Risk assessment:
Hydrogen peroxide:
It is an oxidizer, which is not flammable but may start a fire if put in contact with organic materials, so keep the area well ventilated. The maximum concentration that is safe to use in the laboratory is 20 vol. Make sure eye protection and protective clothing is worn, as hydrogen peroxide is corrosive. In case of any solution getting in contact with the eyes, flush it out with copious amounts of water and seek medical assistance. Any contact with the skin should be avoided but in case there is any contact, wash it off immediately with plenty of water as burns may result. Spillages should be diluted down with lots of water and washed up immediately. Hydrogen peroxide should be stored in a clearly labelled dark bottle, as it decomposes quickly with the presence of light, in a cool place.
pH buffer solutions:
As the solutions are of various pHs between 4.5 to 8.5, they are acidic and basic, so solutions can have corrosive or caustic properties. Wear eye protection and avoid contact with the skin. If there is any spillages, including on the skin or eyes, wash it off immediately with plenty of water, especially if any of the solutions come into contact with the eyes. Store the buffers in clearly labelled bottles.
Glassware:
The syringe plunger slides out easily, therefore secure it to the main part of the gas syringe, but also make sure that this will not restrict any movement of the plunger.
Take care of other glassware, keeping them away from the edge of the workbench to avoid any breakages that may cause injury, especially the boiling tubes as they can roll around.
Potato: It is of low risk, but should be kept in a clearly labelled container.
Products of reaction: The product is water so there is a low risk. However, as the pHs of the buffers present in the mixture of the reaction, in some experiments, have slight hazards, avoid contact with the skin. To dispose of the residue, decant off any liquid present, washing it away down the sink and use a spatula to remove the potato out of the boiling tube to be discarded.
Pilot Study:
Factors to be investigated:
- Mass of potato to use.
- Concentration of hydrogen peroxide that will give a satisfactory rate of reaction.
- Time taken for reaction to complete.
- Verify the range of pH’s to be investigated.
- Suitable volume of pH buffer solution.
- Need for alternative sources of catalase.
Three pHs were chosen to be used in the pilot study; 4.5, 7.5 and 8.5 (the extreme pHs and the optimum.)
Results:
pH of buffer solution: 7.5
Mass of potato used: 5g
Volume of buffer solution:
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5 cm3 with 10 cm3 distilled water
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1 cm3 with no distilled water
Rate: 0.067 cm3 s-1 0.063 cm3 s-1
Mass of potato used: 10g
Volume of buffer solution: 1 cm3 with no distilled water
Concentration of H2O2(aq): 20 vol.
Rate: 0.063 cm3 s-1 0.075 cm3 s-1 0.025 cm3 s-1
Rate is measured by: Volume of O2 produced
Time
The results show that the catalytic reaction is affected by the pH and the data that will be obtained in the full experiment will be able to show the effect of pH on the enzyme.
Conclusion and modifications:
- 20 vol. Hydrogen peroxide should be used instead of 15 vol. To give a faster rate of reaction.
- 10g of potato can provide sufficient catalase for the reaction.
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1cm3 buffer solution is enough to maintain the pH in the potato.
- No distilled water should be added to the potato so that it the concentration of catalase will not be diluted down.
- It is easier to draw the hydrogen peroxide up into the plastic syringe using a crystallising dish then a small beaker.
- pH range is satisfactory.
- There is no need to use an alternative to potato.
- The reaction can be completed within 4 minutes.
Revised Apparatus List:
Same as previous list, except:
- 20 vol. Hydrogen peroxide instead of 15 vol.
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No need for distilled water and 10 cm3 measuring cylinder.
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Use 1 cm3 graduated pipette for greater accuracy then a 10 cm3 graduated pipette as only 1 cm3 solution is needed.
- Use crystallising dish instead of a small beaker to draw up the hydrogen peroxide, as it is easier with a larger open area.
Method:
- Set up the apparatus as shown in the diagram.
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Heat a water bath to a constant temperature of 40oC (the optimum temperature of catalase).
- Grate the potato and weigh out 10g on a watch glass.
- Place in a boiling tube.
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Measure out 1 cm3 of pH buffer solution (of pH 4.5, 5.5, 6.5, 7.0, 7.5 or 8.5) and add it to the potato.
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Place the mixture in the water bath until the temperature of the potato reaches 40oC.
- Take one drop of liquid from the potato mixture and test its pH using narrow range indicator paper to make sure the temperature has not changed the pH in any way and is still at the right pH.
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Draw up 2 cm3 of hydrogen peroxide from a crystallising dish with a plastic syringe. Only pour out the hydrogen peroxide just before starting the catalytic reaction, as the solution will decompose very quickly in light.
- Make sure the gas syringe plunger reads zero and bung the boiling tube of potato. Insert the plastic syringe into the top of the bung.
- Inject the hydrogen peroxide and start the stop clock at the same time.
- Record the volume of oxygen produced by the reaction every 10 seconds for one minute and then every minute thereafter, until 4 minutes has passed.
- Repeat the reaction 3 times and take an average of the volume of oxygen produced at each time interval.
- Repeat the whole experiment with the other different pHs.
- Plot a graph of volume of oxygen produced against time and also of the rate of reaction against pH.
Results:
pH 4.5
pH 5.5
pH 6.5
pH 7.0
pH 7.5
pH 8.5
Summary
1 m3 = 105 cm3
Averages
Rate
Analysis:
Volume against time of various pH
The graph shows that for all the pH’s, the general trend is that the volume of oxygen produced increases quite steadily with time after a slightly more rapid increase for the first 10s. After 10s, up to 120s, similar volumes of gas are produced for all but pH 4.5, which had a slower increase than the other pH’s with the least total volume of oxygen at the end of the experiment. After 120s, the rate at which gas was produced decreased and so the volume of oxygen produced at each pH began to very slowly level off. pH 8.5 showed to most dramatic change in rate at 120s.
Rate against pH
The graph shows an increase of rate with the increase of pH from 0.057 cm3s-1 at pH 4.5 to the maximum rate of 0.099 cm3s-1 at pH 6.5. The rate then takes a sudden decrease at pH 7.0 and rising up again sharply. Above pH7.5, the rate decreases gently to 0.082 cm3s-1 at pH 8.5. The dotted line shows the shape of the curve if the average of only the two higher final volumes were taken to calculate the average rate. This has been done, as the smaller final volume at pH 7.0 is considered as anomalous. The anomaly is the low rate at pH 7.0, as it does not fit the general smooth shape of the curve.
Conclusion:
The hypothesis has been proved to be correct, but the optimum pH found in this experiment is pH 6.5. This is when the charges on the acidic and basic groups of the enzyme, catalase, is the most stable and the shape of the enzyme has not be distorted. With acidic conditions, the enzyme will gain H+ ions, whilst when the conditions are basic, the enzyme will lose H+ ions. This results in the alteration of shape of the enzyme. The change of shape of the enzyme affects its efficiency to bind temporarily with hydrogen peroxide to catalyse its break down. The change of pH alters the charges on the enzyme, which could alter its shape. The rate at which oxygen is produced therefore, in general, increases to a maximum at the optimum pH and then decreases again.
There is the anomaly at pH 7.0 though. It was expected that the rate would be in between the rate of pH 6.5 and 7.5, if the optimum were pH 6.5. The range of the total volume of oxygen produced, for the 3 reactions done at pH 7.0, is very high, with a difference of 7 cm3 between the lowest and highest volume. The large range, makes the average rate at pH 7.0 low, and if the average of the higher two volumes is taken, it can be seen by the dotted line that rate is higher, but still not within the predicted range. This could be due to the uneven distribution of the pH buffer onto the potato, so that not all the potato was at pH 7.0. The volume of oxygen produced each time the experiment is done, therefore varies a great deal. It was also very difficult to keep the water bath at 40oC, so the enzymes may have been below or above its optimum temperature, which in turn affects the rate of reaction as well as the pH. If the temperature was below the optimum, the rate would be low. As the molecules would not have been moving as fast, according to the kinetic theory, the number of collisions between the enzyme and the substrate with enough activation energy for a reaction is reduced with decreased temperature. If the temperature of the catalase exceeded its optimum, the enzyme may have been denatured due to extreme heat and so will not be able to work on the hydrogen peroxide.
There was a massive range of the volume of gas produced at pH 6.5, but the average is still the highest of all the results showing that it is the optimum pH 6.5. The optimum pH in different cells varies, so in potato, the optimum is found to be 6.5 and not 7.6, as stated before the results were obtained. The optimum pH found could also depend on the type of buffer solution used and the ionic strength of the buffer.
Evaluation:
The results are valid to show the general tread of the effect of pH the rate of reaction but not the specific rates at which the enzyme works at as the range of the final volume of gas produced at the end of each run of the experiment of each pH is quite large. The volumes at the end point of each experiment of the pH’s were actually quite similar, except pH 4.5 was lower than the rest. The volume of gas produced will all be the same at whichever pH the enzyme is working at as the concentration of hydrogen peroxide and the mass of potato, and therefore the approximate concentration of catalase was the same. It is only the time it takes all the hydrogen peroxide to be broken down that is the difference between the various pH’s. The time taken for a stable volume of oxygen to be reached should therefore have been measured to calculate the rate of reaction instead of the volume of gas produced. This could be too time consuming though and the end point is not completely definite. The following factors led to the limited reliability of the method as shown by an anomaly at pH 7.0.
Limitations to the experiment include the treatment of the potato used, and the accuracy of the apparatus. The exact concentration of catalase in each of the experiments was unknown, as only the mass of potato was measured. The concentration of enzymes in each sample of potato could have varied a bit and therefore there has been the variation in the total volume of oxygen collected. As the potato was left in water after being grated to prevent it from going off, some water was weighed out and added to the sample. This would dilute the concentration of enzyme, therefore decreasing the rate of reaction. As the volume of pH buffer used was small, only 1 cm3, not all the potato may have been covered by the solution so that not the whole sample of potato would have been at the certain pH of the buffer. The potato itself should have been neutral, so that the rate of decomposition of hydrogen peroxide would have increased slightly with the more extreme high and low pH’s. The uneven distribution of pH buffer solution also accounts for the large range of volumes obtained for each experiment within each pH. The temperature of the water bath was very difficult to keep constant at 40oC and so would also affect the rate of reaction and explain the large range of results. The effect of temperature on the enzyme has been explained in the conclusion. An electrical water bath, which is able to maintain a constant temperature, can be used instead of heating up one’s own.
Readings of the gas syringe can only be to the nearest division making it difficult to state exactly what the volume of gas is. As the scale is marked in 1 cm3 divisions, so the volumes of the results has an uncertainty of ± 0.5cm3. In drawing up hydrogen peroxide into the small syringe, there were often bubbles of air trapped in them, which could not be removed, and so the volume of solution measured out would have been very slightly under 2 cm3. it has to be made sure that all the air is pushed out of the syringe and the spout is completely submerged in the hydrogen peroxide before drawing it up.
More pH’s could be tested, so that a more exact curve for the rate against pH can be plotted to see an extra detailed effect of pH on catalase. To determine a more accurate optimum pH of potato catalase, a narrower range of pH’s around 6.5 could be used to find the pH with the maximum rate of oxygen produced and therefore the optimum pH.