ICT IN BIOLOGY

Urease is an enzyme which can be extracted from soya bean seeds. It was the first enzyme to be purified and crystallised in 1926 by J. B. Sumner who got the Nobel Prize in 1946. It catalyses the hydrolysis of urea (also called carbamide):

You are asked to investigate the effect of two other chemicals on its activity: thiourea, CS(NH₂)₂ (which is also called thiocarbamide) and lead nitrate, Pb(NO₃)₂ .

A. Calibrating the pH probe

MATERIALS

1. Plug in the probe to Channel 1 of the LabPro interface. Connect the LabPro to the USB port of the computer. Then connect the transformer to the LabPro and plug it into the mains.
    

2. Switch on the computer and open LoggerPro, it will automatically recognise the pH probe and set up a spread sheet plus a graph.
    

3. Prepare a beaker with pH 4 buffer and another with pH 7 buffer. Carefully remove the container of electrolyte from the end of the probe and store it in a safe place. Wash the probe with the wash bottle.
    

4. Select the menu in LoggerPro. Then – Ch1 pH probe Next click on .
    

5. Place the probe in the pH 4 buffer and stir it. Type “4” in the Reading 1 box. When the voltage stabilises click on .
    

6. Wash the probe again in distilled water. Place it in the pH 7 buffer. Type “7” in Reading 2 and when the voltage stabilises click on . Finally, click on .
    

7. Wash the probe again and replace it in the electrolyte until you need it for measurements.
    

B. Using the pH probe to observe an enzyme controlled reaction

MATERIALS

Method

1. Prepare a flask with a magnetic stirrer. Add 2cm3 pH 4 buffer, 4cm3 of urease and 10cm3 distilled water. Put 2 to 3 drops of pH indicator in the flask to provide a visual check.
2. Remove the probe from the electrolyte and clamp it in a stand so that it dips into the liquid in the flask
3. Click on setup icon in LoggerPro to open the data collection menu. Select and choose Time Based. The default settings are 10 samples every 1s for 10s. Type in a time interval for the measurements in minutes. The experimental length should be about 8 minutes for a trial run and the frequency of measurement should be 60 per minute.

4. Press to return to the main screen.
    

5. Start the stirrer, collect 10cm³ urea solution in a syringe and start the recording by selecting . Record the pH for a few seconds, then add the urea.
    

6. When the run is complete remove the probe and wash it off. Clean out the flask, recuperate and wash off the magnetic flea. Add 2cm³ pH 4 buffer, 4cm³ of urease and 5cm³ distilled water and 5cm³ thiourea. Put 2 to 3 drops of pH indicator. Start the stirrer, collect 10cm³ urea solution in a syringe and start the recording by selecting . A dialogue box will open and you should store the latest data run. The second run will start. Let it run for a few seconds and add the urea.
    

7. Repeat the recordings using: (see the table below for the combination of liquids)
    

   10cm3 thiourea 5cm3 lead nitrate + 5cm3 distilled water 10cm3 lead nitrate

Summary

Warning Lead nitrate is toxic, dispose of it in the container provided, not in the sink.

8. Discuss and evaluate this investigation.