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Experiment: Chymotrypsin Catalysis Prelab Assignment 1. 12 points) The table provided in Step 1 of Day 1's procedure outlines four different experimental "runs". a. What type of information will you obtain and record in your notebook during each of your runs? Dependent value: Independent value: b. What is the most important difference between these four runs? c What trend do you expect to observe in your results in response to that difference? 2 12.5 points) The table provided in Step 1 of Day 2's procedure outlines ten additional experimental "runs". a. What data will you observe and record for each run? b. What is the most important difference between the "a" and "b" runs? C. What trend do you expect to see in response to that difference? d. What is the most important difference between runs 5a, 6a, 7a, Ba, and 9a? e. What trend do you expect to see in response to that difference? 3. (2 points] Using your Day 1 data, you will generate a graph and use it to calculate one of chymotrypsin's important kinetic parameters. Indicate what you will plot on x and y of the final graph from the Day 1 data (INCLUDE YOUR UNITS AND INDICATE THE APPROPRIATE NUMBER OF SIG FIGS) and identify that kinetic parameter. Kinetic Parameter: X # of sig figs: Y: # of sig figs: 4. (3.5 points] The pH you use in each of your runs will impact the reaction rate you observe. You will take the pH of the solution into account when calculating the total amount of p-nitrophenol. (a) Write the equation that you will use to calculate the total amount of p-nitrophenol and highlight the part that takes pH into account. a. Write the equation (& highlight, as asked): Value b. Write the value you'll substitute into the equation for each run: Run Value Run Day 1 #1 Day 2 #5a Day 1 #2 Day 2 #6a Day 1 N3 Day 2 #7a Day 184 Day 2 #8a Day 2 99a C. WHY do you have to take pH into account to calculate the total amount of p-nitrophenol? Experimental Procedure Day 1 1. The stock enzyme concentration is 5.00 mg/mL. Keep enzyme solution on ice. All other assay solutions should be kept at room temperature. Sequentially prepare the following mixtures in a glass cuvette. Run 1 is a control for the non-enzymatic hydrolysis of p-nitrophenyl acetate at pH 7.00. mL 0.10 M phosphate, pH 7.0 mL delonized water mL chymotrypsin stock in 0.0010 M HCI Run 1 2.00 0.90 0.0 Run 2 2.00 0.70 0.20 Run 3 2.00 0.50 0.40 Run 4 2.00 0.10 0.80 2. For each solution, cover cuvette with parafilm and mix rapidly by inverting several times. 3. For each run, zero spectrophotometer at Ako (single wavelength mode, see Page 21) with solution from step 2. After you zero the mixture, observe absorbance for approximately 1 min to make sure there is no time dependent increase in absorbance due to contamination 4. To start the reaction, add 100. AL 0.0300 M NPA substrate with an automatic pipet to the step 2 solution. Immediately start timer (zero time) and rapidly mix by inversion. 5. Place cuvette in spectrophotometer and record A. after 30 sec and at subsequent 30 sec time intervals for 5 min. 6. Thoroughly rinse cuvette with deionized water, dry and start next run. Day 2 (pH Profile) 1. Sequentially prepare the following mixtures in a glass cuvette 6b 7a 70 Ba 8b 9a 9b 5a 5b 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 ml 0.10 M phosphate, pH 6.0 mL 0.10 M phosphate, pH 6.5 mL 0.10 M phosphate, pH 7.0 mL 0.10 M phosphate, pH 7.5 mL 0.10 M phosphate, pH 8.0 mL deionized water mL chymotrypsin stock mL 0.0010 M HCI . 0.50 0.50 0.50 0.50 0.50 0.40 0.50 0.40 0.50 0.40 0.50 0.40 2.00 2.00 0.50 0.50 0.40 0.40 0.40 0.40 - 0.40 0.40 2. For each run, follow procedure for Day 1, Steps 2-6 except use Biospec-1800 in kinetic mode to determine rates. See the following page for parameter settings (CHY). The "b" runs serve as controls for the non- enzymatic hydrolysis of NPA, and are necessary because the rate of aqueous hydrolysis of NPA is strongly pH dependent Experiment: Chymotrypsin Catalysis Prelab Assignment 1. 12 points) The table provided in Step 1 of Day 1's procedure outlines four different experimental "runs". a. What type of information will you obtain and record in your notebook during each of your runs? Dependent value: Independent value: b. What is the most important difference between these four runs? c What trend do you expect to observe in your results in response to that difference? 2 12.5 points) The table provided in Step 1 of Day 2's procedure outlines ten additional experimental "runs". a. What data will you observe and record for each run? b. What is the most important difference between the "a" and "b" runs? C. What trend do you expect to see in response to that difference? d. What is the most important difference between runs 5a, 6a, 7a, Ba, and 9a? e. What trend do you expect to see in response to that difference? 3. (2 points] Using your Day 1 data, you will generate a graph and use it to calculate one of chymotrypsin's important kinetic parameters. Indicate what you will plot on x and y of the final graph from the Day 1 data (INCLUDE YOUR UNITS AND INDICATE THE APPROPRIATE NUMBER OF SIG FIGS) and identify that kinetic parameter. Kinetic Parameter: X # of sig figs: Y: # of sig figs: 4. (3.5 points] The pH you use in each of your runs will impact the reaction rate you observe. You will take the pH of the solution into account when calculating the total amount of p-nitrophenol. (a) Write the equation that you will use to calculate the total amount of p-nitrophenol and highlight the part that takes pH into account. a. Write the equation (& highlight, as asked): Value b. Write the value you'll substitute into the equation for each run: Run Value Run Day 1 #1 Day 2 #5a Day 1 #2 Day 2 #6a Day 1 N3 Day 2 #7a Day 184 Day 2 #8a Day 2 99a C. WHY do you have to take pH into account to calculate the total amount of p-nitrophenol? Experimental Procedure Day 1 1. The stock enzyme concentration is 5.00 mg/mL. Keep enzyme solution on ice. All other assay solutions should be kept at room temperature. Sequentially prepare the following mixtures in a glass cuvette. Run 1 is a control for the non-enzymatic hydrolysis of p-nitrophenyl acetate at pH 7.00. mL 0.10 M phosphate, pH 7.0 mL delonized water mL chymotrypsin stock in 0.0010 M HCI Run 1 2.00 0.90 0.0 Run 2 2.00 0.70 0.20 Run 3 2.00 0.50 0.40 Run 4 2.00 0.10 0.80 2. For each solution, cover cuvette with parafilm and mix rapidly by inverting several times. 3. For each run, zero spectrophotometer at Ako (single wavelength mode, see Page 21) with solution from step 2. After you zero the mixture, observe absorbance for approximately 1 min to make sure there is no time dependent increase in absorbance due to contamination 4. To start the reaction, add 100. AL 0.0300 M NPA substrate with an automatic pipet to the step 2 solution. Immediately start timer (zero time) and rapidly mix by inversion. 5. Place cuvette in spectrophotometer and record A. after 30 sec and at subsequent 30 sec time intervals for 5 min. 6. Thoroughly rinse cuvette with deionized water, dry and start next run. Day 2 (pH Profile) 1. Sequentially prepare the following mixtures in a glass cuvette 6b 7a 70 Ba 8b 9a 9b 5a 5b 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 ml 0.10 M phosphate, pH 6.0 mL 0.10 M phosphate, pH 6.5 mL 0.10 M phosphate, pH 7.0 mL 0.10 M phosphate, pH 7.5 mL 0.10 M phosphate, pH 8.0 mL deionized water mL chymotrypsin stock mL 0.0010 M HCI . 0.50 0.50 0.50 0.50 0.50 0.40 0.50 0.40 0.50 0.40 0.50 0.40 2.00 2.00 0.50 0.50 0.40 0.40 0.40 0.40 - 0.40 0.40 2. For each run, follow procedure for Day 1, Steps 2-6 except use Biospec-1800 in kinetic mode to determine rates. See the following page for parameter settings (CHY). The "b" runs serve as controls for the non- enzymatic hydrolysis of NPA, and are necessary because the rate of aqueous hydrolysis of NPA is strongly pH dependent