To examine the effect of Substrate Concentration (Hydrogen Peroxide) on the rate of an enzyme catalysed reaction.

5. Check the gas syringe is set to 0. If the gas syringe does not start at 0 there is the possibility that more gas will be evolved than the gas syringe is able to measure, or that readings will not be taken accurately.

6. Using the graduated pipette, 10 cm3 of hydrogen peroxide should be obtained and placed in the syringe. The graduated pipette is to ensure accuracy and to make sure the concentrations of the substrate used are constant throughout the experiment. Place the syringe back into its original position.

7. Apply pressure to the syringe containing the hydrogen peroxide until all the contents have been added to the conical flask. All the substrate must enter the flask to ensure that the test is fair and the exact amount needed is in contact with the potato sample. This is to ensure consistency and accuracy of results throughout the experiment.

8. Immediately start the stopwatch at the most readily available time to ensure that evolution/time is being monitored correctly. Inaccuracy at this point could devoid the results and show that the optimum rate of reaction corresponds to a different concentration.

9. Take readings of oxygen evolution from the gas syringe every 20 seconds and record in a table. Stop recording after three minutes. (180 seconds) As the catalase enzyme breaks down the hydrogen peroxide, oxygen and water will be produced. When the reaction has been fully catalysed there will be no more oxygen evolution and the reaction will be over. The concentration at which the time is lowest will be the optimum substrate concentration for this reaction.

0. Wash and dry the entire system and repeat the above steps different concentrations of substrate as stated below. This is to make sure there is no contamination from previous experiments.

1. Three sets of results should be obtained for each substrate concentration specified to minimise error and inaccuracy. This range had been chosen to include the full range of concentrations to be able to identify the point where the rate of reaction is highest, when the enzyme is working as fast as possible and the substrate concentration no longer affects the rate of reaction. Once the optimum rate of reaction has been found the experiment will be repeated at closer concentrations around this optimum point to determine the actual optimum point more accurately. I suggest maybe closer intervals of 2% differences in substrate concentration at this point rather than the 10% for accuracy.

2. The reason to why I have not included the agitation of the conical flask during the experiment is because I feel that I could not assure that I would be able to generate a constant, consistent swirling motion.

If 10cm3 of 10 Vol. H2O2 is used 100 cm3 of Oxygen should be produced. Therefore is different concentrations of the hydrogen peroxide are needed I will need to dilute it to different amounts. I will dilute the Hydrogen peroxide with water according to the table below.

Volume of 10 Vol. H2O2 in cm3

0

9

8

7

6

5

4

3

2

0

Volume of distilled water in cm3

0

0

2

3

4

5

6

7

8

9

Volume of buffer solution in cm3

0

Final concentration of H2O2 in %

00

90

80

70

60

50

40

30

20

0

0

As an initial pre-test I have decided to use a solution of 0% hydrogen peroxide. This is the highlighted in the table; no repetitions of this reaction are needed because it is aimed to show that it is the hydrogen peroxide releasing the oxygen and not anything else. Another pre-test that I will do is following my proposed method once through for a check that the amounts of substances, equipment and timings chosen are sufficient for me to be able to carry out the experiment and obtain an answer for the brief. This is the point whereby I will make any necessary amendments to my procedure before I continue.

The results I obtain will be of oxygen evolution (cm3) over 180 seconds for each substrate concentration. These will have been recorded in a table. I will calculate an average using the mean where repetitions have been carried out. Then I will plot graphs of oxygen evolution against time. It is then necessary to draw lines of best fit for each set of results and mark them with a key. From here I will obtain values for the rate of reaction by measuring the gradients of each of the graphs to allow me to plot a separate graph showing the rate of reaction against concentration. This final graph will allow me to compare all my results and will enable me to answer my brief, as it will show the actual effect of changing concentration against the rate of reaction. To obtain these gradients from the evolution/time graphs it is necessary to draw a line against the curve of the graph where it beings to level off. I will then draw two more lines on each one, one vertical and one horizontal to form a triangle so I can see exactly how much oxygen was produced over a set period of time. These values will be substituted into the equation y/x to give me the values for the rate of reaction to enable to plot me the second graph. Y is equal to the volume of oxygen produced and x to the time taken for this volume of oxygen to be produced.

I have chosen to obtain the results at hydrogen peroxide concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%. This will enable me to have a wide range of results to help satisfy the brief better. Also I aim to repeat the experiment three times so that an average set of results will be obtained to minimise the effect of uncontrolled variables.

There are several variables that I feel may affect the validity of my experiment and therefore need to be controlled as effectively as possible. The hydrogen peroxide that I am using as the substrate in this experiment may be slightly decomposed so it might not evolve the amount of gas it should do. Therefore I would suggest that fresh hydrogen peroxide should be used for each experiment, or at least from the same batch so it has effectively decomposed the same amount. It should be stored in cool conditions to minimise decomposition.

I know that temperature affects the rate of enzyme activity. Higher temperatures increase the kinetic energy of the reaction molecules leading to more frequent collisions between substrate and enzyme. More enzyme/ substrate complexes would be formed per second and the rate of catalysis would be increased and I would be at risk of invalidating the experiment. For my experiment I have decided to use a water bath for the reaction to take place at a specified temperature of 30oC. This is because I might not necessarily be able to carry the experiment out on the same day so I would risk fluctuation between temperatures on different days. I have chosen the specific temperature of 30oC for my experiment for several reasons. At this temperature it is not raising the energy of the reacting molecules to a state where the reaction would take place too quick for me to monitor, it is not so high that I would risk the possibility of denaturing the enzyme and also it is also not too low risking the possibility of reducing the rate of reaction to a point where it would be inhibiting my accuracy as I would have less time to carry out successful repetitions of my experiment lowering the chance of being able to identify possible anomalous results, which would in turn affect the outcome of the brief. Because I need the reactants to be thoroughly heated to exactly 30oC before carrying out the experiment I will leave the enzyme, substrate and apparatus which will be involved to stand in the water bath for a period of 10 minutes to ensure that the specified temperature is reached successfully.

I have chosen to use potato as my source of catalase because there are catalase enzymes within the potato and we can easily control the surface area and quantity of potato. This factor is important because both the surface area and quantity of the potato would be detrimental to the outcome of the experiment if they were not controlled. In regarding quantity/volume of the specimen, if different masses of potato were used then enzyme concentration would be affected. A larger piece of potato (with greater mass/volume) would consequently have a higher enzyme concentration than a smaller piece (smaller mass/volume) of potato. If enzyme concentration was increased there would be more active sites available for the reaction so again collisions would occur more frequently between enzyme and substrate and this would increase the number enzyme/substrate complexes formed per unit time. By using pieces of potato of the same mass/volume that are of the same tissue type we are effectively ruling out possible inconsistencies that may be caused due to varying enzyme concentration. In view of this factor I have chosen to weigh the pieces once they have been cut to ensure mass/volume remains constant.

Just controlling the volume/mass of the potato specimen is not sufficient as if surface area is not controlled and kept constant throughout the experiment it could have a substantial effect on the authenticity of the results. A sample with a considerably larger surface area to volume ratio than the others would mean the surface area would increase the area for which collisions between substrate and enzyme would be able to occur. More enzyme/substrate complexes would form than usual so the rate of reaction would be faster. This is why I have chosen to control this variable by using a size 6 borer to cut out the sample from the potato and then cut it down to an exact length of 6mm to ensure a fair reaction takes place.

Through research I have discovered that enzymes have different optimum pH values at which they work best. pH changes from the optimum can affect enzyme activity adversely in two ways. The Enzyme molecule will change shape and no longer fit to that of the substrate so enzyme/substrate complexes will not form and a reaction will not occur. Where binding sites within the active site are charged ions, some pH values are inhibitory as they cause the ions to re-associate. It is very unlikely that pH will affect my experiment. However I am going to make precautions. I have substituted 1cm3 of buffer solution for 1 cm3 of water where water is needed to dilute the hydrogen peroxide to its specific concentrations for use in the experiment. The buffer will stop any changes in pH. Where the buffer cannot be used i.e. where a concentration of 100% hydrogen peroxide is needed and buffer cannot be added- (as it would effectively dilute the solution) I will use litmus paper just to maintain that pH is correct at the beginning of the experiment.

These are all the variables I feel that need to be controlled. I have decided to record my results at 20 second intervals, these are regular intervals so that it is easier for me to compare my results. It is important that the results are obtained at the same frequency so I can see how oxygen evolution occurs across the whole experiment. Obtaining results at shorter intervals I feel would be too hard to achieve as there would not be enough time to monitor oxygen evolution properly at this speed and the reaction will not occur so fast that a shorter interval time would give a greater degree of accuracy. Similarly if the time gap between results is too large I will not be able to monitor how the oxygen evolution varies with obtaining only a couple of readings. Therefore I deem 20-second intervals sufficient for this experiment. In the pre-test if this theory proves to be wrong and the time intervals needs altering I will be able to change them more accordingly.