6.1: Pre-Lab Assignments

Learning Objectives

Process Learning Objectives:
At the end of this experiment, you will be able to …

1.  Analyze and manipulate equations and graphical representations to appropriately model experimental data/results. 
   1.  Fit a nonlinear function to data.
   2.  Convert a nonlinear relationship to a linear form.
   3. Extract relevant information from the results (i.e. slope, intercept, coefficients).
2.  Choose experimental conditions to obtain appropriate data for analysis. 

Content Learning Objectives
At the end of this experiment, you will be able to …

3. Describe the Michaelis-Menten mechanism for enzyme catalysis, including the meaning of the Michaelis-Menten parameters.
4. Determine which apple has the slowest rate of browning, making it the best choice for a fruit salad. 

Pre-Lab Assignments

Read this entire module. This module has several parts - each part will be completed on a different day. Please complete the appropriate written assignment for each date (to be turned in on Canvas/Gradescope). The "general" prelab work should be completed and turned in along with the Part I assignment on the first scheduled day of this module. In addition, please complete the following and submit the work prior to the lab meeting.

Pre-lab assignment for Part I

Files to support this experiment are here (click). 

Please submit this assignment prior to the first scheduled lab period for this module. Submit it along with the general pre-lab assignment.

1. Look up the formulas and structures of catechol and 1,2-benzoquinone (“quinone”). Draw the structures in your lab notebook.  Write the balanced chemical equation for the oxidation of catechol to quinone.
2. Briefly define an enzyme and describe in general terms how enzymes affect reaction rates.
3. Enzyme catalyzed reactions typically follow the "Michaelis-Menten mechanism," which may be represented by
   \[\mathrm{E}+\mathrm{S} \rightleftarrows[\mathrm{ES}] \rightarrow \mathrm{E}+\mathrm{P}\]
   Define E, S, and P for this mechanism in terms of the catechol reaction in question 1.
4. Suggest an experimental method for monitoring the progress of this reaction, and explain your reasoning.
5. Based on your suggested experimental method, what data will be collected, and how will you determine the initial rate of reaction from this data?
6. Read through the protocol. In your lab notebook before class, answer the following questions.
   1. How might you extract the juice from the apple puree?
   2. How might you make the stock catechol solution?  Be specific.  Consider the volume needed for the number of people doing the experiment.
   3. What liquid should be used for the reference spectrum (the “blank”)?
   4. Sketch a curve showing the expected absorbance versus time graph for a solution containing catechol and enzyme. Explain your prediction.
   5. Describe the procedure used to obtain the designated amount of each component in the reaction mixture.  Explain why the solutions are added in a specified order. 
7. Create a table in your lab notebook that approximately follows the format below. An example of an electronic version of a table is here (click).  Fill in as much of the table as you can using the information in the procedure below.

Run#	File name	Comment	Volume of Enzyme	Volume of Substrate	Volume of Buffer or Water
Run 1	YYYYMMMMDD_AppleVariety_Run1	Part 1A, Catechol only
Run 2	YYYYMMMMDD_AppleVariety_Run2	Part 1A, Catechol + Enzyme
Run 3	YYYYMMMMDD_AppleVariety_Run3	Part 1A Catechol + E + Buffer
Run 4	YYYYMMMMDD_AppleVariety_Run4	Part IIA
Run 5		Part IIA

(add more rows as necessary...)

Pre-lab assignment for Part II

Files to support this experiment are here (click). 

Please submit this assignment prior to the second scheduled lab period for this module. Submit it along with the general pre-lab assignment.

Although different varieties of apples (and other fruits) brown at different rates, they all turn brown eventually. To preserve fruit, a chef may choose a specific apple variety and add something (an inhibitor) to slow down or inhibit the rate of the browning reaction. Inhibitors are often listed as preservatives in recipes or on labels of fruits sold in cans or jars. 

1. Suggest at least two possible inhibitors for this system. Begin by looking for the preservatives used in recipes or on labels. Also, consider the results obtained in the first cycle of this experiment. Prepare to discuss your choice of inhibitors with your instructors.
2. Select a substrate concentration that falls in the (approximately) linear portion of your Michaelis-Menton plot, and write in your lab notebook the volume of substrate in the reaction mixture that resulted in this concentration.
3. The optimal concentration of inhibitor must be determined experimentally; prepare to record the concentration of the stock inhibitor solution in your lab notebook. Create and complete a table similar to the one below (an example of an electronic version of a table is here (click)). The volume of substrate should be the same in each trial and should correspond to the volume you chose above (in pre-lab question 2). Try using concentrations of inhibitor in each mixture that vary by factors of 10. Get approval from your instructor before you actually use these for experiments. Depending on the selection of inhibitor, you may need to run the reactions without buffer.

   Trial	Volume of Enzyme	Volume of Substrate	Volume of Inhibitor	Volume of Buffer or Water
   1	0.50 mL
   2	0.50 mL
   3	0.50 mL

4. Each trial will be carried out in a cuvette. What is the best order in which to combine the enzyme, substrate, inhibitor, and buffer (if needed)? Explain your reasoning.

Adapted from Cole, Renée S., Marc Muniz, Erica Harvey, Robert Sweeney, and Sally Hunnicutt. “How Should Apples Be Prepared for a Fruit Salad? A Guided Inquiry Physical Chemistry Experiment.” Journal of Chemical Education 97, no. 12 (December 8, 2020): 4475–81. https://doi.org/10.1021/acs.jchemed.0c00517.