BROWNING OF APPLES: A STUDY OF ENZYMES
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BROWNING OF APPLES PRE-LAB
1) Define a catalyst.
2) What is the reason some reactions require catalysts?
3) What are the advantages of using a catalyst?
4) What is an enzyme? Why are enzymes useful?
5) Explain how enzymes recognize their substrates specifically?
6) What is an enzyme inhibitor? How does it work?
7) What is the main component in the following substances?
a) Vinegar
b) Baking soda
c) Lemon juice
d) Milk of magnesia
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8) What is the right time to cut the apples?
9) What precautions must you take take to ensure you do not make mistakes?
10) Why are tyrosinases important to human kind?
OVERVIEW OF ENZYMES
Chemical reactions may be fast or slow. Some chemical reactions are very slow and need the help
of substances called catalysts to increase their speed. Catalysts are substances that increase the
rate of a chemical reaction without being altered themselves. So essentially while catalyst
participates in a chemical reaction and increases its speed, it remains unchanged during that
chemical reaction.
Enzymes are biological catalysts that are present in living things to speed up biochemical
reactions. The enzymes are known for their specificity and their sensitivity to changes in pH and
temperature. If any one of these factors changes, the enzyme will not function as normal. There
are a plethora of enzymes that are needed to keep the human body functioning smoothly and the
absence of an enzyme can have serious consequences.
Enzymes are proteins, which are folded in a specific 3-dimensional configuration to allow them to
perform a specific function. If this 3-dimensional structure is destroyed, the enzymes cannot
function properly. Enzymes are present in all living things. During the course of this experiment we
will the studying an enzyme that causes the browning of apples and the various factors that affect
this enzyme.
The enzyme that causes browning in apples is referred to as Polyphenol oxygenase (PPO). Fruits
and vegetables contain many compounds that can act as substrates for this enzyme to generate
the black pigment melanin, causing the formation of black spots on the plant tissue. The reaction
requires the presence oxygen to proceed and the oxidation of the poplyphenol substrates is
manifested as browning.
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There are two types of PPOs – Catechol oxidase and Tyrosinase. Tyrosinase is the more
accomplished enzyme that is capable of doing more complex reactions and can also carry out
some of the same reactions as catechol oxidase. Tyrosinase enzymes are responsible for
producing the main pigment in the human skin, melanin.
Tyrosine is a non-essential amino acid that is used in creating several neurotransmitters like
epinephrine, nor-epinephrine, serotonin and dopamine, which help neurons communicate with
each other and also influence mood. All the proteins in our body including the enzymes are built
out of the same 20 amino acids, one of which is tyrosine. Tyrosine is also heavily involved in
regulation and secretion of the hormones from the adrenal, pituitary and thyroid glands. Last but
not the least, tyrosine is involved in the production of the pigment melanin which is responsible for
hair and skin color. Since tyrosine is involved in pigmentation, obviously it would have a role to play
in hyper-pigmentation (Dark spots or age spots) and so currently there is a lot of research being
done to find substances that will inhibit tyrosinase activity. Shown below is the structure of the
enzyme tyrosine.
MATERIALS
1 large apple
Baking soda– available at any
grocery store
Milk of magnesia (available at
any pharmacy)
water
Stove for heating
Refrigerator for cooling
1 dozen plastic cups
Plastic knife
Vinegar – available at any grocery store
Lemon Juice
PROCEDURE
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Day 1
1. Create a solution of baking soda in water by mixing a teaspoonful of baking soda in
about half a cup of water and stirring it until it dissolves. Add more water if needed, little
by little until it dissolves.
2. Create a solution of milk of magnesia in water by mixing one part of milk of magnesia
with one part of water (the ratio is more important that the total volume). Please ensure
that the solution flows readily. We do not want to end up with a thick slurry.
3. Take 6 disposable cups and label them as follows
Cup 1: Water
Cup 2: Lemon Juice
Cup 3: Vinegar
Cup 4: Baking soda
Cup 5: Milk of magnesia
Cup 6: Lemon juice (again)
Cup 7: Water
Cup 8: Empty
4. Heat some water on the stove until it boils.
5. NOTE: Please do not cut the apple until the water is boiling.
6. Cut the apple into 10 or more slices carefully using a plastic knife making sure you do
not hurt yourself. Place one apple slice on a plate separately and keep an eye on it
while you continue with your experiments. Note how long it takes for the apple slice to
turn brown.
7. After observation drop this into a cup and leave overnight.
8. Drop one of the slices into the boiling water carefully and let it boil for about 5 minutes.
9. Place one apple slice in each of the cups 1-6.
10. Pour approximately 50 mL or one-fourth cup of the appropriate solution into each of the
cups. Please ensure that the apple is coated with the solution thoroughly. You may
have to swirl the contents around to ensure an even coating of the apple slice in each of
the cups.
11. Note the appearance of all the apple slices immediately following the treatment before
you set them aside for a day.
12. Leave cups 1-5 at room temperature for a day.
13. Place cups 6 and 7 in the refrigerator for a day.
14. Place an apple slice in cup 8 and place that in the fridge for a day.
15. Remove the slice of apple that was boiling and let it cool. Take a picture of the slice
after it is removed from the boiling water
16. In addition to making general observations on the boiled slice, also observe how long it
takes for this slice to brown. Observe it for a whole day.
17. Work quickly and carefully. Please ensure you label everything carefully.
.
Day 2
Remove the apples that were refrigerated. Observe all the apple slices and make your
observations in the table below. Notice the color of the apples (white/pink/light brown/dark
brown/black), their texture (dry/slimy/slippery), their appearance (smooth/wrinkled) and any
odors if present. DO NOT TASTE THE APPLES.
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Take a well labeled picture of each apple slice after 24 hours.
You can dispose of the cups and their contents once you have ensured that you have
recorded all the data you need.
OBSERVATIONS
Insert pictures of how each of the apple slices looked after a day. Please ensure that each
of the pictures is well labeled to reflect what treatment was done on the apple. Draw a table
showing how each treatment affected the apple.
Length of time it took for the apple left out on the counter to turn brown = _______
Table 1
(Add more rows if necessary)
Treatment on the
apples
None (Apple left
out on counter)
Boiled apple
Refrigerated
apple
Cup 1
Cup 2
RESULTS AND CONCLUSIONS
Before working on the results determine what kind of substance was present in each cup and what
are its chemical properties? This may help in understanding and explaining the results better?
1. Summarize which of the treatments were successful in slowing down the browning of
apples and which were not?
2. Among with ones that were successful did you notice a trend? What was this trend if any?
Explain your answer.
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3. What effect if any did temperature have on the browning of apples? Why?
4. What did you notice in all the apple slices that were cooled? Did all of them behave the
same? Why? Why not? Explain and elucidate what you think happened?
5. Which apple slice browned the most? Why do you think this was?
6. Which apple slice browned the least? Why do you think this was?
7. What are the factors that inactivate the enzyme?
8. What role would an inhibitor play in this process? What kind of industries would be
interested in developing an inhibitor for tyrosinase?
Uma Swamy
Revised June 2012
Florida International University
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