77PHYWE Systeme GmbH & Co. KG · D-37070 Göttingen 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 cellrespiratory 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 1Power supply, 12 V 12151.99 1Software Cobra3 - Pressure 14510.61 1Measuring module, pressure 12103.00 1Support clamp for small case 02043.10 1RS232 data cable 14602.00 1Support base, variable 02001.00 1Support rod, l = 500 mm, stainless steel 02032.00 1Bosshead 02043.00 1Universal clamp 37715.00 1Magnetic stirrer, mini 47334.93 1Magnetic stirrer bar, l = 50 mm 46299.03 1Erlenmeyer flask, narrow neck, PN 29 36424.00 1Rubber stopper, d = 32/26 mm, 1 hole 39258.01 1Glass tubes, straight, 80 mm, d = 7 mm, 10 pcs 36701.65 1Rubber tubing, d = 6 mm, 1 m 39282.00 1Graduated cylinder, 100 ml 36629.00 1Mortar with pestle, 150 ml, porcelain 32604.00 1Strainer, fine mesh, d = 60 mm 40968.00 1Graduated pipette, 1 ml 36595.00 1Graduated pipette, 10 ml 36600.00 2Glass beaker, 250 ml, tall form, DURAN 36004.00 2Tripod, ring d = 100 mm, h = 180 mm 33299.00 1Wire gauze 33287.03 1Bunsen burner, natural gas 32165.05 1Safety gas tubing, 1 m 39281.10 1Tubing clip, d = 8...12 mm 40996.01 2Test tube, 100 mm, d = 12 mm, 1 only 36307.10 1Glycerol, 99%, 100 ml 30084.10 1Dropping bottle, plastic, 50 ml 33920.00 1Hydrogen peroxide, 38%, 250 ml 31710.25 1Hydrochloric acid, 1 molar, 500 ml 48454.50 1Sodium hydroxide, 1 molar, 500 ml 48329.50 1Ice cubes, Chicken liver or pig liverDistilled waterPC, Windows® 95 or higher

What you need:

The enzymatic activity of catalase P4120611

What you can learn about

� Enzyme catalase� Decomposition of H2O2

� Poisonous by-product of cellrespiration

� Influence of temperature andpH

Result when acidic

Related topicsEnzyme catalase, decomposition of H2O2, poisonous by-product of cell respiration, influence of temperature andpH

Principle and taskTo examine the enzymatic decomposition of hydrogenperoxide, 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 theBasic-Unit)Cobra3 Basic Unit 12150.00* 1Power supply, 12 V 12151.99* 1Software Cobra3 - Pressure 14510.61* 1Measuring module, pressure 12103.00* 1Support clamp for small case 02043.10* 1RS232 data cable 14602.00* 1Support base, variable 02001.00 1Support rod, l = 500 mm, stainless steel 02032.00 1Bosshead 02043.00 1Universal clamp 37715.00 1Magnetic stirrer, mini 47334.93 1Magnetic stirrer bar, l = 50 mm 46299.03 1Erlenmeyer flask, narrow neck, PN 29 36424.00 1Rubber stopper, d = 32/26 mm, 1 hole 39258.01 1Glass tubes, straight, 80 mm,d = 7 mm, 10 pcs 36701.65 1Rubber tubing, d = 6 mm, 1 m 39282.00 1Graduated cylinder, 100 ml 36629.00 1

Mortar with pestle, 150 ml, porcelain 32604.00 1Strainer, fine mesh, d = 60 mm 40968.00 1Graduated pipette, 1 ml 36595.00 1Graduated pipette, 10 ml 36600.00 2Glass beaker, 250 ml, tall form, DURAN 36004.00 2Tripod, ring d = 100 mm, h = 180 mm 33299.00 1Wire gauze 33287.03 1Bunsen burner, natural gas 32165.05 1Safety gas tubing, 1 m 39281.10 1Tubing clip, d = 8...12 mm 40996.01 2Test tube, 100 mm, d = 12 mm, 1 only 36307.10 1Glycerol in a dropping bottle, 25 ml 48159.04 1Hydrogen peroxide, 38%, 250 ml 31710.25 1Hydrochloric acid, 1 molar, 500 ml 48454.50 1Sodium hydroxide, 1 molar, 500 ml 48329.50 1Ice cubesChicken liver or pig liverDistilled waterPC, 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 abovethe Erlenmeyer flask

— Ease the glass tube, wetted with a drop of glycerol,into the rubber stopper and connect it to the pressuremodule with a short piece of rubber tubing

— Place the Erlenmeyer flask on the magnetic stirrer andhold 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 Göttingen 41206 153

LEB12.06

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 goto measurement with CONTINUE

— Put the chicken liver in the mortar, add a little distilledwater, then use the pestle to crush it and decant thejuice off through the strainer and into a beakerExperiment 1:

— Pour 15 ml of 3% hydrogen peroxide solution (10 mlof 30% H2O2 solution to 90 ml of distilled water) intoa 100 ml graduated cylinder and make it up to 100 mlwith distilled water

— Pour this solution into an Erlenmeyer flask, insert themagnetic stirrer bar and place the flask on the mag-netic stirrer

— Add 1 ml of liver juice and immediately close the flaskwith the rubber stopper

— Start the measurement (running time 150 s)Experiment 2:

— Procedure as in Experiment 1, but with the addition of10 ml of 1 molar sodium hydroxide or 10 ml of 1 molarhydrochloric acidExperiment 3:

— Procedure as in Experiment 1, but previously pour theliver 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 therubber stopper was blown off of the flask (Fig. 3)

— Experiment 2: When sodium hydroxide is added, thepressure curve increases far less sharply than undernormal conditions (Fig. 4). With hydrochloric acid,there is no increase in pressure (Fig. 5).

— Experiment 3: After a 5 minute heat treatment, thepressure in the flask remains constant (Fig. 6). After a5 minute iced water treatment, the pressure increasesas 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 H2O2, a poisonous by-product of cell respiration, to water and oxygen. Whenone mixes blood, for example, with H2O2, the forma-tion of small bubbles of oxygen can be seen.

— Enzymes are pH dependent. Experiment 2 shows thatcatalase prefers alkaline conditions. It is not activeunder acidic conditions.

Phywe Series of publication • Laboratory Experiments Biology • © PHYWE SYSTEME GMBH & Co. KG • D-37070 Göttingen41206154

LEB12.06

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 aredenatured at high temperatures. This is the reasonwhy there is no pressure increase after the 5 minuteheat treatment in Experiment 3. The enzyme proteinsare destroyed. On the other hand, a cold shock onlytemporarily inactivates catalase. When the tempera-ture increases, the enzyme again acts optimally.

NoteThis experiment can also be carried out using the hand-held measuring instrument pressure 07136.00, pressuresensor 07136.01, software pressure 14416.61, data cable07157.01 and boss head 02043.00. In this case, theentries in the equipment list (Fig. 8) which are marked withan asterisk are not required.

Phywe Series of publication • Laboratory Experiments Biology • © PHYWE SYSTEME GMBH & Co. KG • D-37070 Göttingen 41206 155

LEB12.06

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 Göttingen41206156

LEB12.06

The enzymatic activity of catalase