ii) pH - Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.
In this experiment, the pH should be kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (potato tissue).
iii) Substrate Concentration - When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate.
To control the substrate concentration, identical quantities of the substrate were used for each reading. To ensure that this was measured precisely, 5ml syringes were used to accurately gauge to exact quantities.
iv) Inhibition - Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affected this investigation, however, as none were added.
Enzyme cofactors - cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Coenzymes also influence the functioning of enzymes although are not bonded to the enzyme.
Unless enzyme cofactors were present in the potato tissue containing the Catalyse, they were not included in this investigation and therefore would not have affected the rate of reaction and the results of this experiment.
v) Enzyme Concentration - Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect.
I varied the enzyme concentration by altering the number of equal sized discs of potato that contain the Catalyse, in the reaction. The greater the number of discs, the greater the enzyme concentration.
Method
1) Using the cork borer, cut four cylinders from the potato. Make each 4 cm long. It is important to get exactly 4cm long in order to make it a fair test. A great deal of care has to be taken into account when using cork borer.
2) Place 5cm3 of H2O2 into each of four test tubes. The volume of H2O2 is constant and so this is called a control variable.
Wash H2O2 off skin or clothes, if split. As H2O2 is irritant.
3) Add one drop of washing up liquid to each tube, this stops the bubbles leaving the tube. Label the tubes 5°C, 20°C, 35°C, 70°C
4) Prepare a water bath (a beaker) at 5°C. Only half fill the water bath with water to avoid spillage when placing tubes in beaker.
5) Place one cylinder of potato directly into the water bath and also one of the prepared tubes. This is to maintain the same temperature and allows the tissues to equilibrate to the surrounding area.
6) Leave for 5 min, keeping the water bath temp as constant as possible since the temperature is one of the control variables.
7) Add the potato cylinder to the tube and leave for exactly one and half minutes. As time is another control variable.
8) Maintain the temperature of the reactants otherwise temperature of the individual experiments is no longer constant.
9) Measure the height of foam up the tube with a rule.
10) Repeat the procedure for the three other temperatures. Note this is the feature under test, is the uncontrolled or key variable.
Results
Group Results
Analysis
My results proved my prediction to be correct. The breakdown of Hydrogen Peroxide accelerates as the temperature increases until the optimum temperature after which it begins to slow down. Temperature influences the rate of enzyme activity. Usually a 10°c rise doubles the rate of enzyme activity. This is only true up to an optimum temperature, however beyond this point (usually 40°c) the 3D shape of the active site becomes distorted and the enzyme becomes inactive.
Cooling or even freezing does not destroy enzymes, though it slows down their activity. From studying the graph and our results table we can see the enzymes optimum temperature (35°c). It was after roughly 50°c where we could see a decrease in the enzymes activity. A rise in temperature increases the rate of most chemical reactions and a fall in temperature will slow them down. In many cases a rise in 10°C will double the rate of reaction in a cell. This is particle theory. We investigated the temperature at which reactions occurred. We predicted that an increase in temperature would result in an increase in kinetic energy. We were correct, since the speed of particles increases, they should collide more often and therefore the speed of reaction increases. The particles will also have more energy thereby speeding up the reaction even more. It has been suggested that for every 10 degree rise in temperature, the speed of the reaction will double.
At low temperatures particles of reacting substances do not have much energy. However, when the substances are heated, the particles take in energy. This causes them to move faster and collide more often. The collisions have more energy, so more of them are successful. Therefore the rate of reaction increases.
The more successful the collisions are the faster the reaction.
The same can be said for reactions controlled by enzymes, but because enzymes are proteins if the temperature exceeds 50°C the enzyme will be denatured and will no longer work. For this reason few cells can tolerate temperatures higher than approximately 45°C.
Enzymes are specific in the reactions they catalyse, much more so than inorganic Catalysts. Normally, a given enzyme will Catalyse only one reaction, or type of reaction. The enzyme has an active site that helps it to recognise its substrate in a very specific way. Just like a key only fits into a specific lock, each enzyme has its own specific lock, each enzyme has its own specific substrate. This is called the lock and key theory. The enzymes never actually get consumed in the process, they just increase the rate of reactions.
When enzymes denature the heat starts to destroy their shape and structure. The shape of the enzyme is so important to its working that any change in the shape of the molecules will make them less effective or stop them working completely. Therefore I predict that by heating the Hydrogen Peroxide, when it reacts with the enzyme the shape of the enzyme will be ruined due to the high temperature.
The shape of the graph is as I predicted showing that as enzyme concentration increases so does the rate of reaction. This is because at a greater enzyme concentration, there are more free active sites available for the substrate and so more products can be made in a shorter length of time. However, it is not possible to take precise readings from the graph between the plotted points since insufficient readings were taken. To be able to do this, intermediate enzyme concentrations would have to be measured so that the shape of the graph would be more exact.
Comparing my results with the class averages, my results were slightly higher. This may due to my inaccurate eye observation since it was difficult to see where the start of the foam is.
Evaluation
In the boiling tubes it was clear that a reaction was taking place by the observation of bubbles of oxygen gas being released creating a foam in the boiling tubes.
In order to decide how varying the enzyme concentration affected the decomposition of hydrogen peroxide, the rate of reaction was measured. To do this accurately, the time taken for a specific quantity of oxygen gas (a product of the reaction) to be released was determined. This was achieved by observing the time taken for the foam to travel from the mark of the level of H2O2.This was an accurate measure of how the enzyme concentration influenced the breakdown of hydrogen peroxide, as the quantity and speed of gas produced is dependant on the rate of reaction. The marked points remained the same distance apart for each reading for different enzyme concentrations so that they could be accurately compared and the trend observed.
All measurements were taken so that the stopwatch was started once the potato cylinder was dropped into the beaker. To judge accurately, the point at which the fluid reached the marked line, it was examined at eye level and the measurement taken when the bottom of the meniscus was lined up to the mark. This was the same for every reading.
The data obtained from this investigation has been recorded in a table showing the time, enzyme concentration and rate of reaction. This means that the results of the experiment are presented in a clear and orderly fashion that allows patterns in the results to become more obvious.
The rate of reaction was calculated by dividing the time taken for the quantity of gas to be produced from the reaction. By calculating the rate of reaction instead of merely using the time readings, the quicker reactions will be represented as a greater value for the rate of reaction rather than a small time value. This makes the graph clearer and easier to analyse.
Patterns within the results collected from the experiment, are best shown on a graph. This is because overall trends between the enzyme concentration and rate of reaction can be portrayed more effectively and become more obvious.
In this investigation, I measured the rate of reaction at varied temperature. As a precaution, I have limited my contact with the boiling tubes, as my body heat will raise the temperature, increasing the rate of reaction or expanding the foam in the test tube.
I also monitored the temperature using a thermometer to ensure that it remained constant and not disrupt the results of the experiment by affecting the activity of the Catalase. Maintaining at the constant temperature is the most difficult part of the experiment, in order to improve this; an electrical water bath can be use instead of individual beaker.
A pH buffer should be used to maintain a consistent pH level in the boiling tubes. This way there was no variation in pH that might have resulted in an increase or decrease in the rate of reaction. However this was not available at the time of the experiment.
Laboratory coats were worn during the investigation to prevent chemicals from spoiling clothes. Care was also taken whilst handling the chemicals as hydrogen peroxide is corrosive. Whilst using the cork borer, care was also taken as it may cause cuts on skin.
The precision of this experiment, generally, was very limited since insufficient readings were taken. More different temperature such as 45°C, 55°C should be taken in order to produce a more accurate set of results and the graph would then can be used to predict further temperatures.
In this investigation each reading should be repeat so that an average rate of reaction for each enzyme concentration could be calculated. This could be improved by repeating the reading more frequently thus reducing the extent of any anomalies further, once averaged.
For further work, we are alter the controlled and key variables of varies experiment, such as using different concentration of alcohol, since alcohol can break down lipids that are within the cell surface membrane, which will also should the effect of alcohol on membrane
permeability.