substrate (starch) is added. 5. Added 2 mL of 0.25% starch solution to the tube. Immediately started timer and mixed. 6. At 30 second intervals transferred 100 µl from “0°C” experimental set-up (using the micropipette) and added sequentially to Lugols-containing wells of the microtiter dish (96 well plate). Continued adding 100 µl of “0°C” in 30 second intervals until starch was not detectable (“no color change”) for 3 consecutive wells. Recorded the number of wells and the total time it takes to reach “no color change” in Table 3. 7. Repeated the protocol using the following experimental conditions in place of the 0°C temperature: 20°C, 37°C, 53°C, and 80°C. The results are shown below and data has been recorded in Table 3. 6 Table 3. Temperature Results Tube # # of wells to “no color change” Time to “no color change” (min) 0°C >12 >6 20°C 5 2.5 37°C 4 2.0 53°C >12 >6 80°C >12 >6 If you were assigned temperature as your environmental factor, answer the questions below. 1. Copy your hypothesis from Part I into the space below. Evaluate the results against your hypothesis. Do the experimental results support or refute your hypothesis? 7  2. Determine which type of graph will best present the data in Table 3 (# of wells or time, not both). Construct the graph using google sheets and paste the graph into the space below. What can you conclude from the experimental results? Everyone must respond to the following: Report in words the results of each environmental factor on amylase activity: pH: Stach was only located in 2 of the rows present. The reset remained blue with the Lugol’s reagent added. In pH levels 5 and 7 starch was located almost immediately. NaCl: 8 Starched was located in almost every set of wells present. It presented itself in less wells especially in the 1%, 5%, and 10% of NaCl. Temperature: Starch was detected the most in 20 and 37C, it was not detected at all at 80C. 0 and 50C are very close in amounts in well changes. Based on the results, what is the composite optimal environment for amylase activity? NaCl is the most optimal environment for amylace. Post-lab questions 1. Based on what you learned in this lab, generalize and summarize how chemical reactions in cells are dependent on the presence and activity of specific enzymes. What, exactly, can happen to an enzyme’s physical structure when its environment changes? How can this effect the rate of a chemical reaction? What is the term “denaturation” and how does this process alter the enzyme’s ability to function? 2. Discuss whether you think all enzymes have the same optimal environment. What would contribute to them having similar requirements for their optimal environments and what could contribute to them having different requirements for their optimal environments? With regard to the factors that can affect enzyme activity that were tested in this lab, which factors are most likely to vary in the human cellular environment and which are least likely to vary? 1. Enzymes are a catalyst to change in the cell. An enzyme can lose its shape, it can also speed up or slow the reaction. Denaturation is when the enzyme loses shape and stops functioning. 2. I don’t think enzymes have the same optimal environment. I think having similar properties, like temperature, pH level, and substrate concentration. They could have 9 different requirements if the temperature, pH level, and substrate concentration differ. In the human cellular environment I believe pH levels are the least likely to vary. 10