8.3: Experimental

In this lab excecise, you will monitor the the oxidation of L-DOPA catalyzed by tyrosinase.

Materials:

Chemicals: You will be provided with the following stock solutions.

1. pH 6.5 phosphate buffer prepared as follows: Weigh 6.8000 g \(\ce{KH2PO4}\) into a 1 L volumetric flask and add 500 ml of deionized water to dissolve. Add 139.0 ml of 0.1 M NaOH and make up to the mark with deionized water.
2. Solution of L-DOPA in buffer (5.0±0.1 mg/50.00±0.06 ml) (M.W. of L-DOPA = 197.19 a.m.u.).
3. Solution of tyrosinase in buffer (150,000 units/liter). The activity of tyrosinase is listed on the flask. The molecular weight of tyrosinase is 128,000 a.m.u.

NOTE: These are ideal concentrations; your actual stock solutions will be close but not identical to these values. You must get the actual concentrations from the flasks containing the stock solutions BEFORE performing any calculations.

Instruments: You will use two types of UV-vis spectrometers in this module.

1. Full-spectrum UV-visible spectrometer: Cary 60 or Cary 100 spectrometer
2. Single-wavelength spectrometer: A Thermo Genesys 20 spectrometer. (Instructions are linked here.)

Sample Cell: You will use a standard 1-cm cuvettes. For the full-spectum scan, you will use quartz cuvettes. For the rest of the experiment 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 fagile. 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:

I. Range-Finding: Determination of an appropriate wavelength for monitoring the reaction

Before you set out on a kinetics study, you must decide which wavelength (\ \Lambda\) you will use to monitor the reaction. To do this, you will scan the entire UV-visible light range using a spectrometer and determine the best wavelength to use. The ideal wavelength will be one where only one species is absorbing at the maximum wavelength of either the reactant or product. Further, it is ideal that the wavelength is in the visible range if possible, so that you can use glass cuvettes for the remaiing steps. If you determine that the ideal wavelength is in the UV range, then you must use the quartz cuvettes (which are more expensive and fragile!).

1. Create the following solutions: (if you have three quartz cuvettes, you can carefully mix the solutions directly in the cuvettes)
   1. REACTANT: 1.71 ml L-DOPA and 1.29 ml buffer.
   2. ENZYME: 1.29 ml tyrosinase and 1.71 ml buffer.
   3. PRODUCT: 2.00 ml L-DOPA and 1.50 ml tyrosinase.*
      *This solution will produce dopachrome. The molar absorptivity (\(\varepsilon\)) of dopachrome at the peak maximum of the visible band is \(3500 \mathrm{M}^{–1} \mathrm{cm}^{–1}\).²
2. Calibrate the Cary spectrophotometer using the validate software and the appropriate wavelength accuracy test for your instrument (either deuterium emission or xenon emission).
3. Examine the absorption spectra, and choose a wavelength that will allow you to measure either the disappearance of substrate or the appearance of product without interference from the other components.

II. Collection of Kinetic Data

TABLE 1: Combine the DOPA, BUFFER, and TYROSINASE in that order to make solutions A,B,C,D.

Solution	DOPA, mL	Buffer, mL	Tyrosinase, mL
A	0.50	1.50	1.00
B	0.70	1.30	1.00
C	1.00	1.00	1.00
D	2.00	0	1.00

Table 1 gives the reaction conditions for the assay you will perform in this experiment.

1. Turn on the Genesys 20 and let warm up for ca. 15 minutes. Set the wavelength to the value determined from the results of the range-finding part of the experiment.

• 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. See your TA or the lab manager if this is not true in your case.

3. Click on the A vs Time button. Read the Experiment Notes box and then click OK.
4. The Setup box appears: The Experiment Length should be 150 seconds, and the Sampling Rate should be 0.5 points per second. Click OK
5. A Calibration screen appears. 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. 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.)
6. Add the appropriate amounts of Buffer and DOPA to another clean glass cuvette (see Table 1). 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.
7. Open the cover of the instrument. Click the button in the Spectra Pro software. Draw up 1.00 mL of 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. Wipe any fingerprints off the clear (i.e. not frosted) windows of the cuvette surface. Place the cuvette into the instrument with the light path going through the clear windows. Close the cover. (NOTE: Step 6 should be accomplished within ca. 15 – 20 seconds.)
8. Let the data collection run until finished. During the run you should see a smooth increase in absorbance similar to that shown here:

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.

8. Save the data in the data directory (c:\data) as YOURINITIALS-runLN.sbl.

   Note

   NOTE: The use of LN indicates that data are being stored for solution L (where L = solutions A, B, C and D), and run N (where N = 1 or 2). 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.

9. Repeat steps 5 through 9 for runs B, C and D. Then repeat the entire sequence of runs A – D again to have two replicate runs for each substrate concentration used in the assay. (NOTE: The runs are done in the order A – D for the first trial, followed by A – D for the second trial. This order is not random; why do we do it this way?)