Exercise 4 - Properties of Enzymes

STUDENT OBJECTIVE

Students quantitatively assay the activity of the enzyme peroxidase extracted from a turnip or other vegetable. They determine how the rate of an enzyme-catalyzed reaction is affected by concentration of the enzyme, the pH of the reaction, temperature, and inhibitors. 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, 48 ° , 32°, and 4°C
  (A low-temperature bath can be improvised by placing a water-
  filled pan in a refrigerator or an ice and water mixture.) 1 each/lab
Spectrophotometers (500 nm) 8/lab
Thermometer 1/water bath
pH meter Instructor use only
MATERIALS
Turnip (alternative: horseradish root or potato) 1 medium
Cuvettes (color coded) 16/lab
Tube racks, nylon or plastic (for cuvettes) 8/lab
Tissues (Chemwipes) 4 boxes/lab
Wax pencil or markers 8/lab
Test tube, 15 ml capacity 72/lab
Test tube rack 8/lab
Pipettes, 1 ml x 0.1 ml or 0.01 ml (color coded) 8/lab
Pipette, 5 ml x 0.1 ml or 0.01 ml (color coded) 32/lab
Suction devices, 10 cc disposable syringe with 1" rubber
  tubing attached (see lab manual fig. 3.3) 16/lab
Pipetters, automatic (optional--replaces pipettes above) 6/lab
Beakers (50 ml) or small disposable plastic cups (color coded) 32/lab
Plastic crisper, 8" x 4" x 12" (supplies container) 8/lab
Spectrophotometer lamps (spare) 4/lab
Bunsen burner, stand, and beaker for boiling water bath or hot plate 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."

    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.)

  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

  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:
    1% NH2OH◊HCl 0.5 g/50 ml distilled water

    Dissolve hydroxylamine in 25 ml water; add 1 M NaOH until a reading of pH 7 is obtained; bring final volume to 50 ml. Odor can be mildly irritating.
    Shelf life: six months

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 refri gerator 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 ´G for ten minutes.)

  2. Hydrogen peroxide preparation:

    Concentrated H2O2 is corrosive: wear goggles and hand protection to avoid injury.
    10 mM H2O2 10 ml H2O2/distilled water to make 1 liter
    Gently stir the peroxide into water and store in brown dispensing bottle.

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 (autoclave) 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.

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 ° C than 23 ° C specified. Therefore, you would expect to see enzyme activity at room temperature to be close to the same as at 32 ° . 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. Mixing pipettes - could introduce the enzyme into the substrate and alter the substrate concentration;
    Enzyme temperature - enzyme must be kept cold to assure maximum activity;
    Timing - crucial, to allow comparison of results;
    Temperature of water baths - must be maintained at prescribed temperature;
    Pipetting accuracy - will directly influence the reaction rates;
    Clerical error - mislabeling of tubes, entering results in wrong place, errors in graphing will all give erroneous results; etc.
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