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Biological Processes
Lab 4
Enzymes
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Lab 4: Enzymes
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Introduc on
Enzymes are specialized proteins that serve as biological catalysts to decrease the ac va on energy
normally needed for a reac on to occur. This means the reac on rate is up to millions of mes faster
than it would be without the enzyme. Most biochemical reac ons require enzymes for them to occur
at fast enough rates to be useful. Typical nomenclature for enzymes follows the pa ern using the
name of the substrate or the chemical reac on it catalyzes, and ends with “ ase”, e.g. catalase, amyl
ase. (In other words, any me you see a word end in “ase” you know it is an enzyme).
Enzymes are extremely selec ve, and are o en described as having a “lock and key” t (Figure 1).
Their shape determines which substrates they bind and interact with. The ac va on site
Figure 1: The speci city of enzymes is controlled by their lock and key t with a spe
ci c substrate.
Concepts to explore:
Enzymes
Selec vity
Catalysts
Ac va on energy
Ac va on site
Reac on rates
Concepts to explore:
Ac vators
Inhibitors
Lab 4: Enzymes
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is the pocket where the substrate a aches and where the reac on occurs. A er the enzyme/substrate
complex forms and catalysis occurs, the “new” substrate is released from the ac ve site, and the en
zyme can repeat the process. Enzymes levels are not reduced or altered during the reac on. This
means they are e cient and can be used repeatedly.
Enzymes determine the rate at which the reac on occurs (not how it occurs). Their ac vity is a ected
by temperature, pH, enzyme and substrate concentra on, and other chemicals that may be present
(such as salts, which can change the protein structure).
Varia ons in temperature and alkalinity can change the shape of the proteins, such as enzymes, which
makes them inac ve (they can no longer bind to their substrate). The pH can alter charge of the pro
tein, once again changing its shape and rendering them inac ve.
The concentra ons of both the enzyme and substrate determine the reac on rate (Figure 2). Remem
ber that high reac on rates do not always translate into rapid me of comple on (it also depends on
the amount of substrate!).
Ac vators are chemicals that bind to the ac ve site of the en
zyme and help it to bind to the substrate. They are some mes
called cofactors or organic coenzymes.
Inhibitors are chemicals that interfere with the binding of the
substrate to the enzyme. There are two types:
Figure 2: Substrate Satura on Curve
Many drugs and poisons are en
zyme inhibitors. For example, aspi
rin inhibits an enzyme that leads to
in amma on.
Lab 4: Enzymes
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Compe ve (can be replaced by the substrate)
Non compe ve (not removed by the substrate)
Normal cellular processes produce toxic substances (waste) such as hydrogen peroxide and free radi
cals that if not eliminated, will kill the cell. Luckily, yeast and other organisms (including humans) have
an enzyme called catalase that breaks down hydrogen peroxide into oxygen and water, both harmless
to cells.
Experiment 1: E ect of enzyme concentra on
Yeast cells contain catalase. The e ect of catalase can be seen when yeast is combined with hydrogen
peroxide (Catalase: 2H2O2 › 2 H2O + O2). In this lab you will examine the e ects of enzyme (catalase)
concentra on based on the amount of oxygen produced.
Procedure
1. Label three test tubes as 1, 2, and 3
with a permanent marker.
2. Fill each tube with 10 mL hydrogen
peroxide.
3. Label three beaker as A, B, and C.
4. Add 1/2 teaspoon yeast (1 g.) to 100
mL of warm water (30 35 °C) in
Beaker A. Mix well by pipe ng.
Materials
Yeast
Measuring Spoon
3 Test tubes
Test tube rack
3 100 mL Beakers
Hydrogen peroxide
10 mL Graduated cylinder
Permanent marker
Ruler
String*
3 Balloons
Watch*
*You must provide
Figure 3: When catalsae is added to hydrogen peroxide, oxy
gen is released.
Lab 4: Enzymes
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5. Make a serial dilu on of yeast solu on. To do this, measure 10 mL of the yeast solu on from
Beaker A and transfer it to Beaker B. Add 90 mL warm water (30 35 °C) to Beaker B. Mix well
by pipe ng.
6. Measure 10 mL of the yeast solu on from Beaker B and transfer it to Beaker C. Add 90 mL
warm water (30 35 °C) to Beaker C. Mix well by pipe ng.
7. Measure and pour 5 mL from Beaker A into the rst test tube.
8. Quickly a ach a balloon to the top of the test tube so that it will ll with the oxygen produced
by the enzyme reac on. It is important to execute this step quickly so that every bit of gas pro
duced will be captured.
9. Swirl each tube to mix, and wait one minute.
10. A er one minute has passed, wrap the string around the center of the balloon to measure the
circumference. Measure the length of string with a ruler. Record the length in Table 1 below.
11. Repeat step 10 a er two more minutes have passed (three minutes total from the start of the
reac on); and again a er two more minutes have passed ( ve minutes total from the start of
the reac on). Record all data in Table 1.
12. If the reac on has not nished, con nue to monitor how long it takes for the reac on to com
plete, and measure the nal balloon circumference.
13. Repeat steps 7 12 for the remaining test tubes (use beaker B for test tube 2 and beaker C for
test tube 3).
Table 1: E ect of enzyme concentra on on the produc on of gas
Ques ons
1. What is the enzyme in this experiment? What is the substrate?
Tube Amount
of yeast
Circumference
(cm)
A er 1 minute
1 0.05 g
2 0.005 g
3 0.0005 g
Circumference (cm)
A er 3 minutes
Circumference (cm)
A er 5 minutes
Time Required
to Complete
Final
Circumference (cm)
Lab 4: Enzymes
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2. Did you no ce a di erence in the rate of reac on in the tubes with di erent concentra ons of
enzymes? Why or why not?
3. What was the e ect of using less enzyme on your experiment?
4. Do you expect more enzyme ac vity if the substrate concentra on is increased or decreased?
Draw a graph to illustrate this rela onship.
5. Hydrogen peroxide is toxic to cells, yet is a common byproduct of the reac ons that occur in
side the body. How can this compound be changed to become non toxic (Hint: Look at the
chemical formula of hydrogen peroxide)?
Experiment 2: E ect of temperature on enzyme ac vity
This experiment looks at the e ect of temperature on enzyme ac vity.
Procedure
1. With a permanent marker, label the test tubes as 1, 2, 3, and 4. Place the test tubes in the test
tube rack for support.
Materials
Yeast
Measuring spoon
4 Test tubes
40 mL Hydrogen peroxide, H2O2
10 mL Graduated cylinder
4 Balloons
2 Water bath containers*
Pot for boiling water*
Stove top*
Hot Pad*
4 Microwave safe cups*
Permanent marker
Test tube rack
Ruler
String*
Watch*
Thermometer
*You must provide
Lab 4: Enzymes
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2. Use the 10 mL graduated cylinder to measure and pour 10 mL of hydrogen peroxide into each
test tube.
3. Fill a pot with approximately 2 3 inches of water and place it on the stove (turned to medium
high se ng). The water should come to a boil (approximately 100 °C).
4. While the water is hea ng, place each tube in separate, microwave safe cups (or beakers).
5. Gather two containers that can be used as hot water baths. Each container should be wide
enough to t the cup with the test tube in it.
6. Pour 2 3 cups of water into a microwave safe container and heat the water un l it has reached
approximately 85 °C (use the thermometer to monitor this). Pour this water into the rst hot
water bath.
7. Using a hot pad, pour the boiling water from the stove into the second hot water bath.
8. Immediate place the cup holding test tube 1 into the boiling hot water bath and the cup hold
ing tube 2 into the hot (but not boiling) water bath. Keep test tube 3 at room temperature, and
place the cup with test tube 4 in the refrigerator. You may need to add weight (e.g., coins, mar
bles, rocks, etc.) to the cups going into the water baths to keep them from pping over into the
water.
9. Record the ini al temperatures of each condi on in the table below. Let tubes sit for approxi
mately 15 minutes, and record the nal temperature.
10. A er the elapsed me, remove the tubes from their respec ve environments.
11. Add 1/4 tsp. of yeast to the refrigerated test tube.
12. Quickly a ach a balloon to the top of the test tube so that it lls with the oxygen produced
from the enzyme reac on occurring in the tube. It is important to execute this step quickly so
that every bit of gas produced is captured.
13. Swirl the tube to mix, and wait one minute.
14. A er one minute has passed, wrap the string around the center of the balloon to measure the
circumference. Measure the length of string with a ruler. Record the length in Table 2 below.
15. Repeat step 14 a er two more minutes have passed (three minutes total from the start of the
reac on); and again a er two more minutes have passed ( ve minutes total from the start of
the reac on). Record all data in Table 2.
16. If the reac on has not nished, con nue to monitor how long it takes for the reac on to com
plete, and measure the nal balloon circumference.
17. Repeat steps 11 16 for the remaining three test tubes.
Lab 4: Enzymes
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Table 2: E ect of temperature on the produc on of gas
Ques ons
1. What is the enzyme in this experiment? What is the substrate?
2. How does temperature a ect enzyme func on?
3. Do plants and animals have an enzyme that breaks down hydrogen peroxide? How could you
test this?
4. How did the boiling water a ect the overall reac on?
5. How can enzyme ac vity be increased?
6. Design an experiment to determine the op mal temperature for enzyme func on, complete
with controls. Where would you nd the enzymes for this experiment? What substrate would
you use?
7. Draw a graph of balloon diameter vs. temperature. What is the correla on?
Tube
Ini al
Temp. °C
Circumference
(cm) A er 1
minute
Refrigerator
Room temperature
Hot water (~85 °C)
Boiling Water (~100 °C)
Final
Temp. °C
Circumference
(cm) A er 3
minutes
Circumference
(cm) A er 5
minutes
Final
Circumference
(cm)
Time Required
to Complete
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We have discussed the details of cell structure and cell function, and now we need to look at how work is done in the cells. Each individual cell contains a myriad of different enzymes with many different jobs to do. Interestingly, enzymes are specialized workers who are destined by the DNA of a given cell to perform one specific duty. All cells continually make new enzymes, whenever they are needed.
Enzymes are one group of proteins; as we know there are very many different kinds of proteins. The building blocks of enzymes are amino acids, small molecules that the body can make with a few exceptions. There are eight so-called essential amino acids for humans, which we cannot synthesize on our own; they must come from the food we eat. If we don’t eat the right kinds of food, we may be short of some amino acids. This is not a good situation, because we then cannot make the proper enzymes that we need for good health. ‘Good health’ here means proper functioning of all parts of the body.
Our specialized workers – enzymes – help to speed up biochemical reactions. They put so-called substrates together, and break them apart. Enzymes are frequently named after the specific substrate they work with; their names always end in –ase. For example, the enzyme that breaks down milk sugar, which is lactose, is called lactase. Amylase is an enzyme in the saliva that begins the process of breaking down carbohydrates in the mouth to smaller carbohydrates. As you know, small carbohydrates (sugars) have a sweet taste; complex carbohydrates (e.g. flour) do not. Try to chew a piece of plain bread (nothing on it!) for a while and discover what happens! Incidentally, whenever you see the ending –ose, you will know that this is a sugar, such as glucose, fructose, galactose and sucrose.
Enzymes can only do their many jobs properly within specific environmental conditions that have a very limited range of pH, temperature, and other chemical factors. Therefore, if the internal part of a body becomes too alkaline or acid, too hot or cold, or if there are other chemical malfunctions, the enzymes will not work and may break down themselves. You can imagine what a chaotic situation may follow!
Enzymes don’t work in isolation, and they actually have helpers, so-called co-enzymes (or co-factors), that literally help enzymes bind to their specific substrates. It’s a little like a person who helps you get into the saddle on a horse’ back. You may have wondered how vitamins work, and that is what they do: Help enzymes!
There are also ‘inhibitors’ that interfere with enzyme functions; one group is called ‘competitive’ (the substrate may replace them), and one group is called ‘non-competitive’ (which is not replaced). Poisons, such as cyanide, and many drugs are enzyme inhibitors.
This week, you will be working with enzyme functions. Before you begin your work, please read the Introduction very carefully. You will be doing two experiments for which your Lab Kit is required. You will need a few additional items: A stove top to boil water, hot pads (= pot holders), 4 microwave safe cups, a pot for boiling water in, 2 water bath containers, some string and a watch.
Experiment 1: Effect of Enzyme Concentration
You will be working with yeast, which contains the enzyme catalase, and hydrogen peroxide. You will test how the amount of catalase concentration affects the reaction, which is oxygen production.
PLEASE read the introduction first so that these experiments will make sense, and then, carefully, read and follow the instructions. It would be a really good idea, if you had an assistant for this experiment, to help you with measuring the balloons. If there is a lot of enzyme activity, you must be careful holding on to your balloon. Keep taking good notes during the experiment to complete Table 1 and then answer the five questions carefully.
Experiment 2: Effect of Temperature on Enzyme Activity
Again, please follow the instructions carefully step by step, and complete Table 2. Then take your time answering the questions. It is good to be creative, but it must make sense in terms of testing enzyme functions.
Also, when you draw your graphs for each experiment, please remember that you are looking at relationships between two variables!
Take your time with each individual experiment and don’t try to do them in one sitting. The more carefully you will handle each experiment, the better the outcome will be. I recommend that you invite a family member or friend to help you at least with the balloon exercises – let them be your co-enzymes!
Have fun with these experiments!
UMUC Biology 102/103
Lab 4: Enzymes
INSTRUCTIONS:
· On your own and without assistance, complete this Lab 4 Answer Form electronically and submit it via the Assignments Folder by the date listed on your Course Schedule (under Syllabus).
· To conduct your laboratory exercises, use the Laboratory Manual that is available in the WebTycho classroom (Reserved Reading or provided by your instructor) or at the eScience Labs Student Portal. Laboratory exercises on your CD may not be updated.
· Save your Lab4AnswerForm in the following format: LastName_Lab4 (e.g., Smith_Lab4). .
· You should submit your document in a Word ( or x) or Rich Text Format (.rtf) for best compatibility.
Experiment 1: Effect of Enzyme Concentration
Table 1: Effect of Enzyme Concentration on the Production of Gas
Tube |
Amount of yeast |
Balloon circumference (cm) After 1 minute |
Balloon circumference (cm) After 3 minutes |
Balloon circumference (cm) After 5 minutes |
Final Circumference (cm) |
Time Required to Complete |
|||
1 |
0.05 g |
||||||||
2 |
0.005 g |
||||||||
3 |
0.0005 g |
Questions
1. What is the enzyme in this experiment? What is the substrate?
2. Did you notice a difference in the rate of reaction in the tubes with different concentrations of enzymes? Why or why not?
3. What was the effect of using less enzyme on your experiment?
4. Do you expect more enzyme activity if the substrate concentration is increased or decreased? Draw a graph to illustrate this relationship.
5. Hydrogen peroxide is toxic to cells, yet is a common byproduct of the reactions that occur inside the body. How cant his compound be changed to become non-toxic (Hint: Look at the chemical formula of hydrogen peroxide)?
Experiment 2: Effect of Temperature on Enzyme Activity
Table 2: Effect of Temperature on the Production of Gas
Initial Temp. (°C) |
Final Temp. (°C) |
Circumference (cm) After 1 minute |
Circumference (cm) After 3 minutes |
Circumference (cm) After 5 minutes |
Refrigerator |
||||
Room Temperature |
||||
Hot Water (~85 °C) |
||||
Boiling Water (~100 °C) |
Questions
1. What is the enzyme in this experiment? What is the substrate?
2. How does temperature affect enzyme function?
3. Do plants and animals have an enzyme that breaks down hydrogen peroxide? How could you test this?
4. How did the boiling water affect the overall reaction?
5. How can enzyme activity be increased?
6. Design an experiment to determine the optimal temperature for enzyme function, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use?
7. Draw a graph using balloon diameter vs. temperature. What is the correlation?
TYPE YOUR FULL NAME: