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An oxidase is an enzyme that catalyzes the transfer of hydrogen from some compound to molecular oxygen in order to form water. Polyphenoloxidase is a copper-containing enzyme that catalyzes the removal of hydrogen, as in the oxidation of dihydroxyphenols to the corresponding quinones. This type of oxidation is accompanied by a color change. Polyphenoloxidases, also known as o-diphenoloxidases, are distributed widely in the plant kingdom (e.g. champignon mushrooms, potatoes, bananas). They are responsible for the darkening of freshly cut surfaces of plants or fruits. For insects, o-diphenoloxidase is important both for melanin formation and for browning and hardening (sclerotization) of the cuticle. In this experiment, the observed color change is used as a measure of extent of the reaction.


Experimental procedure

Part i. preparation of the enzyme

  1. Peel and grind two to three potatoes in a blender.
  2. Add 25-30 mL of water to the mixture, swirl the mixture occasionally, and let it stand for about 10 minutes.
  3. Filter the solution in cheesecloth to obtain a clear solution.
  4. While you are filtering the solution, prepare the water bath that you will use in the following sections.
  5. Use this solution as a source of catalase and o-polyphenoloxidase.

Part ii. the effects of temperature

  1. The gas-collecting apparatus has been prepared for you, and it consists of a large test tube fitted with a rubber tube leading from a one-hole rubber stopper. The other end of the tube should be directed beneath an inverted, water-filled, graduated cylinder in a large beaker filled with water.
  2. Add about 5 mL of hydrogen peroxide, at room temperature, to the tube.
  3. Quickly add 3 mL of catalase extract.
  4. Stopper the test tube, swirl it once to mix, and collect the gas in the graduated cylinder.
  5. Note the time required to collect 5 mL of the gas.
  6. Repeat the procedure using both extract and hydrogen peroxide cooled in an ice bath. Record the temperature and the time required to produce 5 mL of oxygen.
  7. Repeat using extract and hydrogen peroxide warmed in a water bath to 37 ° C size 12{°C} {} . Again, record the temperature and the time required to collect 5 mL of oxygen.
  8. From your results, answer the question: What effect does temperature have on enzyme activity?

Part iii. the specificity of enzyme activity

In this part of the experiment, you will evaluate the specificity of the substrate structure required for enzyme activity. The substrate structure varies to demonstrate the need for:

  1. an aromatic or aliphatic ring
  2. one or two hydroxy groups
  3. proximity of hydroxy groups on the ring

The substrates include the following:

  1. deionized water
  2. cyclohexanol
  1. 1,4 cyclohexanediol
  1. phenol
  1. catechol (o-hydroxyphenol)
  1. resorcinol (m-hydroxyphenol)
  1. hydroquinone (p-hydroxyphenol)

Caution: Some of the solutions used in the next step are toxic and can be absorbed through the skin. Avoid skin contact.

  1. Prepare seven labeled 6-inch (15-cm) test tubes with labels and contents as follows:

Tube 1: 1.0 mL distilled waterTube 2: 1.0 mL 0.01 M cyclohexanolTube 3: 1.0 mL 0.01 M 1,4-cyclohexanediolTube 4: 1.0 mL 0.01 M phenolTube 5: 1.0 mL 0.01 M catecholTube 6: 1.0 mL 0.01 M resorcinolTube 7: 1.0 mL 0.01 M hydroquinone

To each tube add an additional 3.0 mL of distilled water. (These are the substrate tubes.)

  1. Place all the labeled substrate test tubes into a 37 ° C size 12{°C} {} water bath.
  2. Transfer 3.0 mL of vegetable extract into each of another set of seven test tubes, and then place them into the constant temperature water bath. (These are the enzyme tubes.)
  3. Allow the two sets of test tubes to remain in the constant temperature bath for five minutes.
  4. Quickly add the contents of the enzyme tubes to the substrate tubes.
  5. Remove all the test tubes from the water bath and compare the intensity of any developed color to Tube 1, which contains distilled water.
  6. Arrange the test tubes in progressive intensity of color and record the sequence. Tube 1 (blank) is assigned a value of 0; the other tubes are then ranked 1, 2, 3, and so on.
  7. What can you conclude about the development of a color?
  8. On your report form, list the substrates that do not react with the enzyme.

Part iv. the change in enzyme activity due to change in ph

The final part of the experiment will involve an investigation into the effect of pH on enzyme activity.

  1. Prepare five small test tubes with labels and contents as follows:

Tube 1: 4 mL of pH 4.0 buffer solutionTube 2: 4 mL of pH 6.0 buffer solutionTube 3: 4 mL of pH 7.0 buffer solutionTube 4: 4 mL of pH 8.0 buffer solutionTube 5: 4 mL of pH 10.0 buffer solution

  1. Add to each of these test tubes 15 drops of 0.01 M solution of the most active substrate and 15 drops of extract solution (enzyme).
  2. Mix gently, and then incubate the test tubes in the constant-temperature water bath set at 37 ° C size 12{°C} {} . While you are waiting 15 minutes for the color to develop, do not start the final section involving copper sulfate.
  3. After 15 minutes, examine each test tube for color changes and record your results.
  4. From your results answer the question: What is the optimum pH range for maximum activity of the o-diphenoloxidase enzyme?

Part v. the effect of adding copper sulfate

  1. Place 5 mL of hydrogen peroxide in a test tube.
  2. Place 5 mL of the catalase solution in a second test tube.
  3. Add 5-10 drops of copper sulfate solution to the second test tube.
  4. Place both test tubes in a water bath at 20 ° C size 12{°C} {} for 5 min.
  5. Mix the test tubes and measure the amount of oxygen produced in 5 min.
  6. Record your observations on your report form.

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Source:  OpenStax, General chemistry lab spring. OpenStax CNX. Apr 03, 2009 Download for free at http://cnx.org/content/col10506/1.56
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