Bio 205 Lab W/8:00
Enzyme II Write-Up
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 unheated (control) enzyme was also kept on ice. We again zeroed the Spec 20 with 5.0mL of our substrate. Then we added 100μL of the control enzyme to this tube, which was quickly covered in Parafilm, inverted until well mixed, and inserted into the Spec 20. As the tube was inserted, the timer was started as before. Again, ten readings were taken at 10-second intervals. This was repeated for a total of 3 trials, and the same procedure was used to perform 3 trials at 40°C, 60°C, and 95°C.
For each reaction, we wanted to determine the turnover number in order to quantify the enzyme activity. In order to do this, we would have to divide the rate of the reaction by the enzyme concentration (Department of Biology, WWU, 2011). The rate of reaction is calculated by using a plot of absorbance vs. time. The slope of the trend line of all the points plotted is the rate of reaction in absorbance units per second. For our purposes, the rate of reaction should be expressed in moles per liter per second. This requires a conversion. We divided the slope of the trendline by the molar extinction coefficient of tetraguiacol, the product of our enzyme and substrate’s conjunction. This coefficient is 26,600 at 400nm. Now we needed to calculate the enzyme concentration. To determine this, we multiplied the concentration of our stock enzyme solution, 2 micrograms per milliliter, by the inverse molecular weight of horseradish peroxidase, which is 1/40,000 grams per mole. This value was then multiplied by our volume of enzyme over our volume substrate per tube, to take into consideration the dilution that took place in each tube. Having both the rate of reaction and the enzyme concentration, we could then calculate the turnover number by dividing the former by the latter. The result would tell us the number of substrate molecules converted to product molecules per enzyme unit per unit time, which is abbreviated as sec-1. For specific figures used in the aforementioned calculation,...
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.
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