Temperature Dependence of Enzyme Catalysis

Henrik Kibak x4144

Technical Support: Brenda Deardorff x4075

Overview: During this laboratory exercise student will test the effect of temperature on catechol oxidase, an enzyme extracted from plants.

Background: Catechol Oxidase is a plant enzyme that oxidizes catechol and converts it to benzoquinone. In plants catechol is compartmentalized in the vacuole while the enzyme is in the cytoplasm. When a plant cell is damaged enough to rupture the vacuole, catechol and catechol oxidase come into contact and the reaction proceeds. Benzoquinone is toxic to bacteria and therefore prevents decay in wounded plant tissues.

When you begin handling the Catechol solution you will need to use gloves and goggles as it is a poison. Report all spills to your instructor immediately. Any solution contaminated with catechol must be disposed of in the toxic waste beaker in the hood. Material Safety Data Sheet for catechol.

Checklist of Tasks:

  • Obtain two buckets of ice from cold room. One to cool flasks and the other to supplement the 4° waterbath.
  • Prepare water baths with test tube racks at 4°, RT, 40° and 80° C.
  • Warm up spectrophotometer and set to 470 nm.
  • Obtain seven test tubes and number them 1-7 near the top.
  • Using tape and a Sharpie marking pen, label a 125 mL Ehrlenmeyer flask "Cold dH2O." Fill it with about 100 mL of deionized water and put it on ice.
  • Label one 50 mL Ehrlenmeyer flask "Crude Extract." Label a second 50 mL Ehrlenmeyer flask "Dilute Enzyme." Keep those flasks on ice as well.
  • Peel and wash a refrigerated medium sized baking potato (the below-ground stem of Solanum tuberosum).
  • Chop the cleaned potato into small 2 cm pieces.
  • Place the potato pieces in an ice-cold blender with 40 mL ice-cold deionized (reverse osmosis) water.
  • Blend the pieces from your group's potato at the highest speed for one minute. Sometimes it goes more smoothly to work your way through the buttons quickly to the top speed.
  • Filter the homogenate through two layers of cheese cloth supported by a powder funnel (80 mm) into a chilled 250 mL beaker on ice.
  • Transfer about 45 mL filtered homogenate into a centrifuge tube on ice. The centrifuge tubes are graduated so it's pretty easy to see. Label the screw cap top with a small piece of tape and your group name. The instructor will bring you and your ice bucket to the centrifuge, where he or she will balance the class tubes before centrifugation.
  • Centrifuge five minutes at 500 x g in a refrigerated centrifuge.
  • Transfer 10 mL of supernatant to a chilled Ehrlenmyer flask on ice labelled "Supernatant." This is a crude extract. We will save this since each potato is different and we might need to use it "straight."

 

  • Mix 1 mL of the supernatant (crude extract) with 19 mL of ice cold deionized water (see bottle in refrigerator) to make 20 mL of dilute extract. This will be your enzyme solution so keep it on ice.
  • Set vortexer to "touch" and the speed to between 5 & 6.
Temperature Dependence of Catechol Oxidase - Experimental Set Up
Tube
Label		 Deionized H2O Enzyme Solution 1% Catechol Solution Temperature Final Volume
1 2 mL 0 0 RT (record)  
2 1 mL 0 1 mL RT (record)  
3 1 mL 1 mL 0 RT (record)  
4 0 1 mL 1 mL RT (record)  
5 0 1 mL 1 mL 4° C  
6 0 1 mL 1 mL 40° C  
7 0 1 mL 1 mL 80° C  
Set up tubes ahead of time... Wait!
Adding this starts
the reaction!		 Racks and baths...
  • Add deionized water and enzyme solution ("dilute extract") to the tubes listed in the table above. Use parafilm to cap the tube and mix well. Keep the little parafilm squares on the tube to slow evaporation.
  • Place the labelled tubes in racks at the appropriate temperature. Allow to equilibrate at least 5 min. before adding the 1 % catechol solution.

    How to use a spectrophotometer if students have not used one before...

Plan how your group will carry out the next steps because each of you will need to perform differnt tasks!

  • You will zero only Tube #1, and only zero once!
  • Note that you will add the catechol solution only to tubes 2, 4, 5, 6, and 7 (why?). At 30 second intervals add the 1% catechol solution to the tube, start the timer, mix well, and record the absorbance at T=0 min. for each tube.
  • Note that one person will have to walk back and forth to the water baths... so groups of only two students should set up on tables closer to the water baths.
  • One person records the absorbances in a lab notebook along a time course. Afterwards each participant can copy the data into the table below.
Example Data Table - Absorbance @470 nm
Tube 0 min 5 min 10 min 15 min 20 min
1          
2          
3          
4          
5          
6          
7          

 

 

 

Clean Up:

  • Pour all catechol contaminated liquids into waste bottle in hood.
  • Pour potato supernatant down sink while running some water.
  • Solid waste such as pipette tips that may have come in contact with the catechol may be put in plastic trash bags on bench top.
  • Glass pipettes that may have come in contact with catechol should be rinsed using a deionized water squirt bottle.
  
  • All other materials such as funnels and flasks should be rinsed three times with warm tap water and then once with distilled water before being left to dry.
  • Dispose of potato homogenate and used cheese cloth in ordinary trash.
  • Return racks, thermometers, timers and other non-glassware to original location.

Assignment:

Answer these questions as briefly and simply as you can:

What was the substrate, the enzyme, and the product of the reaction?

Plot the absorbance change over time for each of the tubes. In other words, the horizontal axis should be time ( 0, 5, 10, 15, 20), and the vertical axis should be absorbance.

What was the purpose of each of the tubes? In other words, what were you testing in each of the tubes?

According to your data, what was the optimal temperature for this enzyme?

Why do you think the enzyme does not work equally well at all temperatures?
Group equipment:   Class equipment:
  • Ice buckets w/ice (2 per group)
  • 50 mL Ehrlenmyer flask (2 per group)
  • 125 mL Ehrlenmyer flask (1 per group)
  • 50 mL plastic graduated cylinders (1 per group)
  • Test tubes (8 per group) Must fit in Spec 20 and be able to mix 2 mL.
  • Test tube racks (2 per group)
  • P1000 autopipette and tips
  • Thermometer (1 per group)
  • Timer (1 per group)
  • Vortexer (1 per group)
  • Spec 20 @ 470 nm
  • Funnel (1 per group)
  • Centrifuge tubes (50 mL)
  • Secondary containment for Catechol vials (smallest flat-bottomed tupperware)
  
  • 20 mL scintillation vial
  • Blender (glass portion in refrigerator) (2 minimum) One blender and three pitchers may work.
  • Four square test tube racks per waterbath.
  • Balance for centrifuge tubes.
  • Plastic squirt bottle with diH2O to balance tubes.
  • Rack for 50 mL Falcon centrifuge tubes (styrofoam).
  • Centrifuge (50 mL buckets for Falcon tubes)
  • Water bath at 40 degrees.
  • Water bath at 80 degrees.
  • Goggles
  • Parafilm & scissors
  • Gloves
  • Catechol waste bottle
Group materials:   Class materials:
  • Potato, medium baking (1 per group) refrigerated.
  • Catechol, 1% in H2O (10 mL) refrigerated.
  
  • Cheese Cloth w/Scissors 20 cm x 20 cm squares

Ideal group size is 3 students for this lab. Groups of 2 students will be under stress.