Core Practical 12 – From Topic 5 (On The Wild Side)
To investigate the effect of temperature on the initial rate of an enzyme-catalysed reaction.
Temperature of each enzyme solution (water baths at 10°C, 20°C, 30°C, 40°C and 50°C will be used)
Time taken for the solution to turn pink (measured in seconds using a stopwatch)
- Volume of enzyme solution – 1cm³ of the lipase enzyme solution will be used each time, measured using a syringe
- Volume of milk – 5cm³ of milk will be used each time, measured using a measuring cylinder
- Volume of sodium carbonate solution – 7cm³ of the solution will be used each time, measured using a measuring cylinder
- Volume of phenolphthalein added- 5 drops will be added each time using a pipette
- Colour of solution – the stopwatch will be stopped each time when the solution is completely colourless
- Water baths
- Milk (full-fat)
- 5% lipase solution
- 0.05M sodium carbonate solution
- Hot water baths
- Test tube racks
- 5 beakers
- 5 test tubes
- Marker pen
- Measuring cylinder
- Glass rod
A set-up can involve carrying out and timing the reaction at room temperature as a negative control. The results can then be compared to this standard.
- Set up the water baths at 10°C, 20°C, 30°C, 40°C and 50°C and put a beaker of lipase, containing a 2 cm3 syringe into each water bath.
- Label a test tube with the temperature to be investigated.
- Add 5 drops of phenolphthalein to the test tube.
- Measure out 5 cm3 of milk using a measuring cylinder and add this to the test tube.
- Measure out 7 cm3 of sodium carbonate solution using another measuring cylinder and add this to the test tube. The solution should be pink.
- Place a thermometer in the test tube. Take care as the equipment could topple over.
- Place the test tube in a water bath and leave until the contents reach the same temperature as the water bath.
- Remove the thermometer from the test tube and replace it with a glass rod.
- Use the 2 cm3 syringe to measure out 1 cm3 of lipase from the beaker in the water bath for the temperature you are investigating.
- Add the lipase to the test tube and start the stopwatch.
- Stir the contents of the test tube until the solution loses its pink colour. Stop the watch and note the time in a suitable table of results.
Results & Calculations
A graph can be made where initial rate (1/T) can be plotted on the y-axis and temperature on the x-axis. You should see a graph looking similar to this:
A Q10 value can also be calculated for this reaction using the data below the optimum temperatures. The equation for Q10 is as follows:
(Rate of reaction at temperature + 10°C) ÷ (Rate of reaction at temperature T)
A Q10 value of 2 means that for every increase in 10°C, the initial rate doubles. Likewise, a value of 3 means that every increase in 10°C triples the initial rate and so on. This deduction only works for temperatures up to the optimum temperature.
The peak of the graph indicates the optimum temperature. This is when enzymes have the greatest amount of kinetic energy they can have whilst maintaining their protein structure. At this point, the enzymes are working most efficiently and effectively.
Beyond this optimum temperature the protein structure starts to change. Large amounts of kinetic energy overcome the hydrogen bonds in the tertiary and secondary structures of the proteins. This causes the enzyme to change shape and so the shape of the active site is also altered. For this reason, fewer substrates can bind to form enzyme-substrate complexes. This is why increasing the temperature beyond a certain point slows down the rate of reaction.
- Inconsistency when stopping stopwatch (random error) – the stopwatch may not have been stopped at the exact same stage of the reaction each time. In future, a colorimeter can be used instead to time the reaction until a certain absorbance reading is reached.
- Incomplete shape of graph (systematic error) – a wider range of temperatures should be tested so that you can observe the optimum temperature and rate decline on a graph as shown above.