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STUDENT OBJECTIVE

Students quantitatively assay the activity of the enzyme peroxidase extracted from a turnip or other vegetable. They form hypotheses regarding how the rate of an enzyme-catalyzed reaction is affected by concentration of the enzyme, the pH of the reaction, temperature, inhibitors, and conduct experiments to test their hypotheses. Collected data are compiled in tables and graphs for use in a lab report.

EQUIPMENT	AMOUNT (Class of 24 with 8 groups)
Constant temperature water baths, 48o, 32o, and 4oC (A low-temperature bath can be improvised by placing a water-filled pan in a refrigerator or an ice and water mixture.) Spectrophotometers (500 nm) Thermometer pH meter	1 each/lab 8/lab 1/water bath Instructor use only
MATERIALS
Turnip (alternative: horseradish root or potato) Cuvettes (color coded) Tube racks, nylon or plastic (for cuvettes) Tissues (Chemwipes) Wax pencil or markers Test tube, 15 ml capacity Test tube rack Pipettes, 1 ml x 0.1 ml or 0.01 ml (color coded) Pipette, 5 ml x 0.1 ml or 0.01 ml (color coded) Suction devices, 10 cc disposable syringe with 1" rubber tubing attached (see lab manual fig. 4.3) Pipetters, automatic (optional—replaces pipettes above) Beakers (50 ml) or small disposable plastic cups (color coded) Plastic crisper, 8" x 4" x 12" (supplies container) Spectrophotometer lamps (spare) Bunsen burner, stand, and beaker for boiling water bath or hot plate	1 medium 16/lab 8/lab 4 boxes/lab 8/lab 72/lab 8/lab 8/lab 32/lab 16/lab 6/lab 32/lab 8/lab 4/lab 2/lab

FOR INSTRUCTOR USE IN MAKING EXTRACT

Cheesecloth
Blender or mortar and pestle with fine sand
Aspirator
Buchner funnel
Erlenmeyer sidearm flask, 500 ml
Erlenmeyer flask, 500 ml
Rubber hose
Rubber stopper
Filter paper, Whatman #1 circles
Standard pH solutions—pH 4, pH 7, pH 10 for pH meter
Centrifuge (optional)

SOLUTIONS

Extract of turnip (peroxidase)
10 mM hydrogen peroxide (H₂O₂)
25 mM guaiacol (2—CH₃OC₆H₄—1—OH) (Sigma)*
1% hydroxylamine (NH₂O₂H · HCL) (Sigma)*
0.1 M citric acid (C₆H₈O₇ · H₂O)
0.2 M sodium phosphate, dibasic heptahydrate (Na₂HPO₄ · 7H₂O)
1M NaOH
Citrate-Phosphate Buffers: pH 3, 5, 7, and 9

PREPARATION

One Week before Lab

1. Buffer preparation:
Stock Solution

"A" 0.1 M citric acid, monohydrate	21.01 g/distilled water to make 1 liter Shelf life: three months
"B" 0.2 M sodium phosphate, dibasic heptahydrate	53.65 g/distilled water to make 1 liter Shelf life: one month

Check each prepared buffer with a pH meter; obtain desired pH by adding stock "A" or "B."
Buffer "B" takes time and stirring to go into solution. Make well ahead of time needed.
pH 3 buffer

397 ml "A" stock solution
103 ml "B" stock solution
500 ml distilled water

pH 5 buffer

242 ml "A" stock solution
258 ml "B" stock solution
500 ml distilled water

pH 7 buffer

88 ml "A" stock solution
412 ml "B" stock solution
500 ml distilled water

pH 9 buffer

"B" stock solution alone (If necessary, add 1 M NaOH to increase the pH.)

Buffer solutions may turn cloudy. Filtering may be necessary.

2. Guaiacol preparation:

25 mM guaiacol 3.08 g/1 liter distilled water

Warm very gently and stir to dissolve. (Prepare in fume hood to avoid lingering odor.) Store in a brown bottle in the dark.

Shelf life: three to four months
If using liquid guaiacol solution from Sigma, use 2.8 ml/1 liter distilled water.

3. Sodium hydroxide preparation:

1 M NaOH 40 g NaOH/ distilled water to make 1 liter

Slowly add NaOH pellets to the water and stir until completely dissolved. Wear goggles and be aware of heat of solution! It is best to store in a nonglass container.

Shelf life: indefinite

4. Hydroxylamine preparation:

Day of Lab

1. Extract preparation:

   1 g turnip (horseradish or potato)/100 ml pH 7 buffer

   Grind the vegetable with a small amount of sand and buffer in a mortar and pestle; add remaining buffer and filter through several thicknesses of cheesecloth. The activity of the enzyme should be checked by performing a trial standardization. At 500 nm, the activity of 1 ml of extract in the assay mixture at pH 5 should cause a change from 0 to 1.0 absorbance units within 120 seconds. Dilute with buffer if overactive; add more vegetable if activity is low. The resulting extract can be stored in the refrigerator for 8-10 hours.

   (Alternative: Blend with 70 ml buffer for 15 seconds in a blender. Vacuum filter with a Buchner funnel in a sidearm Erlenmeyer flask and use a second flask as a trap; rinse blender with remaining buffer and filter. The solution can also be clarified by centrifuging at 5000 x G for ten minutes.)

2. Hydrogen peroxide preparation:

CLASSROOM SUGGESTIONS

1. To prevent students from using the wrong pipettes in the wrong solutions, all containers and pipettes should be color coded with colored tape. All equipment should be stored in a plastic crisper next to the spectrophotometer. Explicit instructions for withdrawing the needed amount of solution should be clearly visible beside each stock solution since students are inexperienced in estimating volumes. As an alternative laboratory setup, guaiacol, hydrogen peroxide, and buffers can be dispensed from a side shelf in automatic pipetters marked with the appropriate color code.

   A good substitute for the 50 ml glass beakers are 4 oz disposable cups. This reduces cleanup at end of lab.

2. Many vegetables have peroxidase activity. For example, even the ever-prolific zucchini will give results. However, 25 times as much tissue is needed to give similar results to turnips. If the instructor wanted to do an investigative-type lab, several vegetables could be assayed for activity per gram of tissue with only slight changes in directions to the students.
3. This enzyme experiment works well and students should find that the optimum temperature for peroxidase is 32°C and optimum pH is 5. Hydroxylamine treated and boiled peroxidase will show a dramatic decrease in the rate of reaction.
4. This exercise requires three hours. Students can be divided into groups and assigned to perform the experiments with the following variables: (a) standardization, pH and inhibitor (b) standardization, temperature and boiled enzyme. All the data can be shared at the conclusion of the laboratory. This allows time to summarize the activities of the exercise.
5. A minimum homework assignment might be to hand in graphs of absorbance change as a function of the time for each of the variables investigated. The rate of the reaction is linear over the two—minute reading period. This is a good time to introduce the concept of slopes and how to calculate them. We often use this as the model exercise for teaching students how to write reports in a scientific format.
6. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. None, really. In the first part of the experiment you standardized the amount of enzyme to be used for the rest of the experiments. An older turnip may necessitate using more of it to obtain the same enzyme activity as a younger turnip, but this is compensated for in the standardization procedure.
2. On a particularly hot day the room temperature may be closer to 32^oC than 23^oC specified. Therefore, you would expect to see enzyme activity at room temperature to be close to the same as at 32^o. In this case, you may miss the optimum temperature and it would be wise to devise a way to keep one reaction at a lower temperature.
3. A cooked turnip would provide the same results as those from the "boiled enzyme" experiment. Cooking denatures the perioxidase enzyme.
4. Every enzyme has a specific pH range within which it functions optimally. If an acid optimum enzyme is put in an alkaline environment the rate of catalysis slows, possibly stopping. This is due to a change in shape of the enzyme caused by change in charge distriubtion in the enzyme molecule as the constituent amino acids add or lose protons depending on their pk values.

Supplemental Materials