Enzymes
You simply could not live without enzymes. Nearly every chemical reaction in your body is
helped along and speeded up by these catalytic molecules. Reactions such as the digestion of
food and the ability of carbon dioxide to dissolve in your blood are greatly accelerated by the
action of enzymes. Without them it would take you years to digest a piece of steak, and decades
to produce enough energy to give you the strength to cut it with a knife and fork.
Enzymes speed up the rate of chemical reactions, and in some cases, allow reactions to occur
which probably would not take place in their absence. They are usually large protein molecules
that have an active site into which the reacting molecules, or substrates, can fit. By getting these
molecules close together, a short-lived enzyme-substrate complex is formed. This eventually
separates, leaving behind the products and an enzyme molecule that is ready to do it all again.
Some enzymes break larger molecules down (catabolism) while others help synthesize the
construction of larger molecules (anabolism).
substrate + enzyme ->
enzyme-substrate complex ->
products + enzyme
In this exercise, you will investigate the action of a digestive enzyme and some of the factors that
influence its ability to work.
Objectives
When you have finished this lab, you should be able to
1. explain how an enzyme works, using terms such as catalyst, activation energy,
substrate, product, active site, denaturation, and inhibitor.
2. describe the effect of temperature and pH on enzyme action.
3. recognize most enzymes by their ‘–ase’ ending.
4. interpret the action of some enzymes by their name.
5. define and use intelligently all words that appear in boldface print.
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The Experimental Model
A piece of bread has a large amount of energy stored in it as starch. As soon as you put a
piece in your mouth and begin to chew, the enzyme amylase begins to break it down into its
component parts, simpler sugars. We will use the amylase enzyme system as the model that we
can manipulate.
starch + amylase ->
E-S complex
->
sugars +
amylase
It is easy to determine whether or not the reaction has occurred – we have performed tests for
the presence of simple sugars and starch in an earlier lab. We will use these same tests
(Benedict’s test for sugars and iodine test for starch) in this exercise.
Collection of the enzyme
Procedure: Work in groups of four.
Two members of your group will volunteer to contribute their saliva as a source of the enzyme
amylase.
1. Rinse your mouth with distilled water. Chew on a piece of paraffin or sugarless gum to
induce production of saliva.
2. Collect about 25 mL of saliva (do not include foam in your measurement).
3. Mix the two saliva samples in a small clean beaker to make a total of about 50 mL.
4. Add an equal amount of distilled water (about 50 mL), and mix the contents of the
container.
5. Put several thicknesses of cheesecloth in a funnel and pour the mixture of saliva through
it into a beaker to remove the foam and mucus. This will be the stock solution of the
enzyme (amylase) you will use throughout the rest of the exercise.
6. If more enzyme is needed, do it again!
Effect of Temperature on Enzyme Activity
All chemical reactions are strongly effected by temperature. Generally speaking, cold chemicals
react slowly, while warm reactions proceed much more quickly. You will test the activity of
amylase at several different temperatures.
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Procedure:
1. Label four test tubes from 1 to 4 and place them in a rack.
2. Using the pipettes, add 5 mL of amylase stock solution to each tube.
3. Place the tubes in the temperature designated below. Keep the tubes at these
temperatures for at least 15 minutes (use a timer) to allow for the solution to reach the
desired temperature.
Tube 1: 4oC
Tube 3: 100oC
Tube 2: 37oC
Tube 4: Control: room temperature (@ 20oC)
4. Add 5 mL of the 1% starch solution to each tube (remember to shake the stock bottle),
shake the mixtures gently and return them to their water baths to incubate for 5 minutes.
This allows the entire mixture to reach the temperature of the water bath.
5. Remove a small sample from each tube; place each into a labeled separate depression on
a spot plate and test IMMEDIATELY for the presence of starch by adding 3 drops of
iodine reagent. Remember, the presence of starch will be shown by a dark color. If
starch is present, digestion of starch has not occurred, or is not complete. If starch is
absent, assume that it has all been ‘digested’ into its component sugars.
6. Record your results in Table 1. Indicate whether or not starch was hydrolyzed
(‘digested’) with a yes or no in the last column. (If starch has been digested, you will
have a negative result for the starch test).
7. To test the assumption that all the starch was converted to simple sugars, take 3 mL of a
solution which showed no starch (do not use the control). Add an equal amount of
Benedict’s solution. Warm it in the boiling water bath. If sugar (the product of the
digestion of starch) is present, you should see a color change from blue (the color of
Benedict’s solution) to yellow, orange, green or red. Record your results in Table 1.
8. Wash (with soap and a brush) and rinse the tubes used.
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Table 1. Results of the effect of temperature on enzyme activity.
Tube #
Temperature
1
4oC
2
37oC
3
100oC
4
23oC
Color After Addition of Iodine
Starch present? (yes or no)
Effects of pH on amylase activity.
Amylase begins work on starch in your mouth where the pH is about 7. But when you
swallow, the mixture moves to a pH of 2 in your stomach. The next stop is the small intestine
where the pH is about 8. How well does an enzyme work in these different conditions?
Procedure:
1. Number four test tubes and place them in the rack.
2. Add 3 mL of amylase to each tube.
3. Add the following substances to the tubes as indicated:
Tube 1: 3 mL of buffer solution, pH 4
Tube 2: 3 mL of buffer solution, pH 7
Tube 3: 3 mL of buffer solution, pH 10
Tube 4: 3 mL of distilled water, pH 7
4. Shake and mix the contents and place them in a 37oC water bath for 10 minutes.
5. Add 3 mL of 1% starch solution to each tube, mix, and immediately test with iodine the
contents of each tube for the presence of starch as describe earlier.
6. Continue to incubate the preparations at 37oC and take additional samples at 15 and 30
minutes. Test for the presence of starch as before.
7. Wash and rinse the tubes.
8. Record your results of the tests for starch hydrolysis of substances at different pHs in
Table 2. Indicate whether or not starch was hydrolyzed (‘digested’) with a yes or no in
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the last column. (If starch has been digested, you will have had a negative result for the
starch test – no color change.)
Table 2. Results of the effect of pH on enzyme activity.
Tube #
pH of Mixture
Starch Present? (yes or no)
Immediate
1
buffer, pH 4
2
buffer, pH 7
3
buffer, pH 10
4
dH20, pH 7
15 min
30 min
Summary: Write a brief paragraph summarizing the effect of temperature, pH, and substrate
concentration on the effectiveness of an enzyme.
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