Preparation and Assay of Phenolase and Peroxidase from Sweet and Irish Potato

Title of Lab: Preparation and Assay of Phenolase and Peroxidase from Sweet and Irish Potato
Hypothesis: Polyphenol Oxidase enzyme activity can be detected by change in colour of solution, Inhibitors prevent the reaction of the enzymes with substrates, the enzyme is relatively specific.
Aim: To design and conduct an experiment to demonstrate enzyme activity of Polyphenol Oxidase and Peroxidase when mixed with catechol, caffeic acid, pyrogallad, tyrosine, guacol, and water, to test the effect of inhibitors on these enzymes, to show the specificity of Polyphenol Oxidase and also the effect of amylase on starch.
Theory: Enzymes are large molecules that increase the rates of chemical without themselves undergoing any change (Bettelheim, Brown, Campbell and Farrell 2010). Enzymes are the catalyst of biological processes; they bring the reaction catalyzed to its equilibrium position more quickly than would occur otherwise (Aehle 2007) Enzymes are mainly globular proteins; the tertiary structure has gives the molecule a generally rounded, ball shape. Active sites are cracks or hollows on the surface of the enzyme caused by the way the protein folds itself up into its tertiary structure. Molecules of just the right shape, and with just the right arrangement of attractive groups can fit into these active sites. Other molecules won't fit or won't have the right groups to bind to the surface of the active site. The molecule, which is actually going to react as the reactant. The reactant in an enzyme reaction is known instead as the substrate. (1) Phenolase is a copper containing enzyme that catalyze the oxidation of phenols to the corresponding quinone. Phenolase oxidizes substrates, such as tyrosinase, monophenol oxidase, diphenol oxidase, or catecholase (Logan 2003). Potato polyphenol oxidase (PPO) is an enzyme that is activated upon injury to the potato, e.g., sliced with a knife, cut with a spade, poked with a pitch-fork. It is this enzyme that causes the potato to turn brown where it shows damage. The same enzyme (polyphenol oxidase -- PO) is also found in apples and bananas and grapes (2) Peroxidase is protein-based enzymes that act as catalysts to facilitate a variety of biological processes. Peroxidase activity involves donating electrons to bind to other substrate substances, such as ferrocyanide and ascorbate, in order to break them down into harmless components (3). Hydrogen peroxide (H2O2) is a common end product of oxidative metabolism and, being a strong oxidizing agent, could prove toxic if allowed to accumulate. To prevent this, eukaryotic cells have enclosed the enzymes producing peroxides within a membrane-bound organelle, the peroxisome, which is similar in size and appearance to a lysosome. Peroxisomes also contain high concentrations of peroxidase whose function is to reduce the peroxide to water, rendering it harmless (4).

Diagram: The Polyphenol Oxidase Experiment

Procedure: Polyphenol Oxidase
Macerate the sweet potato using water and blend at of 0-4 degrees Celsius. Centrifuge the solution at low speed (x1000g) for ten minutes to remove cell debris and chloroplasts. The supernatant can be used as a source of enzyme or purified even further by using solvent fractioning.
Add 1ml of water to a test tube of 1ml of the enzyme and observe and record results. Repeat this using the solutions catechol, caffeic acid, pyrogallad tyrosine and guacol.
Add 2 drops of ascorbic acid to a test tube of 1 ml of catechol , then add 1 ml of enzyme and observe and record results.
Boil 1ml of enzyme in a boiling tube, then add it to 1ml of catechol in a test tube and observe and record results

Peroxidase
1ml of water was added to a test tube of 1ml of enzyme and results were observed and recorded. 1ml of caffeic acid was added to a test of 1ml of enzyme, and then 1ml of hydrogen proxide was added. Result were observed and recorded. This was repeated using the solutions catechol, pyrogallad tyrosine and guacol.
2 drops of ascorbic acid was added to a test tube of 1 ml of guiaicol, then 1 ml of enzyme and 1ml of hydrogen peroxide and results were observed and recorded.

Amylase
1ml of starch was added to a test tube, and then a few drops of iodine were added. Results were observed and recorded. 1ml of starch was added to a test of 1ml of enzyme, and then a few drops hydrogen iodine was added. Results were observed and recorded

Results:
Table 1: Theoretical Results of Polyphenol Oxidase reactions
Tube Test Observation
1 Catechol Dark brown colour solution
2 Caffeic Acid A pale orange solution
3 Pyrogallol A deep yellow solution
4 Tryosine No change
5 Guiaicol
6 Water No change
7 Catechol and Ascobic Acid No change
8 Denatured enzyme ad Catechol No change

Table 2: Experimental Results of Polyphenol Oxidase reactions
Tube Test Observation
1 Catechol Intense brownish cloudy colour
2 Caffeic Acid A pale yellowish cloudy colour
3 Pyrogallol Yellow colour
4 Tryosine Stayed the originally colour cloudy off-white
5 Guiaicol Very light yellow cloudy colour
6 Water Cloudy off white
7 Catechol and Ascobic Acid Cloudy off white
8 Denatured enzyme ad Catechol Cloudy off white

Table 3: Results of Peroxidase reactions
Tube Test Observation
1 Catechol Red colour solution and layer of bubbles
2 Caffeic Acid Brown colour solution and a layer of bubbles
3 Pyrogallol Yellow solution and small layer of bubbles
4 Tryosine Light cream colour with layer of bubbles
5 Guiaicol Orange solution
6 Water No colour change, layer of bubbles
7 Guiaicol and Ascobic Acid Off-white solution

Table 4: Results of Amylase reactions
Tube Test Observation
1 Starch Reddish black solution
2 Starch and Amylase Pale yellow solution

Discussion: The control, water and the enzyme did not change colour because there are no phenols to oxidase.
Catechol is a dihydroxyphenol. The catechol reacted the enzyme and give a intense brownish colour this occurs because Catechol is oxidized by the enzyme to form 0-benzoquinone (a quinone) then converted through a series of spontaneous reactions to produce a heterogeneous group of polymers called melanins) the two hydrogen from the hydroyl groups of the phenol are remove to form water. As the polymers gets larger, their colors deepen from pink/ gold through orange-brown and finally to an intense brown-black color. The larger molecules are less soluble in water so eventually precipitate from the solution (Logan 2003).

Phenolase spontaneous spontaneous
Catechol -----> 0-benzoquinone------> pink/gold melanin ------> brown melanin +1/2 O2 +H2O

The reaction catechol, ascorbic acid and the enzyme had no colour. Ascorbic acid was used as an inhibitor so this prevented the catechol and sweet potato from reacting.
The reaction denatured enzyme and catechol had no colour change. This occurred because the change in shape of enzyme. The bonding between the amino acids R-groups in the protein is broken because the high temperature is over the optimum temperature of the enzyme. Thus the active site of the enzyme is changed permanently and the catechol is unable to bind with it (4).
The position of the hydroxyl substituents are integral in the formation of the enzyme-substrate complex. Progallol is trihydrophenol its structure is similar to catechol, so it react with the enzyme in almost the same way (4).
Caffeic acid is a hydroxyl group at the 2- and 3- positions of the aromatic ring and a bulky side group at the 5- position. Relative to Catechol the positions of the hydroxyl groups are significantly different. Enzymes are very substrate specific so would thus not react to a large extent with a substrate that was so different from a substrate, which was so readily oxidised by the polyphenol oxidase. The bulky side group at the 5- position would also prevent proper binding to the active site. It would serve as a steric hindrance to the enzyme (4).
Tryosine is a hydroxyl group at the 1- position which is perhaps able to bind to the active site of the enzyme, the side group at the 4-position of the aromatic ring would act as a steric hindrance to binding to the active site of the enzyme. This explains the lack of reaction of Tyrosine with the enzyme (4).

Perioxdase oxidizes the hydroxyl groups of substrate to produce water.
Caffeic acid was not easily oxidased by the peroxidase because of it structure. The bulky side group at the 5- position prevents proper binding to the active site. (4)
Pyrogallol reacted rapidly with the enzyme Peroxidase to form a yellow product. The active site of the enzyme Peroxidase was perhaps more specific to the structure of Pyrogallol than it was to that of Caffeic acid (4).
Catechol reacted with the enzyme Peroxidase to form a red product. Catechol was thus more complementary to the active site of the enzyme than Pyrogallol or Caffeic acid. (4).
Tyrosine reacted to with the enzyme, Peroxidase to give a very light cream product. This occurred because of tyrosine structure the side group at the 4-position of the aromatic ring would act as a steric hindrance to binding to the active site of the enzyme (4).
Iodine reacted with the starch to form iodine complexes with starch to form a reddish black product (5). Starch reacts with amylase and is broken down into simpler form maltose. Therefore when iodine is added not reaction takes place (6). Sources of errors in this experiment are pyrogallol is decolourize the present of light so this could a have affect the colour change of the solution

Conclusion: Enzyme activity of poylphenol oxidase can be detected by change in solution. Inhibitors prevents enzyme and substrate from reacting and polyphenol oxidase is relatively specific.

Additional Discussion:
1) Phenolase and Peroxidase belong to oxidoreductases since catalyze the transfer of electrons from one molecule to another molecule and they react in a similar way.

2) When the enzyme is boiled during the enzyme preparation the enzyme becomes denatured. The change the shape of enzyme because the bonding between the amino acids R-groups in the protein is broken because the high temperature is over the optimum temperature of the enzyme. Thus the active site of the enzyme is changed permanently and the substrate is unable to bind with it.

3) Examples of phenolase activity in everyday situations are browning of fruits and some vegetables and this is control by sprinkling lime/lemon juice on them because the ascorbic act in the juice act as an inhibitor and prevents the brown quinone product from being formed.

4) Competitive inhibitors were used the concentration of affect the degree off inhibitions