76 PHYWE Systeme GmbH & Co.

KG D-37070 Gttingen Laboratory Experiments, Biology

12.05 Enzyme inhibition (poisoning of enzymes)
Biochemistry LEB 12
Principle and tasks:
The enzymatic hydrolysis of urea in
aqueous solution liberates carbon
dioxide and ammonia. The ions of
these compounds increase the con-
ductivity of the solution. Conductivi-
ty measurements enable the rate of
hydrolysis of urea by the enzyme
urease to be determined. The addi-
tion of an appropriate inhibitor poi-
sons the enzyme, so that it no longer
converts substrate.
Conductivity-time plot for the hydrolysis of urea with urease before and after
poisoning
What you can learn about
Poisoning of enzymes
Enzymatic hydrolysis of urea
Silver nitrate inhibition of
urease
Conductivity measurement
Equipment (for the experimental procedure using the CHEM-UNIT)
Cobra3 CHEM-UNIT 12153.00 * 1
Power supply, 12 V 12151.99 1
Data cable 2 x SUB-D, 9 pin 14602.00 1
Software Cobra3 CHEM-UNIT 14520.61 * 1
Conductivity-temperature electrode 13701.01 * 1
Magnetic stirrer mini, plastic (ABS) 47334.93 1
Magnetic stirring rod, cylindrical, l = 30 mm 46299.02 1
Magnetic bar separator made of PTFE 35680.03 1
Balance SAS 51, 200 g / 0.01 g, RS232 45990.93 1
Support rod, l = 500 mm, stainless steel 02032.00 1
Support base, variable 02001.00 1
Right angle clamp (bosshead) 02043.00 1
Universal clamp 37715.00 1
Beaker, DURAN, tall form, 100 ml 36002.00 8
Beaker, low form, 250 ml, plastic 36013.01 1
Volumetric pipette, 1 ml 36575.00 1
Volumetric pipette, 20 ml 36579.00 1
Safety pipettor Flip 36592.00 1
Microliter syringe, 100 ml 02606.00 1
Pasteur pipettes, l = 145 mm, 250 off 36590.00 1
Rubber caps, 10 pcs 39275.03 1
Spoon with spatula end, l = 150 mm, steel, micro 33393.00 1
Wash bottle, 500 ml 33931.00 1
Urea, 250 g 30086.25 1
Urease solution in 50% glycerine, 1000 U/ml, 10 ml 31924.03 1
Silver nitrate, 15 g 30222.00 1
Distilled water, 5 l 31246.81 1
PC, Windows 95 or higher
Changes in the equipment required for use of the Basic-Unit:
(Instead of * above-mentioned)
Cobra3 Basic-Unit 12150.00 1
Software Cobra3 conductivity 14508.61 1
Measuring module conductivity 12108.00 1
Conductivity probe, glass shaft l = 12 cm 18151.02 1
Temperature sensor, semiconductor type 12120.00 1
What you need:
Enzyme inhibition (poisoning of enzymes)
with Cobra3 Basic-Unit P4120511
Experimental set-up using the Basic-Unit (P4120540)
Enzyme inhibition (poisoning of enzymes)
with Cobra3 Chem-Unit P4120540
Related topics
Poisoning of enzymes, enzymatic hydrolysis of urea, silver
nitrate inhibition of urease, conductivity measurement
Principle and task
The enzymatic hydrolysis of urea in aqueous solution lib-
erates carbon dioxide and ammonia. The ions of these
compounds increase the conductivity of the solution.
Conductivity measurements enable the rate of hydrolysis
of urea by the enzyme urease to be determined. The addi-
tion of an appropriate inhibitor poisons the enzyme, so
that it no longer converts substrate.
Equipment (for the experimental procedure using the
CHEM-UNIT)
Cobra3 CHEM-UNIT 12153.00* 1
Cobra3 power supply 12151.99 1
Data cable 2 x SUB-D, 9 pin 14602.00 1
Software Cobra3 CHEM-UNIT 14520.61* 1
Conductivity-temperature electrode 13701.01* 1
Magnetic stirrer mini, plastic (ABS) 47334.93 1
Magnetic stirring rod, cylindrical, l = 30 mm 46299.02 1
Magnetic bar separator made of PTFE 35680.03 1
Balance SAS 51, 200 g / 0.01 g, RS232 45990.93 1
Support rod, l = 500 mm, stainless steel 02032.00 1
Support base, variable 02001.00 1
Right angle clamp (bosshead) 02043.00 1
Universal clamp 37715.00 1
Beaker, DURAN, tall form, 100 ml 36002.00 8
Beaker, low form, 250 ml, plastic 36013.01 1
Volumetric pipette, 1 ml 36575.00 1
Volumetric pipette, 20 ml 36579.00 1
Safety pipettor Flip 36592.00 1
Microliter syringe, 100 l 02606.00 1
Pasteur pipettes, l = 145 mm, 250 off 36590.00 1
Rubber caps, 10 pcs 39275.03 1
Spoon with spatula end, l = 150 mm,
steel, micro 33393.00 1
Wash bottle, 500 ml 33931.00 1
Urea, 250 g 30086.25 1
Urease solution in 50% glycerine,
1000 U/ml, 10 ml 31924.03 1
Silver nitrate, 15 g 30222.00 1
Distilled water, 5 l 31246.81 1
Hazards and safety measures
Silver nitrate is a highly corrosive solid substance. Dust
from it irritates the respiratory organs. It liberates nitric
acid under the influence of moisture and light. The silver
which is so formed causes typical black stains. In the case
of fire, silver nitrate acts fire promoting by liberating oxy-
gen.
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41205 149
LEB
12.05
Enzyme inhibition (poisoning of enzymes)
Fig. 1: Experimental set-up using the Chem-Unit
Do not inhale dust. Avoid contact with eyes and skin.
When working with it, wear appropriate protective appar-
el, protective gloves and safety glasses. Store it dry and
excluded from light.
Must be kept out of the reach of children.
First aid: Thoroughly rinse the affected skin or eyes
(opened wide) with plenty of water. When eyes are affect-
ed, seek immediate medical attention. After accidents or
on feeling unwell, fetch medical attention at once. When
inhaled: Fresh air, keep respiratory tract free. On respira-
tory distress: Transport to medical attention in a half-sit-
ting position.
Waste disposal: Collect solutions containing silver ions in
a correspondingly labelled container and submit them for
recycling or suitable waste disposal.
Preparatory work
Prepare the solutions required for the experiment as fol-
lows:
1.6% urea solution: Weigh 2.00 g of urea into a 250 ml
beaker and dissolve it in 123.00 g of distilled water
(prepare this fresh before each experiment).
1% silver nitrate solution: Weigh 0.5 g of silver nitrate
tion into a 100 ml beaker and dissolve it in 49.5 g of
distilled water.
Note: The urease solution must always be kept in a re-
frigerator.
Set-up and procedure (using the Chem-Unit)
Set the apparatus up as shown in Fig. 1
Connect the conductivity-temperature probe to the
appropriate input of the Chem-Unit
Call up the Cobra3 Measure programme in Windows
and assign the Chem-Unit as measuring instrument
Set the measurement parameters as shown in Fig. 2.
In the <Displays...> menu, set both Digital display 1
and Diagram 1a (range 0-600 s) to conductivity (range
0 to 1000 S/cm)
Set the temperature compensation in the <Preferen-
ces / Conductivity> menu to linear with Pt 1000 tem-
perature probe
Fix the universal clamp to the rod of the support stand
with a right angle clamp
Use the 20 ml volumetric pipette twice to pipette
40 ml of the 1.6% urea solution into a 100 ml beaker
and add a magnetic stirrer rod
Place the beaker on the magnetic stirrer and immerse
the conductivity electrode in the solution
Adjust the stirrer to a medium stirring speed (Take
care: Do not allow the magnetic stirring rod to hit
against the pH electrode)
Add 50 l of the urease solution with the microliter
pipette and then start the measurement without any
delay by clicking on <Continue> and <Start measure-
ment>
The course of the reaction with time can be visually
followed on the screen
300 seconds after the start of the reaction, use the
volumetric pipette to add 1 ml of 1% silver nitrate to
the solution in the beaker and wait until the pro-
gramme ends the measurement after 600 seconds
At the end of the measurement, save the data in menu
<File> <Save measurement as...>
Results and evaluation
Fig. 3 shows an example of a conductivity-time plot, as is
displayed by the programme after ending the measure-
ment.
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41205 150
LEB
12.05
Enzyme inhibition (poisoning of enzymes)
Fig. 2: Measurement parameters (Chem-Unit) Fig. 3: Conductivity-time plot for the hydrolysis of urea
with urease before and after poisoning
Urease acts on urea to decompose the substrate to car-
bon dioxide and ammonia. The ions so released into the
aqueous solution enable the progress of the reaction over
time to be followed by measuring the conductivity of the
solution. During the first 300 seconds, the conductivity
continually increases as a result of the enzymatic activity.
The addition of silver nitrate first leads to a drastic
increase in the conductivity, because of the silver and
nitrate ions. The continuing course of the reaction curve
shows no further increase in conductivity, however. The
reason for this is the irreversible inhibition of urease by sil-
ver ions, i.e. the enzyme has been poisoned. No further
transformation of substrate is possible.
Experimental procedure using the Basic-Unit
The experiment using the Chem-Unit described above
can be analogously carried out using the Basic-Unit. For
this, the entries in the list of materials which are marked
with an asterisk must be replaced by the materials listed
below. The set-up and procedure are then also slightly dif-
ferent (see below, in particular Fig. 4 and Fig. 5).
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41205 151
LEB
12.05
Enzyme inhibition (poisoning of enzymes)
Fig. 4: Experimental set-up using the Basic-Unit
Fig. 5: Measurement parameters (Basic-Unit)
Changes in the equipment required for use of the Basic-Unit:
Cobra3 Basic-Unit 12150.00 1
Software Cobra3 conductivity 14508.61 1
Measuring module conductivity 12108.00 1
Conductivity probe, glass shaft l = 12 cm 18151.02 1
Temperature sensor, semiconductor type 12120.00 1
Set-up and procedure using the Basic-Unit
Prepare the set-up as in Fig. 4
Connect the conductivity electrode to the conductivi-
ty measuring module and the temperature sensor to
input S2 of the Basic-Unit
Call up the Cobra3 Measure programme in Windows
and assign Cobra3 conductivity as measuring instru-
ment
Set the measurement parameters as shown in Fig. 5
Carry on from here as with the Chem-Unit
Note
The measurement can also be carried out without a tem-
perature probe, but then the temperature of the solution
must be entered by hand in the menu <Preferences /
Conductivity>, or, when using the Basic-Unit, in the field
<Temperature compensation>.
This experiment can also be carried out using hand-held
measuring instrument conductivity 07138.00, conductivi-
ty/temperature probe 13701.01, conductivity software
14418.61 and data cable 07157.01.
Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D-37070 Gttingen 41205 152
LEB
12.05
Enzyme inhibition (poisoning of enzymes)