CHE 341 Lab 2 Tyrosinase Extraction

Procedures

PART 1 Mushroom Dissection and Extraction

Materials

• One mushroom
• Razor blade
• Weigh boat
• Liquid nitrogen
• Mortar and pestle
• Extraction buffer: sodium phosphate buffer, 50 mM, pH = 6.5
• Ice bucket
• 50 mL high speed centrifuge tube
• Beckman Avanti J-26 XPI centrifuge
• Thermo Scientific Genesys 20 Visible Light Spectrophotomer

 

Methods

1. As a class we will decide how to anatomically divide-up a mushroom into as many parts as there are groups. Each student will get a mushroom to completely dissect. Use a razor blade to perform the dissection following the template in Figure 3 (see Introduction).

2. One member of each pair should collect the portion they will be analyzing, e.g. the gills, from every student in the room. If you have a lot of mushroom material, use about 9 grams for the following steps. It is critical to record how much you choose to use for your experiment.

3. Place the collected mushroom material into a weigh boat, chop your sample into small pieces, and freeze with liquid nitrogen (liquid nitrogen will be provided by your TA). This freezing step will crack the cells. Upon thaw, the cells will easily release their contents.

4. Pour the frozen mushroom material, including a small puddle of liquid nitrogen, into a clean mortar and grind with a pestle to pulverize with a circular motion of the pestle for 1 minute.

5. Add 30.0 ml of pH 6.5 phosphate extraction buffer. Homogenize with a circular motion of the pestle for 1 minute.

6. Pour your extract into a high-speed centrifuge tube. Put your tube on ice and take it to the back room where your instructor will demonstrate how to use a two-pan balance to match the weight of your tube to another group's tube for centrifugation. If you need to add extra buffer to your get it balanced, record the exact amount added as this affects your concentration.

7. Centrifuge at 20,000 rcf for 20 minutes and 4 °C.

8. Collect six 1.5-mL aliquots of the supernatant. Label the tube caps with your section number and group number. Freeze five of these samples at -20 °C to maintain activity. You will use them over the next four weeks. Use the sample that you do not freeze for the next part of this experiment.

 

PART 2 Enzymatic Activity of Mushroom Extracts

Materials

• Pipettors, pipet tips, and microcentrifuge tubes
• Spectrophotometers (note model in lab) set to measure 475 nm
• Extraction buffer: sodium phosphate buffer, 50 mM, pH = 6.5
• L-DOPA solution (10.0 mM in extraction buffer)
• SDS soution (10.0% in extraction buffer)
• Stopwatch
• Rectangular cuvette

An enzyme unit (U) of tyrosinase activity is defined as 1 μmol of dopachrome formed per minute. To compare our extracts, everyone will measure activity under the same conditions of 5.0 mM L-Dopa in pH 6.5 buffer at 23 °C (the temperature of our lab room). The molar extinction coefficient for dopachrome at pH 7.0 is ε₄₇₅ = 3.6 × 10³ M^-1cm^-1. Every reaction will have a final volume of 900 μL. For each sample, you will calculate the required volume of L-Dopa solution, enzyme solution, and buffer to obtain your desired concentrations at the 900 μL final volume.

Methods

1. Warm up the spectrophotometer at 475 nm. Record the model and manufacturer of the spectrophotometer (you will need this for your report). Clean the cuvette with water.

2. Check that the SDS is thawed and has no solid precipitate. If there is any solid in the SDS tube, vortex the tube until all solid is dissolved. 

3. In your notebook, set up a protocol table for variation of tyrosinase concentration while maintaining L-DOPA at a saturating concentration of 5.0 mM and a final volume of 900 μL for each sample. Copy Table 1 below into your notebook sideways. Calculate the ingredients for one reaction without SDS assuming 50.0 μl of enzyme extract and one reaction with SDS assuming 5.0 μl of enzyme extract. For the reaction without SDS, each tube should contain:

• x μl of L-DOPA for final concentration of 5.0 mM from a 10.0 mM stock solution. (Hint: this is a dilution calculation - use C₁V₁ = C₂V₂.)

• y μl of enzyme extract. A good starting volume is 50 μl.

• 900 - x - y μl of buffer (for a final assay volume of 900 μl)

You should plan to make at least four kinetics measurements of enzyme extract. You may need to vary your actual extract volume after the first measurement (see note in boxed Note 1) so skip three lines in your table and calculate the volumes for a reaction with 0.1% SDS and 5.0 μl of enzyme extract:

• x μl of L-DOPA for final concentration of 5.0 mM from a 10.0 mM stock solution

• y μl of SDS for a final concentration of 0.100% from a 10.0% stock solution

• z μl of enzyme extract. A good starting volume for extract in these samples is 5 μl.

• 900 - x - y - z μL of buffer (for a final volume of 900 μL)

 

4. To a microcentrifuge tube, add the volumes of L-DOPA and buffer that your calculated in the last step. Before you add the enzyme extract, be prepared to execute the next two steps efficiently.

5. Add the enzyme extract, immediately mix by inverting the sample a few times in the microcentrifuge tube, pour sample into the cuvette, and place the cuvette in the spectrophotometer. Make sure your cuvette is free of smudges, lint, and bubbles. Mix gently to avoid introducing air bubbles that may scatter light in the cuvette.

6. Zero the spectrophotometer. When you see a reading of 0.000, start your timer and record the absorbance at 15-second intervals over 2 minutes. Your start time should not start at the moment of mixing

7. Repeat steps 3 - 5 to obtain a total of four replicate measurements.

8. Repeat steps 3 - 6 for enzyme assays that include a final concentration of 0.1% SDS. In Step 3, you will add the appropriate volume of L-DOPA, SDS, and buffer. In Step 4, take care to mix gently by inversion since SDS is very prone to become foamy if shaken.

Table 1. Suggested data table format for keeping track of your experiment conditions.