6.6: Experimental Procedure for Parts I and II

This is the protocol for using the Genesys 20 UV-vis to monitor the progress of an enzyme-catalyzed reaction.

Materials:

Caution

*Catechol is toxic and is an irritant. Wear PPE and avoid contact.

Chemicals and reagents: You will be provided with the following stock solutions:

1. Potassium phosphate buffer, 100 mM, pH 7.4 (~50 mL)
2. Solution of substrate (catechol) in water. (~50 mL)
3. Solution of substrate (catechol) in 100 mM phosphate buffer, pH 7.4  (~50 mL)
4. Apples of different varieties (or alternate fruit/vegetable)
5. Ice bucket with ice
6. Ultrapure water (~250 mL)

Instruments and equipment:

1. A Thermo Genesys 20 single-wavelength spectrometer.
2. A regular kitchen blender
3. A container to collect apple extract
4. Filter paper (coffee filter)
5. A funnel (to be used for gravity filtration)
6. Automatic Volumetric pipettes
7. Pipette tips of the appropriate size
8. At least two glass cuvettes (see sample cell below*)

*Sample Cell: You will use a standard 1-cm cuvettes. We prefer that you use glass cuvettes when possible (glass is optically transparent to visible light, but not UV light).

Policy on use of Cuvettes

The glass and quartz cuvettes are expensive and fragile. You will treat them carefully and follow the policy below so that these precious tools do not become damaged.

• At all times, cuvettes should be held in a rack at least 12 inches away from the edge of a bench. Never place a cuvette directly on the bench.
• When pipetting, the cuvette is held at eye level so that the user can see that they are not damaging the cuvette with the pipette tip.

Procedure:

Gather all the materials listed above. Each group should choose a different variety of apple so that different types of apples can be compared.

I. Preparation of Enzyme solution from Whole Apples

1. Prepare a solution of catecholase from apples (or alternate fruit/vegetable) in cold water.
   1. Peel and core one apple (or alternate fruit/vegetable), and put its white flesh into a blender. Add about 200 ml (total) of crushed ice and water to blend the apple flesh.
   2. Puree the apple and ice until the fruit is pulverized.
   3. Pour the puree immediately into a beaker, rinse the blender so the next group can use it.
   4. Extract the juice from the puree. 
   5. Keep the extract on ice to slow the browning. (You may store the apple extract in the refrigerator over the week break between your lab periods).
2. Use pH paper to check the acidity of the solution in the cuvette, and record the results.

II. Collection of Kinetic Data

he cuvette should be clean and dry before adding solution (or buffer or water for the blank). In the measurements that follow, the absorbance should never fall below zero or go above 3.  If it does, record a new blank.  

Prepare the spectrometer

Follow the instructions below to prepare the spectrometer for monitoring absorbance at 540 nm and to set up the software so that it is running in absorbance vs. time mode.  Record the spectrum of the blank according to the instructions for your spectrophotometer.  T

1. Turn on the Genesys 20 and let warm up for ca. 15 minutes. Set the wavelength to the appropriate value using the buttons on the instrument (see instructions).

• Start Spectra Pro on the computer by clicking the icon. The main screen appears.
• Notice the buttons:
• Notice that the software recognizes the instrument and puts the name (20 Genesys) in the button bar. Alert your instructors if this is not true in your case.

2. Click on the A vs Time button. Read the Experiment Notes box and then click OK.
3. The Setup window appears:
   Set up the spectrometer to record absorbance every 2 seconds for about 180 seconds.  Note that to collect every 2 seconds, the Sampling Rate should be 0.5 point per second.
   Click OK.

   

   Figure \(\PageIndex{1}\): Setup window in SpectroPro software. (CC-NC-BY-SA; Kathryn Haas)

4. A Calibration window appears. Read the instructions.

   

   Figure \(\PageIndex{2}\): Calibration window. (CC-NC-BY-SA; Kathryn Haas)

    
   1. Set the desired wavelength using the instrument control buttons (if you have not already done so).
   2. Insert a "blank" cuvette: Fill a clean glass cuvette with buffer. Wipe any fingerprints off the clear (i.e. not frosted) windows of the surface. Insert the cuvette into the Genesys with the light path going through the clear windows; close the cover.
   3. Press the button on the 20 Genesys to zero the instrument. Click Done in Spectra Pro. Remove the “blank” cuvette from the instrument and set it aside for later use. NOTE: these cuvettes cost ca. $100 each; please handle them with care and respect.
   4. Press Done.

Note: Sample Prep

The total volume of liquid in the cuvette must be 2.5 ml.  Pipette the appropriate volume of stock catechol solution into the cuvette. Add the desired volume of buffer or water.  Then later, quickly add 0.50 ml of enzyme solution (juice) to the same cuvette, and rapidly mix well using the tip of a Pasteur pipette.

Begin recording absorbance data just before mixing the final component of each solution.

5. Ready the solution (but do not add enzyme yet):
   Add the appropriate amounts of Buffer and Substrate to another clean glass cuvette. Again insert the “blank” cuvette (containing only buffer) into the instrument to check the zero (absorbance should read 0.000). Re-zero the instrument if necessary.
6. Ready the instrument: Open the cover of the instrument. Click the button in the Spectra Pro software.
7. Add enzyme to substrate solution and monitor reaction:
   1. Draw up the enzyme solution. In a smooth, efficient but not rushed manner, add the enzyme to the cuvette. Draw the reaction mixture up into the pipet tip two or three times to thoroughly mix the contents.
   2. Wipe any fingerprints off the clear (i.e. not frosted) windows of the cuvette surface.
   3. Place the cuvette into the instrument with the light path going through the clear windows.
   4. Close the cover. (NOTE: Step 6 should be accomplished within ca. 15 – 20 seconds.)
8. Collect and SAVE data: Let the data collection run until finished. During the a run with enzyme, you should see a smooth increase in absorbance similar to that shown in the figure below. Notice that there is always a “Live” readout of the absorbance in the box at the lower right of the screen, and that the graph and data table is generated in real-time during the run. Notice also that in the run shown here, the first 20 seconds of the run has zero absorbance. This is the time it took to add the enzyme, mix the contents and insert the cuvette into the instrument.

   

   Figure \(\PageIndex{3}\): Copy and Paste Caption here. (CC-NC-BY-SA; Kathryn Haas)

9. Save the data in TWO FILE FORMATS (just in case!) within the data directory (c:\data\Chemistry 301L):
   1. Spectro Pro file using file name YYYYMMDD_Part#_APPLEVARIETY_Run#.sb
   2. Text file using file name YYYYMMDD_Part#_APPLEVARIETY_Run#.txt

      Note

      NOTE: To save a text file, choose Export Data from the File menu and save a .txt copy of the data, using the same root filename (i.e., runLNX.txt). This file should also be saved in the data directory.

10. From the Data menu (top) choose Store Latest Run. Notice that this moves the latest data to the right in the Table Window. 
11. Repeat the steps above as necessary for each sample.