Lab 1 • Enzyme Activity LabQuest Colorimeter Procedure edited by Genesis Perez-Melara

Materials Required:

❏ 2.5 mL Syringes (x3) ❏ 10 mL syringe (x1) ❏ 5 Test Tubes ❏ 1 mL Guaiacol ❏ 1 mL Turnip peroxidase solution ❏ 2 mL Dilute hydrogen peroxidase solution ❏ 4 mL pH 7 buffer solution ❏ Timer

❏ One P-1000 and tips ❏ LabQuest Tech

❏ Colorimeter ❏ 5 (small rectangular plastic containers) with Caps

❏ parafilm Part 1 - Procedure: Structured Inquiry

1. Label the test tubes and syringes, as follows: 2.5 mL syringe labeled ‘E’ for enzyme - turnip peroxidase solution 2.5 mL syringe labeled ‘P’ for product as represented by indicator. ***Guaiacol reacts with free O2 (product) to form brown color*** 10 mL syringe labeled ‘NB’ for neutral buffer - pH 7 2.5 mL syringe labeled ‘s’ for substrate - 0.1% H2O2 labeled ‘SPNB’ for mixture A (SPNB - Substrate + Product + Neutral Buffer) Test tube labeled ‘ENB’ for mixture B (ENB - Enzyme + Neutral Buffer) 2. Fill and prepare the labeled test tubes with the appropriate solutions using the respective syringes (provided): a. Dispense the following reagents in “Tube SPNB”: 2 mL of ‘S’ substrate 1 mL of ‘P’ indicator for product 1 mL of ‘NB’ neutral buffer b. Cover tube ‘SPNB’ with parafilm and gently invert two times to mix c. Dispense the following reagents in “Tube ENB”: 1 mL ‘E’ enzyme (turnip peroxidase) 3 mL ‘NB’ neutral buffer d. Cover tube ‘ENB’ with parafilm and gently invert two times to mix 3. Set aside tubes and begin to set up the colorimeter/Labquest (a more detailed description of how to set up the LabQuest can be seen in the step-by-step explanation towards the end of this #Hinton20 • Harvard Medical School • v2.0 lab manual): a. Set the colorimeter to display Absorbance Values b. Using the arrows on the colorimeter, set the to 430nm c. Change the mode to "Time Based" d. Set the interval to 2s/sample e. Set the length to 300 seconds (or 5 Minutes) **Reach out to a tutor or lab assistant if you are having trouble!!** 4. Calibrate the colorimeter (you only need to do this once} a. Put at least 2mL of water into a clean b. Place the. cuvette in the colorimeter with the clear side facing the arrow and press "CAL"

c. Remove cuvette 5. Next, add 1 mL of the mixture ‘ENB’ to a new clean cuvette 6. Place this cuvette in the colorimeter so that the arrow lines up with the CLEAR sides of the cuvette 7. Do these next steps quickly! a. Add 1 mL of the mixture "SPNB" to the cuvette b. Close the cover of the colorimeter c. Press start on the lab quest to start collecting data 8. Make observations and record data (draw a sketch of what you see on the LabQuest screen) a. Note: The color intensity is used as a way to quantify the amount of oxygen that is produced in the enzymatic reaction. The brown color is produced when the guaiacol reacts with oxygen (the product of the enzyme-substrate reaction). Therefore, the more intense the color, the more oxygen is produced in the reaction. 9. Establish the rate of enzyme reaction using the data collected on LabQuest

Part 2 Guided Inquiry: Variables that affect the rate of enzyme reaction

10. Before proceeding, record the baseline rate in your notebook or sheet that you established at the end of part 1. 11. Rinse the two cuvettes used in part with tap water 12. Next, Label the five cuvettes as follows:

***Use washi tape to label the cuvettes - cut a small square piece of tape, stick it to the cap of the cuvette, and label the tape with a sharpie***

Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette 5

pH3 pH5 pH6 pH8 pH10

**We will not be using a pH 7 buffer because the ‘NB’ in part 1 already had a pH of 7**

#Hinton20 • Harvard Medical School • v2.0 13. Now, label 5 test tubes as: ‘SPNB’ 14. In each of the tubes, dispense: 2 mL ‘S’ solution (substrate) 1 mL ‘P’ solution (product indicator - Guaiacol) 1 mL ‘NB’ neutral buffer pH 7 **Note all of these tubes contain the substrate**

15. Next label 5 test tubes as follows:

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 pH 3 pH5 pH6 pH8 pH10

16. In tube ‘pH3’, dispense the following volumes of reagents: 1 mL of turnip peroxidase solution, using the syringe labeled ‘E’ 3 mL of pH 3 solution, using the ‘NB’ syringe (Rinse this syringe before using)

17. Repeat step 16 for tubes ‘pH5’, ‘pH6’, ‘pH8’, and ‘pH10’ with their respective pH buffers 18. Do these next steps quickly! a. Add 1 mL of the mixture "SPNB" (from any tube that is labeled ‘SPNB’) to cuvette labeled as ‘pH3’ b. Next, add 1 mL of the mixture from Tube ‘pH 3’ to this same cuvette c. Close the cover of the colorimeter d. Press start on the lab quest to start collecting data 19. Repeat step 18 for the rest of the pH conditions 20. Record the respective rate of enzyme reaction for each pH condition 21. Answer the assessment questions found at the last page of this lab manual

#Hinton20 • Harvard Medical School • v2.0 Labquest Step-by-Step Handout • CJ Beneduce

The home screen of the lab quest and lab quest two can be seen here. To get started, press anywhere in the red and select “Change Units”

Make sure that the colorimeter is set to display “Absorbance”

Next, press on “Mode”. You should see a screen like this

Clicking on the dropdown bar should give you a list. If it’s not already, make sure the option “Time Based” is selected

#Hinton20 • Harvard Medical School • v2.0

Set the Interval to 2 s/sample. This is how often the instrument will take a reading. Set the length to 300 sec- onds. Click OK

Once your sample is ready, click on the Graph Icon ( ). This will bring you to a screen looking like this. When ready, press the play button in the bottom left corner. The instru- ment will begin taking measurements and will automatically stop after the interval we set above (300 seconds)

Once the run is completed, this icon should become clickable. Clicking on it will save the current run and create a new run for your next sample. Click it after finishing each sample

Clicking on the Table Icon ( ) brings you to this screen. Here you can give each run a de scriptive name so you know which sample it is and so the data can be retrieved later.

#Hinton20 • Harvard Medical School • v2.0

Back on the graph tab, you can select the run icon and select which run you would like to display on the graph. Make sure that you select the new/ current run before starting you next sample. If you don’t you may loose your data.

When finished collecting all your data, click on the File Icon ( ) and select the File tab. In the drop down box, select “Save” and give your data a descriptive name. Something along the lines of “20141020_Enzyme Catalysis_Group 2”

#Hinton20 • Harvard Medical School • v2.0 Part 3: Enzyme Activity - Open Inquiry: Design an Experiment!

What questions occurred to you as you completed your studies of peroxidase activity? Now that you are familiar with enzymatic activity and ways to assess that activity, design an experiment to investigate one of your questions. Questions may involve assessing a range of abiotic factor effects on peroxidase activity, modeling the kinetics of enzyme activity, comparing protein sequence or optimal conditions for peroxidases from different sources, testing potential chemical inhibitors of enzyme activity, comparing sensitivities of different kinds of enzymes to the same abiotic factors, or identifying indicators for different kinds of enzyme activity.

Definition: abiotic factors - nonliving factors that can be chemical or physical. An example can be found in part 2 of this manual. pH is a chemical, nonliving factor.

Use the following steps when designing your experiment:

1. Define the question or testable hypothesis. 2. Describe the background information. Include previous experiments. 3. Describe the experimental designs with controls, variables, and observations 4. Describe the possible results and how they would be interpreted. 5. List the materials and methods to be used. 6. Note potential safety issues.

After the plan is approved by your mentor/teacher:

7. The step by step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc., as well as the weights and volumes used. 8. The results should be recorded. 9. The analysis of results should be recorded. 10. Draw conclusions based on how the results compared to the predictions. 11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables. 12. Further study direction should be considered.

#Hinton20 • Harvard Medical School • v2.0

Part 2 Assessment Questions:

1. Based on the graph and the overall slope of the line, what can you conclude about the effect of pH on reaction rate? Why do you think that occurs?

2. What happens to turnip peroxidase during and after the reaction?

3. What would the reaction look like if you omitted parts of the reaction from the mix?

4. WHat other factors may influence enzyme activity (rate of reaction)?

5. Peroxidase breaks down hydrogen peroxide. WHat other types of enzymes might be needed in an organism?

6. You investigated peroxidase from a turnip/ How might the activity of peroxidase from a mammal be different from that of a turnip?

#Hinton20 • Harvard Medical School • v2.0