3-Specs_and_Enzymes_Lab Report_2023

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Colorado State University, Fort Collins *

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212

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Biology

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Jan 9, 2024

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LIFE 212 Laboratory Report 3 Name:____________________________ Due:_____________________________ Section:____________________________ 50 pts total plus Notebook Conclusion (4 pts) Enzymes I: Spectrophotometry and Partial Purification of Tyrosinase Enzyme from Potato Tubers (50 points) Note: Label your graphs properly; use a descriptive title, label the x- axis and y -axis, and be sure to include all units. Note: Show your work on all calculations in all the problems in the report: 1. Results Table (8 points) Tube # Concentration of Dopachrome (mM) Absorbance A/C 1 0 2 0.125 3 0.25 4 0.5 5 1.0 6 2.0 7 4.0 8 8.0

LIFE 212 Laboratory Report 3 Name:____________________________ Due:_____________________________ Section:____________________________ 2. Plot and tape in the space below your standard curve relating the absorbance readings of dopachrome (A 475 : y axis) versus the concentrations of dopachrome in mM ( x -axis). Include a straight line equation and your regression coefficient (r 2 ) on the graph. If you are using Excel, choose the option that allows you to print the graph and the data sheet on the same page. Excel instructions for plotting a straight line graph are in Appendix I. (10 points) Note: This is your standard curve for dopachrome. You will need it for next week’s lab so make an additional copy of the graph before you turn in your report; bring the graph to the next two labs.

LIFE 212 Laboratory Report 3 Name:____________________________ Due:_____________________________ Section:____________________________ 3. Critical Thinking Question: Calculate A/C for all the dopachrome concentrations and then determine the average A/C value (show your work); this is one way to determine the extinction coefficient (E) for dopachrome. Using the slope of your best-fit line from the standard curve in Question 2 above, what is the extinction coefficient for dopachrome? (Don't forget to include units). Compare the two values for E; which method provides the greatest accuracy in determining the extinction coefficient? Support your choice. (6 points) 4. Problem Solving Question: Assume that you are studying tyrosinase activity data that has been generated in an experiment using reaction volumes of 4 mL instead of 3 mL. You obtained absorbance values for dopachrome of 0.2 after 0.5 minutes, 0.4 after 1 minute, 0.8 after 2 minutes, and 3.2 in 8 minutes. Note: Show your work for each of the following questions. a. Using your standard curve for dopachrome absorbance values vs dopachrome concentrations in Question 2 above, calculate the total amount of micromoles (µmol) at each of the indicated time points. Show your work. (8 points)

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LIFE 212 Laboratory Report 3 Name:____________________________ Due:_____________________________ Section:____________________________ b. Calculate the velocity (V) of the enzyme reaction in Question 4.a above. (Velocity = Rate of reaction =  (µmol of product)/  (time). Show your work. (6 points) c. Repeat the calculations in Question 4.a for a reaction volume of 1 mL instead of 4 mL. show your work. (8 points) 5. Using your data and standard curve in question 2, find and record in your notebook the A 475 at which the tyrosinase reaction rate will produce a total of 2  mol of dopachrome in a 1mL reaction volume or the equivalent of 2.0 mM dopachrome. Show your straight line equation and your work. (4 points) Important Note: You will need this calculated value for next week’s lab: Be sure that you record this information in your notebook to use in next week’s lab.

LIFE 212 Laboratory Report 3 Name:____________________________ Due:_____________________________ Section:____________________________ Notebook Conclusion (4pts) The conclusion should be a concisely written summary of the experimental results using technical language. Describe each objective from your Introduction individually and frame in context with the experimental results. Vague and/or incomplete conclusions will not receive any credit. For example, avoid vague phrasing, eg, "Our objectives were met".

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