NAME: Kimberly George

LAB PARTNERS: Shenese Harrysingh

STUDENT ID NUMBER: 813117977

DEMONSTRATOR: Aneisha

DATE: 18/02/2014
COURSE CODE: BIOL 1362
COURSE NAME: Biochemistry
TITLE: Enzymatic activity in sweet potato extract, Irish potato extract and milk.
AIM:
1. To prepare a crude enzyme extract of Irish potato, sweet potato by maceration and
centrifugation.
2. To determine the enzymatic activity in sweet potato extract, Irish potato extract using
different substrates.
3. To determine the substrate concentration and heat treatment on activity of the enzyme
xanthine dehydrogenase found in milk.
THEORY:
Enzymes are biological catalysts that speed up chemical reactions by providing a pathway with a
lower activation energy. They are large proteins made up of many amino acids that are folded so
that they have specifically shaped binding or active sites. Enzymes are usually named after the
reaction they catalyse. The general features of an enzyme are its catalytic power, specificity and
regulation. The catalytic power refers to the rate of an enzyme- catalysed proceeds faster than
uncatalyzed reactions. Enzymes are highly specific, meaning that their active sites can only bind
to a particular substrate to form an enzyme-substrate complex. The catalytic behaviour of
enzymes can be regulated. Other characteristics of enzymes are:
-

Enzymes are not consumed in the catalysed.

Enzymes are affected by environmental factors such as changes in temperature and pH
because at high temperatures and pH, proteins tend to be denatured. The optimum
temperature of most enzymes in the human body is 37- 40C. (Worthington Biochemical
Corporation). The unfolding of the protein results in loss of biological enzymatic activity
due to the distortion of the active site. Denaturation causes the disruption of the bonds
which make up the tertiary structure.

Catalysis is shown in the diagram below which illustrates the change in free energy from
reactants to products as well as the activation energy needed to reach the transition state between
reactants and products. The higher the activation energy, less substrate molecules are able to
reach the transition state to be turned to products and thus the slower the reaction. However
when an enzyme is present it provides a pathway from substrate to product which has a lower
activation energy than the uncatalyzed reaction.

http://www.uvm.edu/~biology/Classes/011/Lab5.pdf
Two hypotheses that explain the specificity of the enzyme and catalysis are the lock and key and
the induced fit. The lock and key hypothesis explains how the shape of the enzyme is analogous
to the shape of the active site and vice versa. The induced fit hypothesis shows the ability of the
enzymes active site to induce a change in its conformation to fit the substrate. The two types of
specificity are absolute and relative specificity. In absolute specificity, the enzyme acts only one
substrate meaning it will only catalyse one reaction. In relative specificity the enzyme acts on the
substrate similar in structure and contain the same type of bonds.
Inhibitors are any substances that decrease or sometimes stop the velocity or rate of an enzymecatalyzed reaction. There are two types reversible, which bind to enzymes through non-covalent
bonds, and irreversible which bind through covalent bonds. Two types of inhibitors are
Competitive and Non-competitive. These inhibitors decreases the rate of reaction due to them
either competing for an active site (competitive) or binding to the enzyme-substrate complex or
enzyme and causing a conformational change to the active site (non-competitive).
This experiment consists of three parts. Part A is to determine the enzymatic activity of
phenolase enzyme, present in both sweet and Irish potato through a corresponding change in
colour from clear to brown. Phenolase is a copper- containing enzyme that catalyse the oxidation
of phenols to the corresponding quionine. This oxidation of phenol in the presence of the enzyme
phenolase is called enzymatic browning.
Part B is also to determine the enzymatic activity however using the enzyme peroxidase which
is present in Irish and sweet potato also through a colour change from clear to brown. Peroxidase
catalyzes the reaction:
2 H2O 2 H2O + O2
(http://www.chem.purdue.edu/teacher/table_of_contents/spectronic%20educator/enzyme.pdf)

The reaction converts toxic hydrogen peroxide into water and oxygen. The production of oxygen
thus oxidizes the substrate present to give the brown colour which is the process of fruit
browning.
For Experiments part A and B, substrates caffeic acid, catechol, guaicol, pyragallol and tyrosine
is used and for each substrate there are four tubes consisting of i) substrate and water (blank), ii)
enzyme and water(blank), iii) enzyme and substrate, iv) enzyme, inhibitor and substrate
respectively. The substrate and enzyme blank represent the controls in the experiment in which
they are compared to the final colour to observe if there is a colour change and thus enzymatic
activity. Ascorbic acid reduces quinones to diphenols and prevents fruit browning. Therefore it
acts as a reducing agent. Sometimes ascorbic acid is oxidized completely and browning may
occur due to melanin formation. Ascorbic acid acts as a competitive inhibitor of phenolase by
chelating metal ions and reduces oxygen and thus lowers the pH to diminish the enzyme activity.
In part C of the experiment, the effect of substrate concentration and the effect of heat treatment
on enzymatic activity for the enzyme xanthine dehydrogenase, found in fresh milk, is determined
by recording the time taken for the colour change from blue to white to occur. Xanthine
dehydrogenase is a protein found in humans that belong to the group of molybdenum-containing
hydroxylases involved in the oxidation of purines. All the enzymes in this experiment belong to
the class oxidoreductases which catalyze oxidation- reduction reactions.
The limitations to this qualitative method of determining the enzymatic activity are the different
perceptions of colour which will introduce error into the experiment. Also the time taken for the
colour change to be seen may not be accurate and the time lapse between the addition of the
enzyme to substrate and starting the stop clock. Perhaps the use of a spectrophotometer could be
used to determine accurately the colour changes of the product.

MATERIALS AND REAGENTS:

Sweet potato extract

Acetaldehyde

Paraffin

Guaicol

Irish potato extract

Methylene blue

catechol

Pyragallol

Milk

hydrogen peroxide

caffeic acid

Tyrosine

METHOD:
The Irish and sweet potato extract was prepared by maceration using either water or buffer of pH
close to the pH optimum of the enzyme by means of a blender or mortar and pestle at 0-4oC. The
homogenate was then centrifuged at low speed (x1000g) for ten minutes.
In part A, four test tubes were labelled (1-4). To test tube 1, 2ml of substrate (catechol) and water
were added. To test tube 2, 2ml of the enzyme ( Irish potato extract phenolase) and 2ml of
water were added. To test tube 3, 2ml of the Irish potato extract (phenolase) and catechol were
added. Lastly to test tube 4, 2ml of Irish potato extract (phenolase) and catechol and 2 drops of

inhibitor (ascorbic acid) were added. The procedure was repeated using sweet potato extract as
the enzyme phenolase.
In part B, the procedure was similar. Four test tubes were labelled 1-4. To test tube 1, 2mL of
substrate (catechol) and 1mL of water were added. To test tube 2, 1mL of enzyme (Irish potato
extract- peroxidase) and 2mL of water were added. To test tube 3, 1mL of enzyme and 2mL of
substrate were added and lastly to test tube 4, 1mL of enzyme, 2mL of substrate and 2 drops of
ascorbic acid (inhibitor) were added. The procedure was repeated using sweet potato extract as
the peroxidase enzyme.
The procedure was also repeated using the substrates caffeic acid, guiaciol, pyragallol and
tyrosine respectively for both part A and B. It was also repeated using the substrates amylase,
starch and iodine but only using the enzyme and substrate.
In part C (i), 2ml of fresh milk and 1-2 drops of methylene blue were added to a test tube. The
mixture was swirled until well mixed. 1ml of 0.2M substrate (acetaldehyde) was then added to
the mixture, it was then swirled and the test tube was held at an angle while liquid paraffin was
immediately added to it. The liquid paraffin was allowed to run along the sides to form a layer of
the reaction. The time was recorded on addition of acetaldehyde to the enzyme (xanthine
dehydrogenase). The time taken (t) for the reaction mixture to change from blue to white was
recorded using a stop clock. The procedure for part C (i) was repeated using the following
concentrations of acetaldehyde respectively: 0.4M, 0.6M, 0.8M and 1.0M. All results were
tabulated and observations were recorded. A graph was drawn showing the rate of reaction.
In part C (ii), 3 test tubes were labelled (A, B, C) respectively. To tube A, 2mL of fresh milk,
1mL of water and 0.5mL of methylene blue were added. To tube B, 2mL of fresh milk, 1mL of
acetaldehyde and 0.5mL of methylene blue were added. To tube C, 2mL of boiled milk, 1mL of
acetaldehyde and 0.5mL of methylene blue were added. All the tubes were swirled gently and 56 drops of liquid paraffin were added to tubes A-C. The time on addition of methylene blue were
noted for tubes A-C simultaneously. The time (t) taken for the mixture to turn white were noted
1
for all tubes simultaneously. The reaction rate was taken as t .

RESULTS:
Table 1: Enzymatic Activity of Polyphenol Oxidase (Phenolase) Sweet Potato extract
Substrate

Caffeic Acid

Catecol

Guaicol

Pyragallol

Tyrosine

Reaction
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate

Observations
Colourless solution
Pale yellow solution formed
Rusty orange solution formed
Yellow solution formed
Colourless solution
Pale yellow
Brown solution
Pale yellow
Colourless solution
Pale yellow solution
Light Brown solution
Cream
Colourless
Pale yellow solution
Rusty orange solution
Cream
Colourless
Pale yellow
Brown solution
Pale yellow

Table 2: Enzymatic Activity of Polyphenol Oxidase (Phenolase) Irish Potato Extract

Substrate

Caffeic Acid

Catecol

Guaicol

Pyragallol

Tyrosine

Reaction
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate

Observations
Colourless solution
Pale yellow solution formed
Rusty orange solution
Pale cream
Colourless solution
Pale yellow
Brown solution
Light cream
Colourless solution
Pale yellow solution
Rusty orange solution
Cream solution
Colourless
Pale yellow solution
Cloudy brown solution
Yellow solution
Colourless
Pale yellow
Brown solution
Pale yellow

Table 3: Enzymatic Activity of Peroxidase Sweet Potato

Substrate

Caffeic Acid

Catecol

Guaicol

Pyragallol

Tyrosine
Starch
Amylase
Iodine

Reaction
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Enzyme and Substrate
Enzyme and Substrate
Enzyme and Substrate

Observations
Clear/colourless solution
Pale yellow solution
Pale yellow bright orange
Pale yellow solution
Clear/colourless solution
Pale yellow solution
Pale yellow orange solution
Pale yellow light orange solution
Colourless
Colourless
Red-brown solution
Pale orange
Colourless
Colourless
Dark orange solution
Light yellow
Colourless
Colourless
Yellow
Pale yellow
Cloudy
Cloudy
Greenish yellow

Table 4: Enzymatic Activity of Peroxidase Irish Potato

Substrate

Caffeic Acid

Catecol

Guaicol

Pyragallol

Tyrosine
Starch
Amylase
Iodine

Reaction
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Substrate and Water
Enzyme and Water
Enzyme and Substrate
Enzyme, Inhibitor and Substrate
Enzyme and Substrate
Enzyme and Substrate
Enzyme and Substrate

Observations
Clear, colourless solution
Clear, colourless solution
Yellow
Pale Yellow
Clear, colourless solution
Colourless
Light peach reddish brown
Light cream dark peach
Pale orange
Colourless
Dark orange
Cloudy off-white solution
Colourless solution
Colourless solution
Yellow solution
Pale yellow solution
Colourless
Colourless
Dark Yellow solution
Pale yellow
Colourless
Colourless
Yellow/brown (colour of iodine)

Table 5: The Effect of Substrate Concentration on Enzymatic Activity of Xanthine

Dehydrogenase Fresh Milk
Concentration of Substrate
(Acetaldehyde) (M)

Reaction Time (min)

0.0

0.2
0.4
0.6
0.8

26
30.5
46
58

1.0

105.5

Table 6: The Effect of Heat Treatment on Enzymatic Activity of Xanthine

Dehydrogenase Fresh Milk

Tube

Reaction Time (min)

60

B
C

60
60

DISCUSSION:
In part A of the experiment the qualitative results for determining phenolase activity using each
substrate were similar between Irish potato and Sweet potato extracts. For the substrate caffeic
acid, when the phenolase was added (enzyme substrate complex ES) the colour was rusty orange
for both extracts. The enzyme-inhibitor-substrate complex (EIS) in Irish potato was pale cream
however it was yellow in sweet potato. For catechol, the ES complex was brown for both and
the EIS was pale yellow for sweet potato whereas it was light cream for Irish Potato. For guiacol
the ES complex was brown in sweet potato and rusty orange in Irish potato. However the EIS
was cream in both extracts. For pyrogallol, the ES was cloudy brown for both extracts and in the
EIS complex it was cream in sweet potato and cream in Irish potato. Lastly for tyrosine the ES
was brown for both extracts and the EIS was pale yellow for both extracts.
Therefore the results show the occurrence of enzymatic or fruit browning when the polyphenol
oxidase is added to all substrates. This is because the phenolase catalyses the oxidation of the
substrate to its subsequent quionine which is a brown pigment. However when the inhibitor,
ascorbic acid is introduced enzymatic browning either completely stopped in substrates such as

catechol and tyrosine or slowed down as seen in substrates guaiacol, caffeic acid and pyrogallol.
This is because ascorbic acid acts as a strong reducing agent and removes oxygen from the
reaction so no oxidation of the substrate occurs. The ascorbic acid also chelates the metal ions
thus decreasing the pH and reducing the phenolase activity. The enzyme phenolase is very
specific towards substrates tyrosine and catechol this is why the colour went straight to brown..
The following equations show the common reaction of phenolase with substrate tyrosine and
catechol:

http://en.wikipedia.org/wiki/Catechol_oxidase

In part B of the experiment the qualitative results for determining peroxidase activity using each
substrate were fairly similar between Irish potato and Sweet potato extracts. For the substrate
caffeic acid, when the peroxidase was added (enzyme substrate complex ES) the colour was
yellow for Irish potato and bright orange for sweet potato. The enzyme-inhibitor-substrate
complex (EIS) was pale yellow for both extracts. For catechol, the ES complex was rddish
brown for the Irish potato and dark orange for sweet potato. The EIS was light orange for sweet
potato whereas it was dark peach for Irish Potato. For guiacol the ES complex was red brown in
sweet potato and dark orange in Irish potato. The EIS was white in the Irish potato extract and
pale orange in sweet potato extract. For pyrogallol, the ES was yellow and dark yellow for Irish
and sweet potato extracts respectively. For both extracts, in the EIS complex it was pale yellow.
Lastly for tyrosine the ES was dark yellow and yellow for Irish and sweet potato respectively and
the EIS was pale yellow for both extracts. Amylase and starch showed no reaction while iodine
gave a yellow-brown colour for Irish potato extract and for sweet potato, amylase and starch
showed a cloudy solution while iodine gave a greenish-yellow colour. The inhibitor ascorbic acid
also acts as a strong reducing agent to remove oxygen and prevent browning. This was seen in all
substrates however catechol gave a dark peach colour in the Irish potato extract. This suggest
that oxidation of the ascorbic acid could have occurred to form melanin. Peroxidase show

relative specificity to substrates guaiacol and pyrogallol since these two gave colours closest to
brown. Catechol showed enzymatic browning but is particularly specific to the enzyme
peroxidase. The following equations how the reaction of peroxidase with guaiacol and
pyrogallol:

http://pirate.shu.edu/~rawncarr/Enzyme1/Enzyme1NEW.htm

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analyticalenzymes/peroxidase-enzymes.html
The peroxidase catalysed the oxidation of the halide used in this experiment which was iodine to

give its brown pigmentation however it does not oxidise amylase or starch.
In the part C (i) of the experiment, the rate of reaction increased as the substrate concentration
increased. The rate increased gradually from 26 minutes at [S] of 0.2 to 58 minutes at [S] of 0.8.
Then the rate increased exponentially or suddenly to 105.5 minutes at concentration 1.0. In the
beginning when substrate concentration is low the time increases rather slowly due to the
availablility of active site to the substrate so it does not take long to react. However as [S]
increases the time increases exponentially until it comes a maximum time of 105.5 minutes. This
is because the active sites are being occupied with the increase in substrate molecules.
The graph showing the effect of substrate concentration on time of an enzyme-catalysed reaction
was a sigmoidal curve which mean that the enzyme xanthine dehydrogenase is an allosteric
enzyme or a low specificity. The factors that are being kept constant are temperature, enzyme
concentration and pH.
In part C(ii) the rate of reaction of the enzyme (xanthine dehydrogenase) catalysed reaction
remained the same in all the tubes. This result was quite inaccurate with tube B, which contained
the boiled milk since the reaction rate should have increased due to an increase in temperature.
The effect of temperature on an enzyme- catalysed reaction is that as temperature increases, the
rate of reaction increases until an optimum temperature is reached in which after the rate
decreases suddenly. As temperature increases the kinetic energy and collision frequency of the
substrate and active site of the enzyme is increased. This results in more substrates binding with
the active site to form enzyme-substrate complex and eventually being converted into product.

The rate of reaction decreases because after the optimum temperature because denaturation of
the enzymes active site occurs causing it to lose its shape and thus its catalytic power. Thus it is
rendered inactive.
The limitation to part A and B of the experiment is the use of qualitative results to determine
enzymatic activity in phenolase and peroxidase. This introduced error because it was based on
perception of the colour change rather than a more accurate method such as the use of a
spectrophotometer. In part C of the experiment the limitation was the time lapse between
recording the time at which enzyme was added to substrate. This is could have introduced error
into the experiment since the exact time at which the enzyme substrate complex formed could
have been missed. Also the time at which the colour change could have also been recorded either
too early or late again due to the perception of colour by the individual.

ADDITIONAL DISCUSSION:
1. What major class of enzymes does (a) phenolase and (b) peroxidase belong?
The major class of enzymes phenolase belongs to is the oxidoreductases, specifically
polyphenoloxidases. Peroxidase also belongs to the class oxidoreductases.
2. Explain what happens on boiling the enzyme preparation
On boiling the enzyme preparation, the temperature increases to a point in which an
optimum temperature is reached. After this point denaturation of the enzyme occurs in
which it unfolds. The active site loses its shape causing it to become catalytically
inactive. Denaturation is a cooperative process which means as soon as denaturation
begins the entire protein unfolds one time. Denaturation occurs due to the high
temperatures disrupting thee hydrophobic bonding in the tertiary structure of the enzyme.
3. Give examples of phenolase activity in everyday situations. How do we try to control
such activity? Examples of phenolase activity are: the browning of fruits, vegetables and
seafood such as shrimp upon exposure to oxygen. Various techniques of controlling this
-

browning are:
By adding lemon juice and other acids to lower the pH and remove the copper cofactor

which is required for the enzyme to function.

By steaming the fruits, vegetables and seafood to denature the enzymes
By refrigerating these foods exposing them to low temperatures can also prevent

enzymatic browning by reducing rate of reaction.

By exposing the food to an inert gas, like nitrogen, can prevent oxidation of the substrate

thus preventing enzymatic browning.

4. What class of inhibitor was used in your experiment?
The class of inhibitor (ascorbic acid) used was a competitive inhibitor of polyphenol
oxidase. It competed with phenolase for the active site since it possessed the same shape.

REFERENCES:
Books:
Palmer, Trevor, and Philip L. R. Bonner. 2007. Enzymes: biochemistry, biotechnology and
clinical chemistry. 2nd ed. Chichester: Horwood.
Pratt, Charlotte W, and Kathleen Cornely 2004. Essential Biochemistry. Hoboken, NJ: J. Wiley.
Websites:
"Enzymatic browning." Food-Info.net :. http://www.foodinfo.net/uk/colour/enzymaticbrowning.htm (accessed March 25, 2014).