BIOLOGY EXPERIMENTS ON FILETM
ORGANS AND SYSTEMS • 2.01–1
Enzyme Activity And Temperature
Topic
Effect on temperature on enzyme activity
Introduction
Humans need a constant supply of protein to maintain a healthy body. However,
proteins are large molecules and cannot pass through the wall of the intestine
into the bloodstream. To get proteins into the body, the intestine secretes
powerful enzymes called proteases which can break down the protein into the
much smaller amino acids. These amino acids can then be absorbed through the
intestine wall and used to build human proteins.
Developed black and white photographic film (sometimes called the negative)
consists of two layers – a clear celluloid backing layer covered with a layer of
gelatin. The gelatin layer contains small grains of silver, which make the
developed film look black. Gelatin is a protein and can be broken down by
proteases. When the gelatin is broken down, the silver grains fall away from the
celluloid backing and the film becomes clear. In this experiment, you will use this
change (which is easy to see) to investigate the conditions that affect the rate at
which protease breaks down gelatin.
Time needed
40 minutes
Materials
water baths set at temperatures of 10°C, 20°C, 30°C, 40°C, and 50°C
(with suitable frame for supporting test tubes)
5 Pyrex™ test tubes
developed photographic film (about 2.5 × 3 cm) or two 35 mm negatives
timer or stopwatch
25 ml protease solution (1 – 5% as suggested by the manufacturer’s instructions)
5 ml pipette or syringe
25 ml deionized water
waterproof marker pen
scissors
safety glasses
Safety note
Be careful when using the scissors to cut the film.
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2.01–2 • ORGANS AND SYSTEMS
BIOLOGY EXPERIMENTS ON FILETM
Procedure
Note to teachers:
Because protease can degrade on storage, you should test the protease solution
before the experiment to check that the strength is correct.
1. Cut the developed film into five pieces about 1 cm long and 1 cm wide, or cut
the negatives into quarters, i.e., so the piece of film fits easily into a test tube.
2. Use the waterproof pen to label the five test tubes with the temperatures of the
water baths.
3. Place 5 ml of protease solution and 5 ml of deionized water in each test tube.
Place one tube in each of the water baths (see diagram 1 below).
Pyrex™ test
tube
1
Pyrex™
test
tube
water
at 30°C
30°C
solution
thermostat
control
exposed
negative
film
on/off
switch
frame to hold
tube
lead to electrical outlet
Water bath with close-up of a test tube and film strip
4. Wait for 5 minutes for the solutions in the test tubes to reach the temperature
of the water baths.
5. Drop a strip of film into each tube and start the stopwatch.
6. After 1 minute, look at each piece of film and record its condition
(i.e., whether it is still black or if it has cleared) in the data table on the next
page. You may find it helpful to use a key such as B = black and C = clear.
7. Repeat your observation and recording every minute. Continue for 25 minutes
or until all the strips of film in the tubes have cleared.
Analysis
1. Which strip of film cleared first? What does this tell you about the effect of
temperature on protease activity?
2. The best temperature for protease activity is called the optimum temperature.
You can only get a rough idea of the optimum temperature using these results.
How could you get a more accurate figure using the equipment above but
with a slightly changed procedure?
3. At one temperature, none of the strips of film cleared. Suggest a reason for this
and design an investigation to test your explanation.
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BIOLOGY EXPERIMENTS ON FILETM
ORGANS AND SYSTEMS • 2.01–3
4. Proteases are also used in biological stain removers to digest protein-based
stains like egg and blood. The optimum temperature for the enzymes used in
these stain removers is much higher than the protease you have used. Suggest
a reason why.
Want to know more?
Click here to see what we found.
DATA
TABLE
Film in tube at:
Time
(min)
10°C
20°C
30°C
Film in tube at:
40°C
50°C
Time
(min)
1
14
2
15
3
16
4
17
5
18
6
19
7
20
8
21
9
22
10
23
11
24
12
25
10°C
20°C
30°C
40°C
50°C
13
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or transmittal is copyright protected by the publisher.
8.05 • OUR FINDINGS
BIOLOGY EXPERIMENTS ON FILETM
Organs And Systems
2.01 Enzyme Activity And Temperature
1. If the protease is from a plant or a bacterium, the tube held at 30°C will
probably clear first. If the protease is from an animal, the tube at 40°C may
clear first.
2. Repeat the experiment with tubes and water baths at temperatures clustered
around the observed optimum. For example, if the 30°C tube cleared first,
repeat the experiment with water baths and tubes at 24°C, 28°C, 32°C, and
36°C to obtain a more accurate value for the optimum temperature.
3. Proteases are enzymes and thus have all the properties of enzymes, e.g., they
are denatured by boiling or the presence of heavy metal ions, and work best at
a particular pH and temperature (optimum conditions). If none of the films
cleared, repeat the experiment with a range of pH conditions at 25°C (a likely
optimum temperature). If there are still no clear films at the end of 25
minutes, try the experiment again with a fresh batch of enzyme – the original
source may have been damaged or be out-of-date. (The protease most often
used in school laboratory work is a bacterial protease and is fairly stable
under a range of conditions, but it can degrade on storage and become less
effective than expected.)
4. Biological stain removers make use of proteases to breakdown protein-based
stains such as blood and egg. These proteases come from organisms that live
in very hot environments and thus contain enzymes that are stable at higher
temperatures. This means the proteases can be used at higher temperatures in
washing machines without being denatured. The higher temperatures allow
the clothes to be washed more quickly and help the non-enzyme components
in the stain removers to work better (reactions are faster at higher
temperatures). However, biological stain removers do tend to require lower
washing temperatures than non-biological ones.
2.02 Enzyme Activity And pH
1. Results will vary, but typically, the film strip in tube 1 will clear first.
2. The film strip in tubes 3 and 4 also cleared. This is because the dilute solution
of sodium hydroxide dissolves protein.
3. Tubes 2 and 4 act as “controls” to let you see the effect of changes in pH on
enzyme activity by ruling out changes due to other factors. The result from
tube 4 tells you that the result in tube 3 (which implies that a high pH is good
for protease activity) cannot be trusted because sodium hydroxide by itself
breaks down gelatin.
4. The water bath increases the rate of reaction compared with room
temperature. It would have taken much longer to perform the experiment at
room temperature.
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