6.3: Part I, The effect of apple extract on oxidation of catechol

Pre-lab assignment for Part I: Which Apples?

📅 Due before Wet Lab Meeting for Part I 

Read sections 6.1, 6.2, 6.3 and 6.6 of this manual and complete the GENERAL PRE-LAB ASSIGNMENT for this part of the module.  Then complete the following written assignment and submit your work before the deadline.

Files to support this experiment are here (click). 

1. Monitoring the catechol oxidation reaction:
   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. Describe how chemists monitor the progress of a chemical reaction over time. Suggest an experimental method for monitoring the progress of this reaction, and explain your reasoning.
   3. Based on your suggested experimental method, what data will be collected, and how will you determine the initial rate of reaction from this data?
2. The model:
   1. Briefly define an enzyme and describe in general terms how enzymes affect reaction rates.
   2. 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.
3. 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. 
4. 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...)

Procedure

The experimental conditions are described below. The detailed procedure for obtaining appropriate materials, operating the UV-vis instrument, and conducting the experiments  is in Section 6.6.  For Part I, each group will select one type/variety of apple to use in these experiments.  Then the entire class will share data

Experiment 1A: How does apple extract effect oxidation of catechol? Does pH matter?

How does enzyme extracted from an apple affect the rate of oxidation of catechol, and how does buffering affect the rate?  Select an apple varietal (for example, Granny Smith, McIntosh, Pink Lady…). Compare the oxidation of catechol under three conditions: no added apple juice, with added buffered apple juice, and with unbuffered apple juice of the same variety.  

1. Run 1: For reaction mixture 1, mix 2.0 ml of catechol in water with 0.5 ml of water. 
2. Run 2: For reaction mixture 2, mix 0.5 ml apple juice extract (the enzyme) with 2.0 ml catechol in water. 
3. Run 3: For reaction mixture 3, mix 0.5 ml apple juice extract (the enzyme) with 2.0 ml of catechol solution in buffer.  

In your lab notebook before doing the experiment, sketch a graph showing the predicted absorbance vs. time trace for all three mixtures on the same axis.

Thinking About the Data from Experiment 1A

*Note, there is a MatLab template available for data analysis, but you may use any software you prefer.

1. Make a table that summarizes the components of the reaction mixtures (this was part of the pre-lab assignment - update if necessary and add to your notebook).
2. Why is 0.5 ml of water added to reaction mixture 1?
3. Determine the initial rate of the reaction for each mixture, and record the results in your notebook in the same table as above.  
4. Which reaction mixtures contain the catecholase enzyme? Based on your data, explain how the enzyme affects the rate of oxidation of catechol.
5. How does buffering the mixture affect the oxidation rate for the apple variety you selected?
6. Recall that apples contain catechol and catecholase. Based on your results, suggest one way to slow or inhibit the catalyzed browning of an apple.

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Experiment 1B: Does the type of apple matter?

In the next part of this experiment you will compare the browning of your selected apple varietal to a different type of apple.  If more than one team is doing the experiment, decide which teams will work with which apples, and decide whether the experiment should be carried out unbuffered or at pH=6.5?  Provide justification for your answer.  

One goal of physical chemistry is to develop mechanisms that explain the rate of a reaction.  The Michaelis-Menten mechanism is often used to describe the kinetics of an enzyme-catalyzed reaction.  The equation below derives from this mechanism:

\[v_0=\frac{v_{\text{max }}\left[S_0\right]}{K_M+\left[S_0\right]} \label{mm} \]

where \(v_0\) is the initial rate, \(v_{\text{max}}\) and \(K_M\) are the maximum rate and the Michaelis-Menten constants, respectively, and \(\left[S_0\right]\) is the substrate concentration.  

Questions for preparation:

1. Identify the independent and dependent variables in Equation \ref{mm}.  What must be varied to determine \(v_{\text{max}}\) and \(K_M\)?
2. Examine Equation \ref{mm}. Predict the appearance of a graph of initial rate, \(v_0\) versus substrate concentration, \(\left[S_0\right]\). Consider the dependence of the initial rate on low substrate concentration (as \(\left[S_0\right]\) approaches 0) and high concentration (as \(\left[S_0\right]\) approaches \(\infty\)).
3. Create a table similar to the one below in which you will add rows 2 through 7 (this was part of the pre-lab assignment). Keep in mind that the data analysis procedure amplifies the uncertainty in measurements made with low substrate concentrations.  If you decide to run the reactions unbuffered, the last column gives the volume of water, and the substrate solution should be made in water. If you decide to run the reactions buffered, the last column gives the volume of buffer, and the substrate solution should be made in buffer.

   Run #	Volume of Enzyme	Volume of Substrate	Volume of Buffer or Water
   Run 4	0.50 mL	2.00 mL	0 mL
   Run 5 ...	0.50 mL	…	…
   Run 11	0.50 mL	0.125 mL	1.875 mL
   Run 12	0.50 mL	0 mL	2.00 mL
   Run 13	0 mL	2.00 mL	0.50 mL

4. How do Runs 12 and 13 differ from Runs 4 - 11? Why is it important to collect the data in Runs 12 and 13?

Run the Experiment

Follow the protocol using the mixtures from the table.  Then analyze your data and determine the \(v_{max}\) and \(K_M\) parameters for the enzyme extracted from your type of apple. There is a MatLab template provided to help you with this, but you may use any computational software you prefer.

Share your data with the rest of the class using the shared spreadsheet.

Discuss: Which type of apple is best for a fruit salad? What are the \(v_{max}\) and \(K_M\) parameters for the enzyme extracted from each type of apple, and how are these related to the selection of the type of apple that would be best for a fruit salad?

Thinking About the Data: Experiment 1B

*Note that there is a MatLab template to help you through this, but you may use any software you prefer to accomplish this work.

1. Determine the initial rate of the reaction for each mixture and tabulate the results in your notebook. 

2. If needed, calculate a “corrected” rate for trials 1-8 by subtracting the rates determined for the two controls (Runs 12 and 13).  This is usually not necessary.

3. Create a graph showing the dependence of rate on substrate concentration.  Ideally, all the rates should be plotted on the same axes.  Describe in words how the substrate concentration affects rate, comparing your result to your predicted graph.  Team members should reach consensus on a common description.  

4. The kinetic parameters for this reaction may also be extracted directly from your graph of rate versus substrate concentration by fitting the data with a nonlinear equation of the form: \[y=\frac{C x}{D+x} \label{lb}\]

5. Explain the shape of this graph in terms of the Michaelis-Menton Equation. 

6. We can simplify the determination of the kinetic parameters if we linearize the rate expression. Many scientists have found it useful to graph \(\frac{1}{rate}\)  or \(\frac{1}{v_0}\)  versus \(\frac{1}{[S_0]}\) for mixtures with different substrate concentrations but constant enzyme concentration. Such a plot is often referred to as a Lineweaver-Burk plot. Show how the Michaelis-Menton equation can be linearized.

7. What are the slope and intercept of this linear graph?

8. Prepare a Lineweaver-Burk plot to extract the kinetic parameters of the catecholase/catechol reaction using the linearized equation.  

9. Compare the Michaelis-Menton nonlinear fit to the Lineweaver-Burke linearization fit.

   1. Make a graph of [S] vs Initial Velocity with three data series: \(v_0(expt)\) (symbols), \(v_0(MMfit)\) (solid line), and \(v_0(LBfit)\) (dashed line). 
   2. To create the latter, you will need to transform the Lineweaver-Burke fit "back" to a Michaelis-Menton plot. Discuss with your classmates how you can do this.
   3. Create a residual plot for the experimental data compared to the two fits
   4. Discuss the conclusions you make from this graph in your notebook.

10. One person from your team should post the 'best' \(V_max\) and \(K_M\) values (derived from LB or MM) obtained and the type of apple analyzed in the shared data spreadsheet. Please be sure to enter the data carefully on the correct line of the spreadsheet. The other tema members should check the data entries to ensure they are accurate.

11. What are the units for your \({v_{max}}\)  and \(K_M\) values?  Use the extinction coefficient of \(1.03 \times 10^4\) L mol\(^{-1}\) for 1,2-benzoquinone at 540 nm to convert these to concentration units.

12. Compare the values of \({v_{max}}\)  and \(K_M\) determined using the Lineweaver-Burk plot and using non-linear curve fitting.  Are there substrate concentrations for which one method provides a better fit?

13. Tabulate the  \({v_{max}}\)  and \(K_M\) values for each type of apple (or other fruit) studied.  Do these values predict that different types of apples (or other fruit) brown at different rates?  Explain your answer. If possible, compare your tabulated results to those of others from your class, and use the class’ values to support your answer.

14. What does the value of \({v_{max}}\)  tell you about the reaction?  What does the value of \(K_M\) tell you about the reaction?

15. Suppose you want to make a fruit salad with apples. What factors would you consider when preparing the apples and the salad? Discuss based on your kinetics results.

 

 

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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.