Horseradish Peroxidase Write-Up
Bio 205 Lab W/8:00
Enzyme II Write-Up
Methods:
My partner and I ran two experiments to measure the activity of the enzyme horseradish peroxidase under varied conditions. The first of which measured the effects of altered pH levels, while the goal of the second was to examine the effects of varied temperatures. To test the effects of pH on horseradish peroxidase, we began by zeroing a Spec 20 with 5.0mL of substrate (25mM guiacol) at pH 6.5. Once the Spec 20 was accurately zeroed, we added 100μL of the enzyme to this tube. The initial concentration of our enzyme throughout these experiments was 2μL/mL. We then covered the tube with Parafilm and inverted it until well mixed. At this point, we quickly and simultaneously placed the tube in the Spec 20 and started the timer. We took an initial absorbance reading and continued to take absorbance readings every ten seconds for a total of 10 readings between 0 and 90 seconds of activity. We repeated these steps twice more with a pH 6.5 solution of the substrate. After this, we performed three trials each with the same procedure using substrate samples at pH 3.5 and again at pH 9.5. Overall, we had a total of 9 trials using 3 different pH levels. In each trial, the tube in use was carefully dried and properly oriented prior to taking readings. When the enzyme was not in use, it was stored on ice. The next experiment’s purpose was to examine the effects of temperature on ppH activity. Using two small tubes of enzymes, we dispensed 0.45mL of enzyme into three separate 1.5mL plastic tubes. Each tube was labeled with the treatment temperature it underwent and our group name. The labels were necessary because each tube was then placed in its respective temperature zone among tubes from other groups. The heat blocks and water baths were set at 40°C, 60°C, and 95°C. Each enzyme stayed in its specified temperature zone for ten minutes, undisturbed. The enzymes were then immediately placed on ice. The remaining
Enzyme II Write-Up
Methods:
My partner and I ran two experiments to measure the activity of the enzyme horseradish peroxidase under varied conditions. The first of which measured the effects of altered pH levels, while the goal of the second was to examine the effects of varied temperatures. To test the effects of pH on horseradish peroxidase, we began by zeroing a Spec 20 with 5.0mL of substrate (25mM guiacol) at pH 6.5. Once the Spec 20 was accurately zeroed, we added 100μL of the enzyme to this tube. The initial concentration of our enzyme throughout these experiments was 2μL/mL. We then covered the tube with Parafilm and inverted it until well mixed. At this point, we quickly and simultaneously placed the tube in the Spec 20 and started the timer. We took an initial absorbance reading and continued to take absorbance readings every ten seconds for a total of 10 readings between 0 and 90 seconds of activity. We repeated these steps twice more with a pH 6.5 solution of the substrate. After this, we performed three trials each with the same procedure using substrate samples at pH 3.5 and again at pH 9.5. Overall, we had a total of 9 trials using 3 different pH levels. In each trial, the tube in use was carefully dried and properly oriented prior to taking readings. When the enzyme was not in use, it was stored on ice. The next experiment’s purpose was to examine the effects of temperature on ppH activity. Using two small tubes of enzymes, we dispensed 0.45mL of enzyme into three separate 1.5mL plastic tubes. Each tube was labeled with the treatment temperature it underwent and our group name. The labels were necessary because each tube was then placed in its respective temperature zone among tubes from other groups. The heat blocks and water baths were set at 40°C, 60°C, and 95°C. Each enzyme stayed in its specified temperature zone for ten minutes, undisturbed. The enzymes were then immediately placed on ice. The remaining
Cited: Alberts, Bruce; Bray, Dennis; Hopkin, Karen; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith and Walter, Peter. 2010. Essential Cell Biology (3rd ed.). Garland Science, Taylor & Francis Group, LLC, New York. Chattopadhyay, Krishnananda and Mazumdar, Shyamalava 1999. Structural and Conformational Stability of Horseradish Peroxidase: Effect of Temperature and pH. 39: 263-270. Department of Biology, Western Washington University. 2011. Biology 205 – Introduction to Cellular and Molecular Biology Laboratory Manual. Western Washington University, Bellingham, WA. Stahle, Sam and Yang, Pin (2011) [Enzyme II Lab]. Unpublished raw data.