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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.

Questions & Answers

are nano particles real
Missy Reply
Hello, if I study Physics teacher in bachelor, can I study Nanotechnology in master?
Lale Reply
no can't
where we get a research paper on Nano chemistry....?
Maira Reply
nanopartical of organic/inorganic / physical chemistry , pdf / thesis / review
what are the products of Nano chemistry?
Maira Reply
There are lots of products of nano chemistry... Like nano coatings.....carbon fiber.. And lots of others..
Even nanotechnology is pretty much all about chemistry... Its the chemistry on quantum or atomic level
no nanotechnology is also a part of physics and maths it requires angle formulas and some pressure regarding concepts
Preparation and Applications of Nanomaterial for Drug Delivery
Hafiz Reply
Application of nanotechnology in medicine
has a lot of application modern world
what is variations in raman spectra for nanomaterials
Jyoti Reply
ya I also want to know the raman spectra
I only see partial conversation and what's the question here!
Crow Reply
what about nanotechnology for water purification
RAW Reply
please someone correct me if I'm wrong but I think one can use nanoparticles, specially silver nanoparticles for water treatment.
yes that's correct
I think
Nasa has use it in the 60's, copper as water purification in the moon travel.
nanocopper obvius
what is the stm
Brian Reply
is there industrial application of fullrenes. What is the method to prepare fullrene on large scale.?
industrial application...? mmm I think on the medical side as drug carrier, but you should go deeper on your research, I may be wrong
How we are making nano material?
what is a peer
What is meant by 'nano scale'?
What is STMs full form?
scanning tunneling microscope
how nano science is used for hydrophobicity
Do u think that Graphene and Fullrene fiber can be used to make Air Plane body structure the lightest and strongest. Rafiq
what is differents between GO and RGO?
what is simplest way to understand the applications of nano robots used to detect the cancer affected cell of human body.? How this robot is carried to required site of body cell.? what will be the carrier material and how can be detected that correct delivery of drug is done Rafiq
analytical skills graphene is prepared to kill any type viruses .
Any one who tell me about Preparation and application of Nanomaterial for drug Delivery
what is Nano technology ?
Bob Reply
write examples of Nano molecule?
The nanotechnology is as new science, to scale nanometric
nanotechnology is the study, desing, synthesis, manipulation and application of materials and functional systems through control of matter at nanoscale
Is there any normative that regulates the use of silver nanoparticles?
Damian Reply
what king of growth are you checking .?
how did you get the value of 2000N.What calculations are needed to arrive at it
Smarajit Reply
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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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