Title: Preparation and Assay of Phenolase and Peroxidase from Sweet and Irish Potato.

Aim: To determine the degree of specificity of the enzymes Polyphenol Oxidase and Peroxidase;
to determine the effect of inhibitors on enzyme activity and to examine the effect of enzyme
activity on the rate of breakdown of the substrates provided.

Theory: Enzymes are catalysts. A catalyst is a substance which speeds up the rate of a chemical
reaction without itself being used up. Most enzymes are proteins. The catalytic activity of
enzymes is based on the precision of the protein conformation of the enzyme. In particular, this
specificity depends upon the shape of a small part of the enzyme molecule where the enzyme
actually comes into contact with the substrate molecule, called the active site. This is a small
“cleft” in the surface of the enzyme where certain amino acid residues are exposed. These
residues contain substituent groups that bind to the substrate and catalyse its chemical
transformation. The substrate molecule fits into the active site and interacts with these amino
acids by ionic and hydrogen bonding, forming an enzyme-substrate complex. A reaction then
occurs, and the product leaves the active site. If an enzyme is broken down into its component
amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and
quaternary structures of enzymes proteins are essential to their catalytic activity. One of the aims
of this experiment is to examine the effect of enzyme activity on the rate of breakdown of the
substrates provided. This will in turn be used to determine how specific enzymes are their
substrates since the substrates which react most rapidly with the enzyme will be the most
complementary.

Enzymes are highly specific, readily discriminating between substrates with quite similar
structures. This is due to the chemical reactions that take place between substrates and enzymes’
functional groups within the active site of the enzyme.

Procedure: This lab was divided into two parts, the first of which utilized the enzyme
Polyphenol Oxidase (Phenolase) and the second of which utilized the enzyme, Peroxidase.

In the first part of the lab, the effect of enzymes on the rate of substrate breakdown was
examined. Also, the effect of inhibitors on enzyme activity was examined.

For this section of the lab, 5 test tubes were first prepared, each containing 2 mL of the substrate,
Catechol. 2 mL of water was added to the first test tube. 2 mL of the enzyme, Phenolase was
added to the second test tube alone. 2 mL of inhibitor was added to the third test tube alone. 2
mL of enzyme and 2 mL of inhibitor were added to the fourth test tube and to the fifth test tube
was added 2 mL of boiled enzyme. Observations of any colour changes and the time taken for
these colour changes to occur were made and recorded.

Still in the first section of the lab, 3 other test tubes were prepared, each containing a different
substrate. The first of these 3 test tubes contained 2.0 mL of Caffeic acid, the substrate, and 2.0
mL of the enzyme, Phenolase. The second of these 3 test tubes contained 2.0 mL of the substrate,
Pyrogallol and 2.0 mL of the enzyme, Phenolase. The third of these 3 test tubes contained 2.0
mL of the substrate and 2.0 mL of the enzyme, Phenolase. These three test tubes were observed
immediately after mixing the substrates with the Phenolase. These observations were recorded.

The second part of the lab dealt with the enzyme Peroxidase and its oxidation of a variety of
phenols in the presence of hydrogen peroxide. It also dealt with the effects of inhibitors on
enzyme activity by adding 2 drops of inhibitor to a particular substrate and then adding the
enzyme after. It examined the degree of specificity of the enzyme in use by adding the said
enzyme to a number of substrates which were all similar in structure to each other and then
observing the effects of the enzyme on the different substrates.

For this section of the lab, 5 test tubes were prepared, each containing a different substrate. The
first test tube contained 2.0 mL of the substrate, caffeic acid, to which was then added 1.0 mL of
the enzyme, peroxidase and 3 drops of hydrogen peroxide. Observations of the effects of the
enzyme on the substrate were made and recorded. The second test tube contained 2.0 mL of the
substrate, pyrogallol, to which was then added 1.0 mL of enzyme and 3 drops of hydrogen
peroxide. Observations of the effects of the enzyme on the substrate were made and recorded.
The third test tube contained 2.0 mL of the enzyme, catechol to which was then added 1.0 mL of
enzyme and 3 drops of hydrogen peroxide. Observations of the effects of the enzyme on the
substrate were made and recorded. The fourth test tube contained 2.0 mL of the substrate,
tyrosine, to which was then added 1.0 mL of enzyme and 3 drops of hydrogen peroxide.
Observations of the effects of the enzyme on the substrate were made and recorded. The fifth test
tube contained 2.0 mL of the substrate, guiaicol, to which was then added 1.0 mL of enzyme, 3
drops of hydrogen peroxide and 2 drops of inhibitor. Observations of the effects of the enzyme
on the substrate in the presence of inhibitor were made and recorded.

Results:

Table 1: Observations of the effects of the enzyme Phenolase on the substrate Catechol and
other substrates.
Reaction Mixture Observations

Catechol and Water Upon adding water to the Catechol, it remained

colourless. The solution showed no change in
appearance after a long period of time.

Catechol and Phenolase Upon adding the enzyme Phenolase, to the

Catechol the contents of the tube turned a pale
orange colour. After some time, the contents
turned dark brown.

Catechol and Inhibitor Upon adding inhibitor to the substrate, the

contents of the tube remained colourless. The
solution showed no change in appearance or
colour, after a long, period of time.

Catechol, Inhibitor and Enzyme Upon adding Inhibitor and then enzyme, the
solution remained colourless. After a long period
of time, no change in appearance of the contents
of the tube was observed.

Catechol and Boiled Enzyme A white precipitate was observed in a colourless

solution.

Caffeic acid and Phenolase Upon adding the Phenolase to the Caffeic acid, a
pale peach colour was observed which over time
slowly turned to a pale orange colour.

Pyrogallol and Phenolase Upon adding the Phenolase to the Pyrogallol, a

pale peach colour was observed which rapidly
changed to a deep yellow colour.

Tyrosine and Phenolase Upon adding the Phenolase to the Tyrosine, a

pale peach colour was observed. After a long
period of time no change was observed indicating
little or no reaction.

Table 2: Observations of the Effects of Peroxidase on Different Substrates

Reaction Mixture Observations

Caffeic acid, Peroxidase and Hydrogen
Peroxide
Pyrogallol, Peroxidase and Hydrogen Peroxide
Catechol, Peroxidase and Hydrogen Peroxide
Tyrosine, Peroxidase and Hydrogen Peroxide
Guiaicol, Peroxidase and Hydrogen Peroxide,
Inhibitor
Discussion: The purpose of this lab was to examine the function of enzymes in their conversion
of substrates to their products, to examine the effect of an inhibitor on an enzyme and to
determine the degree of specificity of certain types of enzymes with respect their substrates,
Upon adding the Peroxidase to the Caffeic acid
and Peroxide mixture, the contents of the tube
were initially colourless. As time progressed
the mixture went from colourless to pale
orange and a thick layer of fine bubbles were
observed at the surface of the mixture.
Upon adding the Peroxidase to the Pyrogallol
and Peroxide mixture, the contents of the tube
were initially colourless. The mixture then
quickly went from colourless to a yellow
orange brown colour. A thick layer of fine
bubbles were observed at the surface of the
mixture.
Upon adding the Peroxidase to the Catechol
and Peroxide mixture, the contents of the tube
were initially colourless. The mixture then
went rapidly from colourless to pale orange to
a darker, burnt orange to a red brown colour. A
layer of fine bubbles were observed at the
surface of the mixture.
Upon adding the Peroxidase to the Tyrosine
and Peroxide mixture, the contents of the tube
were initially colourless. The mixture then
went slowly form colourless to peach. A layer
of fine bubbles were observed at the surface of
the mixture.
Upon adding the Peroxidase to the Guiaicol,
Peroxide and Inhibitor mixture the contents of
the tube were initially colourless. The mixture
then went rapidly from colourless to red to a
dark, red brown colour. A layer of fine bubbles
were seen at the surface of the mixture.
In the first part of the lab, the enzyme under study was the enzyme, Polyphenol Oxidase
(Phenolase). The substrates of Polyphenoloxidases are mono-, di- , tri and polyhydroxyphenols
which are oxidised to their corresponding quinones by the enzyme.

Polyphenoloxidases catalyse the o-hydroxylation of monophenols to o-diphenols. They can also

further catalyse the oxidation of o-diphenols to produce o-quinones. It is the rapid
polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that is the
cause of food browning.

The first part of the lab dealt with the effect of enzyme activity on the dihydroxyphenol
substrate, Catechol. The structural formula of Catechol is:

A mixture of Catechol and water yielded no visible reaction. Upon addition of the Phenolase
enzyme however, a change was observed as the mixture changed from colourless, to pale orange
to dark brown. This was due to the Phenolase enzyme oxidising the Catechol to its benzoquinone
derivative which is a reddish brown melanoid pigment. In the third test tube, inhibitor was added
to the substrate alone without any enzyme and no reaction was observed. The purpose of
preparing this tube was to show that the inhibitor does not have any direct effect on the substrate
and does not oxidise it. The fourth test tube had a substrate and inhibitor mixture to which was
then added the enzyme Phenolase. No change was observed in this mixture. Since the test tube
which contained the enzyme and substrate had undergone a colour change thus indicating that a
reaction had taken place and since the test tube which contained the inhibitor and substrate alone
had undergone no visible colour change, it can be concluded that the lack of reaction in the
fourth test tube was due to the inhibitor. No other reactants were present to prevent the
oxidisation of the substrate besides the inhibitor. The enzyme was responsible for the oxidation
of the substrate in the second test tube.

The fifth test tube had a reaction mixture comprised of the substrate Catechol and denatured
enzymes which had been prepared by boiling. No change in the substrate was observed i.e a dark
brown colour had not appeared after some time indicating that no reaction had taken place. The
purpose of this tube was to show how important the structure of an enzyme is to its function.

The substrate specificity of an enzyme is determined by the properties and spatial arrangement of
the amino acid residues forming the active site. Boiling enzymes can disrupt the hydrogen, van
der Waals bonds, hydrophobic interactions and electrostatic interactions that maintain the tertiary
and quaternary structure of enzymes. Boiling will thus affect the spatial arrangement of the
exposed residues in the acitive site of the enzyme which is needed in order for the enzyme to
bind to the substrate. Since enzymes are highly specific, slight changes in the active site will
determine whether or not the substrate can bind to the active site or whether or not the enzyme
will be able to carry out its function.

Three other test tubes were prepared in the first part of the lab, each containing a different
substrate but the same Phenolase enzyme.

The first of these three tubes contained Caffeic acid. A pale peach colour was observed which
over time changed to a pale orange colour. The second contained Pyrogallol which rapidly
changed from a pale peach colour to a deep yellow colour. The third of these contained Tyrosine
which underwent no reaction with the enzyme Phenolase.

From these results it can be seen that Pyrogallol was oxidised at the fastest rate and underwent
the most drastic colour change. The structure of Pyrogallol is shown below:

The position of the hydroxyl substituents are integral in the formation of the enzyme-substrate
complex and the fact that there are two possible 2- positions on the aromatic ring to which
hydroxyl groups are bonded increases the chances of this happening. The structure of Pyrogallol
is also quite similar to the structure of Catechol.

Caffeic acid did not react as quickly as the Pyrogallol and only reacted to a small extent, the
colour change not being very drastic. The structure of Caffeic acid is shown below:

There 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 Phenolase.
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.

Tyrosine did not react at all with the enzyme. Its structure is shown below:

Whilst there 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 substrate. This explains the lack of reaction of
Tyrosine with the enzyme, Phenolase.

The results produced by the last three tubes indicate that Phenolase shows relative specificity.
The more similar in structure the substrates were to Catechol, the more rapidly they reacted with
the enzyme.

The second part of the experiment dealt with the enzyme Peroxidase. The enzyme peroxidase
catalyses the oxidation of a variety of phenols and ceratin aromatic amines in the presence of
hydrogen peroxide.

For this part of the lab five test tubes were prepared, four of which contained a different
substrate, hydrogen peroxide and the enzyme, Peroxidase. The fifth contained a different
substrate, hydrogen peroxide, enzyme and inhibitor.

The first test tube contained Caffeic acid which did not react rapidly with the enzyme and which
did not undergo a drastic colour change. It did however undergo some reaction since it went
from colourless to pale orange. The bubbles that were seen at the surface of the mixture were
bubbles of oxygen.

The second test tube contained Pyrogallol which reacted rapidly with the enzyme Peroxidase to
form a dark, yellow brown 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. The bubbles were again
oxygen.

The third test tube contained Catechol which reacted rapidly with the enzyme Peroxidase to form
a dark, red brown product. Catechol was thus more complementary to the active site of the
enzyme than Pyrogallol or Caffeic acid. The bubbles at the surface were again oxygen.

The fourth test tube contained Tyrosine which only reacted to a small or negligible extent with
the enzyme, Peroxidase. The colour change was from colourless to peach and was very difficult
to observe. The bulky substituent group at the 4-position of the aromatic ring prevented the
enzyme from oxidising the substrate in this case as well. The bubbles observed at the surface
were oxgen.

These observations show that Catechol was the substrate most complementary to the active site
of the enzyme Peroxidase with Pyrogallol being the second most complementary.

In the fifth test tube, the substrate that was used was Guiacol. Hydrogen peroxide, the enzyme
Peroxidase and inhibitor were used with this substrate. It was expected that no reaction would
take place since inhibitor had been included in the reaction mixture. However, Guiacol reacted
rapidly with the substrate and a dark, red brown product was produced. The amount of inhibitor
added (2 drops) was not adequate for its effects to be seen.

This part of the lab with respect to the tube containing Guiacol and inhibitor could have therefore
been improve by adding more inhibitor than merely 2 drops.

The substrate Pyrogallol decolourises in the presence of light. This is a possible source of error
since results were largely based on colour changes of the reaction mixtures and it was required
that colour changes be entirely due to the enzymes that were used.

When using the inhibitor, a precaution was taken to add the inhibitor before the enzyme to the
substrate. As was shown in the first part of the lab, the inhibitor does not have a direct effect on
the substrate so would not interfere with the enzyme acting on the substrate in this respect. It
only directly affects the enzyme. If the enzyme was added to the substrate before the inhibitor
some substrate breakdown would take place leading to confusion of results and wrong
conclusions.

Additional Discusion:

1)To what class of enzymes does a) phenolase and b) peroxidase belong?

Both of these enzymes are oxidoreductases since they both react with the same substrates in a
similar manner and both involve the transfer of electrons.

2)Explain what happens on boiling the enzyme preparation.

Boiling temperature is much higher than the optimum temperature of enzymes. High
temperatures change the shape of the enzyme molecule. Enzymes are globular proteins. Their
tertiary structure is maintained by bonds between the amino acid R-groups of a polypeptide
chain. High temperatures cause these bonds to break, damaging the tertiary structure of the
protein. If the three dimensional shape of the active site has been altered, substrate molecules
will be unable to combine with it. The irreversible change in the structure of the enzyme at high
temperatures is called denaturation.
3)Give examplesof phenolase activity in everyday situations. How do we try to control such
activity?

Enzymatic browning of fruits and some vegetables is a chemical process that occurs in everyday
situations. It involves phenolase breakdown of polyhydroxyphenols such as Catechol to their
brown coloured quinones. Enzymatic browning can be prevented by sprinkling lemon juice on
the freshly cut fruits and vegetables. The ascorbic acid in the lemon juice acts as an inhibitor
preventing the breakdown of the substrate to the brown coloured quinone product.

4)What class of inhibitor was used in your experiment?

Competitive inhibitors were used since with competitive inhibitors the degree of inhibition
depends on the relative concentration of substrate and inhibitor. Increasing the concentration of
the substrate will reduce the effect of the competitive inhibitor which is what happened in this
lab. The much greater concentration of substrate made the effects of the inhibitor negligible.

References:

David Hames, Nigel Hooper. Instant Notes in Biochemistry. New York: Taylor and Francis Group, 2005.

David L. Nelson, Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and
Company, 2008.

T. W. Graham Solomons, Craig B. Fryhle. Organic Chemistry. New Jersey: John Wiley & Sons, 2006.