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Computer-Interfaced Experiments - Volume Measurement

Enzyme Kinetics
Enzymatic Decomposition of Hydrogen Peroxide

Objectives: Dependance of Reaction Rate on Temperature and on Concentration of Enzyme or Substrate, First Order Reaction

Peter Keusch


Datalogging using the Program CASSY ® Lab and the Analog-Digital-Converter Sensor-CASSY - LEYBOLD DIDACTIC

German version





Chemicals:
hydrogen peroxide 30 %
catalase from the bovine liver, crystalline suspension in water  (Merck)


Apparatus and glass wares:
magnetic stirrer hotplate
2 magnetic stirring bars
stirring bar remover
crystallizing dish d = 190 mm, h = 90 mm  (for water bath)
100 mL round bottom flask with center neck 29/32 and 2 angled side necks 14/23
pressure equalizing dropping funnel, 14
adapter connecting reducing, 29
contact thermometer
thermometer 0 - 50 °C (resolution: 0.1 °C)
gas syringe 100 mL
micropipette
volumetric pipette 3 mL
volumetric pipettes 30 mL
2 pipette bulbs
motion transducer
silicone tubing
silicone cord
weight
path cords
disposal container


Hazards and safety precautions:

Hydrogenperoxide 30 % is toxic, corrosive - can cause serious burns. Eye contact can cause serious injury, possibly blindness. Harmful by inhalation, ingestion and skin contact.

Safety goggles and gloves must be worn, good ventilation required.


Theoretical background:

Hydrogen peroxide is decomposed by the anzyme catalase to water and oxygen:

equation

The enzymatic decomposition of H2O2 can be followed by measuring the volume of oxygen produced.







Volume measurement

Whenever a gas is produced in a chemical reaction, the best way of determining the reaction rate is to measure the volume of gas evolved at different times. Gas syringes are commonly used to measure gas volumes (Fig. 1). The plunger of the syringe moves outward as gas fills it. The reaction is complete when syringe no longer moves.
The plunger is moistened with graphite lubricant. In order to compensate the friction resistance, a weight acting contra the friction force is fastened at the end of the plunger.

The motion of the plunger is transfered by means of a silicone cord to the potentiometer shaft. In this manner, the linear movement is translated into a rotary movement and converted into a resistance change. The change in resistance is converted to voltage   (Fig. 1 ).  The measured output voltage is proportional to the distance moved by the plunger (see Catalyzed Decomposition of Hydrogen Peroxide using Chromate / Dichromate).

apparatus
Fig. 1: Experiment set-up



Matching of the program CASSY-Lab

The motion transducer connected to the Input A of the interface is supplied with 5 volts direct current.

Via  F5  the window   ‘Settings'  is to be opened, in which the register card  Cassy   is selected. Input A  appears highlighted and is to be clicked. The window  ‘Sensor Input Settings’  opens. Under   Quantity Voltage UA1   is selected and under  Meas.Range  V3 V .. 3 V . The  Instantaneous Values  are to be activated. Clicking the field  Correct, the window   Correct Measured Values  is opened. In order to calibrate the measurement, the plunger of the gas syringe is set to  0 mL. It is to make sure that the variable resistor of the motion transducer is adjusted anti-clockwise at the stop position. Now the first target value  0  V  is entered and  Correct Offset  is clicked. Afterwards the plunger is set to the  100 mL  mark on the syringe barrel. Hence, the internal resistance of the motion transducer is changed. The second target value  1 V  is entered and  Correct Factor  is clicked. Now the window can be closed. The calibration is complete. Also the window ‘Sensor Input Settings’  is closed. Clicking the field  Display Measuring-Parameter,   Automatic Recording   is selected and the   Meas. Interv. is set to  1 s .

matching


Cassy lab measures voltage values, which correspond to the increase of volumes in the gas syringe. In order to follow the change in volume on the screen (Fig. 2), the measured values of voltage are converted into volume values by entering of a formula: In the window  ‘Settings'  the register card   Parameter/Formel/FFT   is selected. The field   New Quantity  is clicked and under   Formula UA1*100   (1 Volt corresponds to a volume of 100 mL) is entered. The volume is signified by the   Symbol  V  and under  Unit  mL  is entered. The other fields remain unchanged.




Experiment 1: Motivation experiment - Decomposition of hydrogen peroxide using grass

Experimental procedure:

A three-necked round bottom flask is fitted with an internal thermometer, a dropping funnel and an adapter connecting reducing. The adapter is connected via a silicone tubing to the gas syringe   (Fig. 1).   Into the flask placed in a water bath, somewhat grass is placed. The grass is covered with 30 ml of dist. water. The dropping funnel is filled with 3 mL of 6% hydrogen peroxide solution. A stopper is placed on the dropping funnel. The stirrer is started. The stopcock of the funnel is opened and hydrogen peroxide solution is allowed to drain from the funnel. When this procedure is finished, the stopcock of the dropping funnel is closed and the valve of the syringe is opened. Immediately the sensing software is started and the reaction temperature is read to the nearest 0.1°C. In a couple of seconds gassing begins and the plunger of the gas syringe is moved forward.

The change in voltage and in oxygen volume is displayed on the measuring screen  (Fig. 2).

measuring screen
Fig. 2: Real time graph - Decomposition of hydrogen peroxide using grass
change in voltage (black)      change in volume of oxygen (red)

Fig. 3: Three dimensional structure of catalase
(iron always in the ferric state)



Experiment 2: Temperature dependance of reaction rate

Experimental Procedure:

Into the flask 30 mL of dist. water are pipetted. 0.01 mL of the catalase suspension are added. The solution is allowed to equilibrate in the constant-temperature bath.
The dropping funnel is filled with 3 mL of 2% hydrogen peroxide solution. The syringe valve is opened and while stirring the hydrogen peroxide solution is added. When the addition is complete the reaction temperature is read to the nearest 0.1°C and the sensing software is started.

Data analysis using Excel:


Excel
Tab. 1: Measured values V(t), conversion according to y = V(t) / V¥ · n(H2O2) / 2


temperature
Fig. 4: Effect of temperature on reaction rate     cS = 0.59 · 10-1 mol / L
21°C  (1)    32.4°C  (2)    52°C  (3)


Experiment 3: Dependance of reaction rate on enzyme conzentration

Experimental procedure:

The experiment is carried out as described above (Experiment 2). The volumes of catalase suspension used are 0.005, 0.010 and 0.020 mL. The volume of 6% hydrogen peroxide solution is 3 mL. The reaction temperature is 21 °C.

Data analysis:


plot
Fig. 5: Effect of enzyme concentration on reaction rate    T = 21 °C     cS = 1.77 · 10-1 mol / L
0.005 mL  (1)     0.010 mL  (2)     0.020 mL  (3)   catalase suspension


Experiment 4: Dependance of reaction rate on substrate concentration and proof of a first order reaction

Experimental Procedure:

The experiment is carried out as described above (Experiment 2). 3 ml of 2 %, 4 % and 6 % hydrogen peroxide solution are used. The volume of catalase suspension is 0.010 mL. The reaction temperature is 23.9 °C.

Data analysis:

An Excel function is used to convert the volume values   (Tab. 2).   In so doing a plot of

- ln(1- (V¥/ Vt)

against t is allowed. Thus, according to   equation (7)   Kinetic equations (Download PDF file)  the rate constant can be determined   (Fig. 7).


Excel
Tab. 2: Measured values V(t), conversion according to y = ln(1 - (Vt / V¥))


Dependance of reaction rate on substrate concentration


volume
Fig. 6: Effect of substrate concentration on reaction rate      T = 23.9 °C
cS = 0.59 · 10-1 mol / L  (1)      cS = 1.18 · 10-1 mol / L   (2)      cS = 1.77 · 10-1 mol / L  (3)


Proof of a first order reaction


first order
Fig. 7: Determination of the rate constant     T = 23.9 °C
cS = 0.59 · 10-1 mol / L  (1)       cS = 1.18 · 10-1 mol / L   (2)      cS = 1.77 · 10-1 mol / L  (3)
y = ln(1 - (Vt / V¥))


Discussion:

·   The decomposition of hydrogen peroxide is very slow in aquaeous solutions but is accelerated by wide variety of catalysts. The reaction is typically first order in catalyst concentration and H2O2. With enzyme catalase the reaction is zero order in H2O2 (rate depends only in amount of catalyst). At low concentrations, Michaelis-Menten kinetics predict that the rate will become first order for the substrate (peroxide).

·   The reaction rate of the enymatic decomposition of hydrogen peroxide increases in the available temperature range with rising temperature.

·   A linear relationship exists between the catalase concentration and the reaction rate.

·   Possible inhibitors for catalase are cyanide, phenols, azide and urea. All these inhibitors are competitive, and connect to the catalase directly, thus reduce the catalase's potential to exercising its catalatic action. Another possible inhibitor is hydrogen peroxide in high concentrations, which poisons the enzyme system. Up to 0.4M H2 O2 concentration, the rate is proportional to the concentration of the substrate. At higher concentrations of H2O2, the rate of the reaction is dropped off.


References:
Catalase Kinetics
  Demonstration Experiment on Video   Decomposition of Hydrogen Peroxide with Catalase


Index of CASSY Experiments






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