Lab Exercise 6 - Enzymes
Rennin, Urease, and Catalase
Introduction:
Enzymes are chemicals within living units that act in the same manner as catalysts in non-living
systems. In non-living units, the catalysts are usually inorganic, while in living units, enzymes are
more often organic substances. That is to say that catalysts and enzymes act to speed the rate of
chemical reactions. A certain amount of energy is required to start any chemical reaction; this is termed
“Energy of Activation”. Enzymes and catalysts act to lower the activation energy requirements of
chemical reactions. Enzymes are proteins, and thus are composed of amino acids.
Enzymes react with other chemical molecules called substrates. The reaction of an enzyme with a
particular substrate is determined by the geometric shape of both the enzyme and the substrate. These
shapes are specific for a particular substrate. In order to interact, the shapes of these two must be
compatible. The enzymes have an area called the active site; this is where the substrate will interact
with the enzyme. When the enzyme is in contact with the substrate, this union is termed an enzymesubstrate complex. The generalized formula for this is as follows:
E (enzyme) +
S (substrate)  ES (enzyme/substrate)  E (enzyme) + P (product)
The enzyme (as is true for all catalysts) is not chemically changed by this union. The substrate however,
may be joined with another chemical attached to a neighboring site on the enzyme and produce a
product, or a substrate might attach to an enzyme and be subsequently broken into two or more parts
producing multiple products. These products will be chemically different from either of the original
substrates.
The shape of enzymes may be altered by various means; this alteration is termed denaturation. There
are optimal conditions of temperature and pH for enzyme activity. Outside of their optimal
conditions, the shape of the enzymes will be altered (denatured). Another means of limiting enzyme
activity is allosteric regulation and feed-back mechanism
Objectives:
1.
2.
3.
4.
5.
6.
Define enzyme and substrate
Describe the generalized reaction for an enzyme and substrate.
Explain the role played by enzymes in living organisms.
Predict the effect of temperature, enzyme concentration and pH on enzyme activity.
Understand the medium in which enzymes and inorganic catalyst are more effective.
Define all bold print and underlined words in the reading.
Rennin:
Rennin is an enzyme found in the lining of the stomach of milk fed mammals. Its main function is to
solidify milk so that it may remain in the stomach long enough to be digested by other protein
digesting enzymes.
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Urease:
Urease is an enzyme that converts urea and water to ammonia and carbon dioxide
Urease
H4CN2O + H2O--------------------- CO2
Urea
water
+
carbon dioxide
2 NH3
ammonia
In the lab activity using urease, the pH of the urea solution has been adjusted to an acid and a pH
indicator phenolphthalein, has been added. As the reaction proceeds, the pH indicator
phenolphthalein turns from colorless to various shades of pink, indicating that the solution is now
basic.
Catalase:
Each enzyme has a pH at which the speed of the reaction is optimum. Any other pH effects
hydrogen bonding and the structure of the enzyme, which will result in reduced activity. Catalase is
an enzyme present in cells that speeds up the break down of hydrogen peroxide (H2O2) a toxic
chemical, to water and oxygen.
Catalase
2H2O2------------------------ 2H2O
Hydrogen peroxide
water
+
O2
oxygen
In a cell, oxygen released by the above reaction is used for other cellular processes, but, when the
reaction occurs in a test tube, oxygen gas bubbles to the surface, producing a layer of foam on the
surface. The amount of foam produced and the speed with which it forms are measures of catalase
activity.
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Lab Activity 6 - Enzymes
Rennin, Urease, and Catalase
Required Materials:
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Marker pencil
Ruler
12 Test tubes
Test Tube Holder
2% Milk
12 droppers
20% Rennin
Ice cubes
3 Beakers
Hot Plate
Tongs
Water Bottle
20% Urea solution containing 5 drops of phenolphthalein
20% Urease Solution
Macerated Potato
Spatula
Hydrogen peroxide
1% HCl
0.1M NaOH.
Liver extract
MnO2 crystals
2 Thermometers
Assignment 1
Effect of temperature on enzyme Rennin:
Procedure:
Using a marker pencil and a ruler, label and mark three clean test tubes at 2cm. Fill and
treat as follows:
Tube 1: Fill to the 2cm mark with refrigerated 2% milk. Add 10 drops of refrigerated
rennin and place in an ice bath.
Tube 2: Fill to the 2cm mark with warmed 2% milk. Add 10 drops of warmed rennin and
place in a water bath at 37oC. Before you do this, you must warm the rennin in a separate test
tube and the milk in a separate test tube. Place both test tubes in a beaker of warm water to
gently heat. Once both tubes are warmed, add the warmed rennin to the warmed milk and place
the test tube in the warm water bath.
Tube 3: Fill to the 2cm mark with warm 2% milk. Add 10 drops of boiled rennin and place
in a hot water bath at 80o to 100oC. Before you do this, you must boil the rennin in a separate
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test tube and warm the milk in a separate test tube. Then the boiled rennin can be added to the
warm milk and that test tube is placed in a hot water bath.
After 30 minutes, examine the tubes and answer questions a, b and c in the Lab Report portion
of this exercise (assignment 1).
Assignment 2
Effect of time and concentration on enzyme Urease
Procedure:
Take 3 test tubes. Label and mark all 3 test tubes at 4 cm and fill to this mark with urea solution.
Tube 1: This tube will contain only 4 cm of urea solution. Record the color of the solution in
the appropriate table of the Lab Report portion of this exercise.
Tube 2: Add 5 drops of urease solution to the urea.
Record your results in the appropriate table of the Lab Report portion of this exercise, noting
the time at five minute intervals until changes in color have occurred.
Tube 3: Add 10 drops of urease solution to the urea.
Record your results in the appropriate table of the Lab Report portion of this exercise, noting
the time at five minute intervals until changes in color have occurred.
Assignment 3
Effects of pH (both acidic and basic) on the enzyme Catalase.
The source of catalase for this experiment will be potato cells.
Procedure:
Label and mark the test tubes 1, 2 and 3. Mark each tube at 2cm and 6cm.
Macerate 3 small pieces of potato with water and a pinch of sand using a mortar and pestle.
Fill as follows:
Tube 1: Fill to the 2cm mark with water. Add 2 scoops of macerated potato Wait 3 minutes. Add
hydrogen peroxide (H2O2) to the 6cm mark. Measure the height of foam layer produced (from the
top of the liquid to the top of the foam layer) and record in the appropriate table.
Tube 2: Fill to the 2cm mark with HCl. Add 2 scoops of macerated potato Wait 3 minutes. Add
hydrogen peroxide (H2O2) to 6cm mark. Measure the height of foam layer produced (from the top
of the liquid to the top of the foam layer) and record in the appropriate table.
Tube 3: Fill to the 2cm mark with NaOH. Add 2 scoops of macerated potato. Wait 3 minutes.
Add hydrogen peroxide (H2O2) to the 6cm mark. Measure the height of foam layer produced (from
the top of the liquid to the top of the foam layer) and record in the appropriate table.
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Caution: HCI is a strong acid and NaOH is a strong caustic base. Exercise care in using these
chemicals. Follow your instructor’s direction for disposal of these tubes. If any acid or base should spill
on your skin, rinse immediately with clean, cool water.
Assignment 4
Action of MnO2 (Manganese dioxide), an inorganic catalyst, and catalase from liver cells
Take 2 clean test tubes and number them 1 and 2.
Fill test tube 1 and 2 as follows:
Tube 1: Add a spatula of MnO2 to the test tube and slowly add one dropper (1 ml) of H2O2 to the
same test tube. After 1 minute measure the thickness of the foam layer (from the top of the liquid to
the top of the foam layer) in millimeters (mm) and record in the appropriate table.
Tube 2: Add 1 dropper full (1ml) of liver extract to the test tube. Slowly add 1 dropper (1ml) of
H2O2 to the same test tube. After 1 minute measure the thickness of the foam layer (from the top of
the liquid to the top of the foam layer) in millimeters (mm) and record in the appropriate table.
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Lab Report 6 - Enzymes
Rennin, Urease, and Catalase
Name: ________________________________
Date:
____________________________
Class Index: ___________________________
Instructor: ____________________________
Before you begin filling out this lab report you must read Exercise 6 - Enzymes in your lab manual. Complete
Assignments 1-5 below. You can use your Lab Manual results and Textbook to complete the information
below.
Assignment 1
Effect of Temperature on Enzyme - Experiment with Enzyme Rennin
a) What is the effect of cold temperature (test tube A) on the activity of the enzyme rennin?
______________________________________________________________________________
b) At what relative temperature cold (tube A), warm (tube B), or boiling (tube C) does the enzyme
rennin solidify milk? ____________________________________________________________
c) What happens to the enzyme rennin when the temperature is raised to the boiling point (test tube
C)? ___________________________________________________________________________
d) According to your readings, where would you find the enzyme rennin?
_________________________________________________________________________________
e) What is the function of enzyme rennin?
_________________________________________________________________________________
e) According to your readings, why is it important for rennin to solidify milk?
_____________________________________________________________________
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Assignment 2
Effect of Time and Concentration on Enzyme Activity - Experiment with Enzyme Urease
Color of urea solution after the following minutes:
Tube #
Content
0 minutes
1
Urea solution
2
Urea solution + 5
drops of urease
Urea solution +
10 drops of
urease
3
5 minutes
10 minutes
15 minutes
20 minutes
a) What is the relationship between enzyme concentration and the rate of the reaction?
______________________________________________________________________________
b) What is the relationship between time and urease activity?
______________________________________________________________________________
Assignment 3
Effect of pH on Enzyme Activity - Experiment with Catalase from potato extract
Tube #
Content
pH
Height of foam
layer in mm
Neutral
________mm
Acidic
_________mm
Basic
_________mm
Distilled water +
1
Potato extract +
H2O2
2
HCl + Potato
extract + H2O2
3
NaOH + Potato
extract + H2O2
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Explanation of Enzyme Catalase
activity with respect to pH.
a) What was the substrate in this reaction?
______________________________________________________________________________
b) What were the products of this reaction?
______________________________________________________________________________
c) At what pH was the foam formation greatest (“optimum" pH)?
______________________________________________________________________________
d) What is the effect of too low or too high pH on enzyme activity?
______________________________________________________________________________
Assignment 4
Action of Manganese dioxide (MnO2), an inorganic catalyst, and an organic catalyst (enzyme from
liver cells)
Tube #
Content
Thickness of foam layer in mm
1
H2O2 + MnO2
_________mm
2
H2O2 + liver extract
_________mm
a) The foam layer of small bubbles results from the production of which gas?
___________________________________________________________
b) Which gives the greater reaction, MnO2 or catalase? _____________________________________
c) What was the source of catalase in this experiment?
________________________________________________________________________________
d) Would catalase be found in your body? Yes/No
If you answered yes, where would it be found?
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