77 PHYWE Systeme GmbH & Co.

KG D-37070 Gttingen Laboratory Experiments, Biology

LEB 12 Biochemistry
The enzymatic activity of catalase 12.06
Principle and tasks:
To examine the enzymatic decompo-
sition of hydrogen peroxide, a cell
respiratory poison, in the liver. To in-
vestigate the influence of tempera-
ture and pH on the metabolic activi-
ty.
Normal result
Equipment (for the experimental procedure using the Basic-Unit)
Cobra3 Basic Unit 12150.00 1
Power supply, 12 V 12151.99 1
Software Cobra3 - Pressure 14510.61 1
Measuring module, pressure 12103.00 1
Support clamp for small case 02043.10 1
RS232 data cable 14602.00 1
Support base, variable 02001.00 1
Support rod, l = 500 mm, stainless steel 02032.00 1
Bosshead 02043.00 1
Universal clamp 37715.00 1
Magnetic stirrer, mini 47334.93 1
Magnetic stirrer bar, l = 50 mm 46299.03 1
Erlenmeyer flask, narrow neck, PN 29 36424.00 1
Rubber stopper, d = 32/26 mm, 1 hole 39258.01 1
Glass tubes, straight, 80 mm, d = 7 mm, 10 pcs 36701.65 1
Rubber tubing, d = 6 mm, 1 m 39282.00 1
Graduated cylinder, 100 ml 36629.00 1
Mortar with pestle, 150 ml, porcelain 32604.00 1
Strainer, fine mesh, d = 60 mm 40968.00 1
Graduated pipette, 1 ml 36595.00 1
Graduated pipette, 10 ml 36600.00 2
Glass beaker, 250 ml, tall form, DURAN 36004.00 2
Tripod, ring d = 100 mm, h = 180 mm 33299.00 1
Wire gauze 33287.03 1
Bunsen burner, natural gas 32165.05 1
Safety gas tubing, 1 m 39281.10 1
Tubing clip, d = 8...12 mm 40996.01 2
Test tube, 100 mm, d = 12 mm, 1 only 36307.10 1
Glycerol, 99%, 100 ml 30084.10 1
Dropping bottle, plastic, 50 ml 33920.00 1
Hydrogen peroxide, 38%, 250 ml 31710.25 1
Hydrochloric acid, 1 molar, 500 ml 48454.50 1
Sodium hydroxide, 1 molar, 500 ml 48329.50 1
Ice cubes, Chicken liver or pig liver
Distilled water
PC, Windows 95 or higher
What you need:
The enzymatic activity of catalase P4120611
What you can learn about
Enzyme catalase
Decomposition of H
2
O
2
Poisonous by-product of cell
respiration
Influence of temperature and
pH
Result when acidic
Related topics
Enzyme catalase, decomposition of H
2
O
2
, poisonous by-
product of cell respiration, influence of temperature and
pH
Principle and task
To examine the enzymatic decomposition of hydrogen
peroxide, a cell respiratory poison, in the liver. To investi-
gate the influence of temperature and pH on the metabol-
ic activity.
Equipment (for the experimental procedure using the
Basic-Unit)
Cobra3 Basic Unit 12150.00* 1
Power supply, 12 V 12151.99* 1
Software Cobra3 - Pressure 14510.61* 1
Measuring module, pressure 12103.00* 1
Support clamp for small case 02043.10* 1
RS232 data cable 14602.00* 1
Support base, variable 02001.00 1
Support rod, l = 500 mm, stainless steel 02032.00 1
Bosshead 02043.00 1
Universal clamp 37715.00 1
Magnetic stirrer, mini 47334.93 1
Magnetic stirrer bar, l = 50 mm 46299.03 1
Erlenmeyer flask, narrow neck, PN 29 36424.00 1
Rubber stopper, d = 32/26 mm, 1 hole 39258.01 1
Glass tubes, straight, 80 mm,
d = 7 mm, 10 pcs 36701.65 1
Rubber tubing, d = 6 mm, 1 m 39282.00 1
Graduated cylinder, 100 ml 36629.00 1
Mortar with pestle, 150 ml, porcelain 32604.00 1
Strainer, fine mesh, d = 60 mm 40968.00 1
Graduated pipette, 1 ml 36595.00 1
Graduated pipette, 10 ml 36600.00 2
Glass beaker, 250 ml, tall form, DURAN 36004.00 2
Tripod, ring d = 100 mm, h = 180 mm 33299.00 1
Wire gauze 33287.03 1
Bunsen burner, natural gas 32165.05 1
Safety gas tubing, 1 m 39281.10 1
Tubing clip, d = 8...12 mm 40996.01 2
Test tube, 100 mm, d = 12 mm, 1 only 36307.10 1
Glycerol in a dropping bottle, 25 ml 48159.04 1
Hydrogen peroxide, 38%, 250 ml 31710.25 1
Hydrochloric acid, 1 molar, 500 ml 48454.50 1
Sodium hydroxide, 1 molar, 500 ml 48329.50 1
Ice cubes
Chicken liver or pig liver
Distilled water
PC, Windows

Set-up (using the Basic-Unit)

Set up the equipment as shown in Fig. 1.
Fasten the support clamp for a small case to the back
of the Cobra3 Basic Unit
Connect the Cobra3 Basic Unit to the PC with the
data cable
Fix the Cobra3 Basic Unit with plugged-on pressure
module to the support rod so that it is directly above
the Erlenmeyer flask
Ease the glass tube, wetted with a drop of glycerol,
into the rubber stopper and connect it to the pressure
module with a short piece of rubber tubing
Place the Erlenmeyer flask on the magnetic stirrer and
hold it with a universal clamp
Procedure (using the Basic-Unit)
Call up the COBRA3 MEASURE programme in Windows
Enter PRESSURE as measuring instrument
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41206 153
LEB
12.06
The enzymatic activity of catalase
Fig. 1: Experimental set-up using the Basic-Unit
Fig. 2: Measurement parameters (using the Basic-Unit)
Set the measurement parameters (see Fig. 2) and go
to measurement with CONTINUE
Put the chicken liver in the mortar, add a little distilled
water, then use the pestle to crush it and decant the
juice off through the strainer and into a beaker
Experiment 1:
Pour 15 ml of 3% hydrogen peroxide solution (10 ml
of 30% H
2
O
2
solution to 90 ml of distilled water) into
a 100 ml graduated cylinder and make it up to 100 ml
with distilled water
Pour this solution into an Erlenmeyer flask, insert the
magnetic stirrer bar and place the flask on the mag-
netic stirrer
Add 1 ml of liver juice and immediately close the flask
with the rubber stopper
Start the measurement (running time 150 s)
Experiment 2:
Procedure as in Experiment 1, but with the addition of
10 ml of 1 molar sodium hydroxide or 10 ml of 1 molar
hydrochloric acid
Experiment 3:
Procedure as in Experiment 1, but previously pour the
liver juice into a test tube and either heat it for 5 min-
utes in a beaker of boiling water or cool it for 5 min-
utes in a beaker of ice cold water containing ice cubes
Results and evaluation
Experiment 1: The pressure curve increases sharply,
but drops down vertically at 1330 hPa, because the
rubber stopper was blown off of the flask (Fig. 3)
Experiment 2: When sodium hydroxide is added, the
pressure curve increases far less sharply than under
normal conditions (Fig. 4). With hydrochloric acid,
there is no increase in pressure (Fig. 5).
Experiment 3: After a 5 minute heat treatment, the
pressure in the flask remains constant (Fig. 6). After a
5 minute iced water treatment, the pressure increases
as under normal conditions. Here again, there is a ver-
tical pressure drop at 1330 hPa (blown-off stopper)
(Fig. 7).
Catalase is an enzyme, which occurs in humans pre-
dominately in the liver and in erythrocytes. It decom-
poses hydrogen peroxide H
2
O
2
, a poisonous by-
product of cell respiration, to water and oxygen. When
one mixes blood, for example, with H
2
O
2
, the forma-
tion of small bubbles of oxygen can be seen.
Enzymes are pH dependent. Experiment 2 shows that
catalase prefers alkaline conditions. It is not active
under acidic conditions.
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41206 154
LEB
12.06
The enzymatic activity of catalase
Fig. 4: Result when alkaline
Fig. 3: Normal result
Fig. 5: Result when acidic
Fig. 6: Result after heating
Enzymes are composed of proteins. Proteins are
denatured at high temperatures. This is the reason
why there is no pressure increase after the 5 minute
heat treatment in Experiment 3. The enzyme proteins
are destroyed. On the other hand, a cold shock only
temporarily inactivates catalase. When the tempera-
ture increases, the enzyme again acts optimally.
Note
This experiment can also be carried out using the hand-
held measuring instrument pressure 07136.00, pressure
sensor 07136.01, software pressure 14416.61, data cable
07157.01 and boss head 02043.00. In this case, the
entries in the equipment list (Fig. 8) which are marked with
an asterisk are not required.
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41206 155
LEB
12.06
The enzymatic activity of catalase
Fig. 7: Result after cooling
Fig. 8: Experimental set-up using the hand-held measuring instrument pressure
Room for notes
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41206 156
LEB
12.06
The enzymatic activity of catalase