Excel Spreadsheet

Highlight the cells containing specific sample calculations by filling those cells in yellow. The purpose of this is to help the grader find the required information. Neglecting to do this will result in points deductions.

1. Create plots of $A_{400}$ vs. time for all your raw kinetics data for the eight catechol concentrations with and without inhibitor. Graph trendlines on each data set to illustrate how well each approximates linear intitial rates. Use these plots to guide any decisions about elimination of later time points. You are not required to label the axes.
2. Clearly label your substrate concentration calculations (the final concentrations in the assay) and highlight the cell of one sample calculation in yellow.
3. Clearly label the slope calculations and highlight the cell of one sample calculation in yellow.
4. Clearly label the relative activity calculations and highlight one sample calculation in yellow.
5. Organize your $[catechol]$, $v_o$ (relative activity) data, and $[catechol]/v_o$ values for your Eadie-Hofstee plot. Highlight one sample calculation for the x-axis calculation in yellow
6. Create Eadie-Hofstee plot and graph a linear trendline of the data. These graphs will be used in your Results.
7. In the LINEST array of your Eadie-Hofstee plot data, the four cells for slope, slope standard error, y-intercept, and y-intercept standard error must be highlighted in yellow.
8. Clearly label the cells containing $V_{max}/K_m$ values and highlight one sample calculation in yellow.
9. Clearly label the cells containing the propagated error for $V_{max}/K_m$ and highlight one sample calculation in yellow.
10. Create an organized summary table of your estimates for $V_{max}$, $V_{max}/K_m$, and $K_m$ with estimated uncertainty. If you organize these data into a formal table, you can simply cut and paste it into your worksheet assignment as Table 1 (see details below).
11. Calculate the inhibitor concentration and use this to calculate your value(s) for $K_i$ in μM units. Clearly label and highlight these cells in yellow.
12. Clearly label the cell(s) containing the propagated error for the $K_i$ value(s). Label and highlight these cells in yellow.

Lab 6 Worksheet

Download and complete the worksheet from the Main Page. Follow the instructions below.

1. Prepare an Eadie-Hofstee plot of your unhibited and inhibited kinetics data. Make sure to include labels and the correct units on both axes. Plot a linear trendline through each dataset, include the equations and $R^2$ values in the caption. Your caption should also name the enzyme, temperature, buffer conditions, the substrate, the inhibitor and its concentration with the correct units. Identify this figure as Figure 1.

2. Create a table summarizing your estimates for $V_{max}$, $V_{max}/K_m$, and $K_m$ for tyrosinase reaction of catechol at pH 6.5 both with and without inhibitor. Include uncertainty for all six parameters. Identify the inhibitor and its concentration. Identify this table as Table 1.

3. Suggest an unresolved problem in the current literature that you will address then state the objective of your investigation and describe how it will help to resolve that problem.

• To help you justify the purpose of this study (there is no one correct justification), it is helpful for you to read the introductions of journal articles that report $K_i$ values for your inhibitor. One such paper (Chiari 2011) is posted with this week's assignment on the D2L main page. From such papers, you will learn why other researchers consider tyrosinase enzyme worthy of study.
• To relate your study to other work in the field, consider your data to be a unique contribution to the current state of tyrosinase research.
• After providing general background, raise an issue that has not yet been studied in the published literature. Some ideas to consider:
• Has the $K_i$ been reported for your inhibitor in Agaricus bispous tyrosinase? If not, your measurement is novel and can be compared to other known $K_i$ values for your inhibitor.
• You studied the inhibition mechanism of your tyrosinase inhibitor with SDS activation. Have any tyrosinase inhibition studies been conducted with SDS activation? Would you predict that SDS changes the mechanism? Does it change the mechanism?
  

4. Interpret the statistical significance of changes to $V_{max}$ and $V_{max}/K_m$ with inhibitor and assign the inhibition mechanism.

• Compare the values with and without inhibitor by considering your reported error for the values in Table 1.
• At this point, simply interpret your table and figure without discussing any problems in your data. You will discuss the quality of your data later.
• Regardless of your results, you must assign a mechanism and calculate $K_i$ to the best of your ability. If your data gave a negative $K_i$, ask your instructor for assistance. They will be able to identify calculation errors or may be able to find a better approach to the analysis. If the best $K_i$ you can report is indeed negative, you must note your awareness that the result is unreasonable.

5. Report and compare your $K_i$ (or $K_{ie}$ and $K_{ies}$) and your inhibition mechanism to published literature.

• You should have already located a literature $K_i$ value for your inhibitor with tyrosinase in Step 11 of Lab 4. This value should be for Agaricus bisporus tyrosinase. Only use a $K_i$ value from a different source organism if no value was reported for Agaricus bisporus.

• If your changed inhibitors since then or now realize that the literature value is not appropriate for comparison, follow the instructions in Step 11 of Lab 4 to locate the value for comparison to your results.

6. Cite reference(s) used for $K_i$ comparison.

• The citation must reference published peer-reviewed literature, not the BRENDA database, and follow the course citation guidleines

7. Report and compare your $K_m$ to published literature.

• Compare your catechol $K_m$ to a published catechol $K_m$ value for Agaricus bisporus tyrosinase. You should have already located this value in Step 10 of Lab 4 and reported it in the Lab 5 Assignment and discussed this comparison in class during Lab 6.
• If your $K_m$ differs significantly from the literature, suggest a reason for why that might be the case.

8. Cite reference(s) used for $K_m$ comparison.

• The citation must reference published peer-reviewed literature, not the BRENDA database, and follow the course citation guidleines

9. Comment on the quality of your data and the impact your data quality has on your conclusions.

• Comment on the quality of your data (including the quality of the trends ($R^2$ for both your rates and Eadie-Hofstee analysis), the number of data points you used, the RSDs for your kinetics parmeters, etc.).
• If you do not trust your results, explain what went wrong.
• If you dropped data points, justify your decisions.
• Be clear about the limits of your ability to interpret based on the magnitude of your uncertainty.
• Comment on whether the agreement or lack of agreement of your $K_m$ value with previously published results impacts your confidence in your measured $K_i$ value.

10. Summarize your conclusions in terms of your stated goals and suggest future experiments.

• Briefly summarize what you learned about your inhibitor in terms of the goals you stated in #3. Comment on your confidence in these conclusions.
• Suggest at least one future experiment or improvement.

Submit

• A printed copy of the Lab 6 Worksheet with the rubric stapled on top.
• Upload to the D2L Submissions folder:
• A digital copy of the Lab 6 Worksheet.
• Your Excel Spreadsheet presented according to the instructions above.