The energy required to initiate the reaction is called the activation energy.
When the substrate reacts, they need to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrates or the product. (Source: www.s-cool.co.uk)
The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sits available. (Biology 1)
In this experiment I will be investigating how the concentration of the substrate, Hydrogen Peroxide, affects the rate of reaction with potato catalase.
Hydrogen peroxide is an end product of energy releasing reactions in the cell. This compound, which is toxic, is split into water and oxygen after reacting with catalase (potato). The volume of oxygen produced per minute will give the rate of reaction. (Enzyme Study Guide)
Variables
The factors that are going to remain the constant in this experiment are the temperature, enzyme concentration, pH, quantity of solution, quantity of potato catalase and the measurement of 5cm marked on the monometer. These variables must all be kept constant in order for the experiment to be a fair test.
I will ensure that the temperature is kept constant by storing everything I use at room temperature. The pH will remain equal if I use the same part of the potato during the experiment, which means not using the skin but the inside of the potato. The other variables can be kept at constant levels by measuring them as accurately as possible.
The independent variable in this experiment is the change of substrate concentration.
Apparatus List
- 150 potato discs (enzyme) – cut from the inside of the potato (not skin) so that the pH remains constant.
- Cork borer
- Knife
- Ruler
- 45cm³ hydrogen peroxide (1 mol concentration) – substrate for dilution
- Distilled water – for diluting the hydrogen peroxide
- Monometer tube approximately 3mm in diameter
- Monometer fluid
- Spring clips – to open and close the monometer, which will bring the monometer fluid back down to its original level when opened.
- Wax pencil – for marking a gap of 5cm on monometer above the level of monometer fluid.
- 2 syringes 5cm³ –to increase the accuracy of measurements
- 5 boiling tubes
- Rubber bung
- Forceps
- Sticky labels
- 5 Beakers 20cm³
- Clamp stand
- Digital stop clock – for gathering more accurate results
Procedure
- Consider safety precautions – handle dangerous equipment carefully, wear lab coat and goggles
- Set up the apparatus as shown below
- Cut 10 potato discs of 1mm thickness.
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Get five 20cm³ beakers, distilled water and 45cm³ hydrogen peroxide to prepare dilutions as followed:
- Label the beakers with their concentrations
- Put 5cm³ of the first prepared substrate concentration into the boiling tube then add 5cm³ of the catalase solution to it.
- Put rubber bung back onto boiling tube as soon as possible because the reaction begins as soon at the two solutions are combined.
- Start timing as soon as the monometer fluid reaches the bottom marking of the 5cm gap, and finish timing as it gets to the end of the 5 cm gap. Start and stop timing as fast as possible so that more accurate results can be obtained.
- Repeat this 3 times for each substrate concentration and record the results with the digital stop clock.
Results
Analysis
The results show that as the concentration of Hydrogen Peroxide increases, the rate of reaction also increases. At first this was at a steady rate but once the reaction rate reached 0.45cm³min the increase in rate began to slow down.
The graph shows that there are two anomalies in the results. Both of them are above the expected rate of reaction. This could be due to one or more of the variables not being constant as they should have been.
The dotted line shows my prediction of the results if higher concentrations of hydrogen peroxide were used. I have predicted that as the concentration increased, the rate at which it increases will slow down and eventually stop. This will mean that the rate of reaction will remain constant because the active sites in the enzymes become saturated.
If the substrate concentration is constantly increased, and the enzyme concentration remains constant, there comes a point where every active site is working continuously. If more substrate is added the enzyme cannot work any faster, because the active sites are saturated. The substrate molecules have to wait until the enzyme–substrate complex (e-s complex) has released the products before it can enter into the active site.
Evaluation
The experiment was not as accurate as it could have been. The factors that affected the reliability of the results included, the thickness of potato discs, time taken to replace the rubber bung and not having high enough substrate concentrations.
There are two anomalies in the results at 4vol and 8vol substrate concentrations. The anomalies could have been due any of the variables not remaining constant.
It was difficult to maintain accuracy during the procedure. This was because it had to be repeated so many times. It was quite a long procedure and it had to be done in total 15 times.
One variable that may have differed was the thickness of the potato discs. As there were 150 discs it was difficult practically to make sure they were all equal in size. Given the time I had to complete the experiment many of the discs were measured subjectively.
The time taken to replace the rubber bung, after the substrate had been added to the potato catalase also affected the reliability of the experiment. Once the bung had been replaced on the test tube the reaction had already begun so the time taken was not measured accurately.
I also had to predict the rest of the graph because there were not enough results. This was due to there not being higher substrate concentrations available.
This experiment could be improved if there was access to higher concentrations of Hydrogen Peroxide. This would have increased the number of results obtained and therefore increased the range of the graph. This would have shown more patterns in the results, which would have produced a more accurate and successful experiment.
