Experiment to Investigate the Effect of Copper Ions on a Solution of Amylase and Starch using Iodine
Experiment to Investigate the Effect of Copper Ions on a
Solution of Amylase and Starch using Iodine
PLANNING
Aim
Copper ions are heavy metals therefore copper sulphate (CuSO4) is likely to be an inhibitor. This is since heavy metals are known to be inhibitors having an effect on enzyme functioning. In this experiment I will investigate how a range of copper sulphate concentrations affects the ability of the enzyme amylase to hydrolyse starch into maltose.
Hypothesis
I predict that as I increase the concentration of CuSO4 the more time it will take for the enzyme amylase to hydrolyse starch into maltose thus the rate of reaction will decrease. This is since the Cu2+ is likely to alter the shape of the active site of amylase hence reducing the enzyme's ability to catalyse the reaction.
I know that as I double the concentration of CuSO4 twice the volume of copper ions will be present for the same given volume. This therefore means that the chance of the copper ions colliding with amylase will increase by a scale factor of 2. The chance of a collision is random but doubling the concentration of CuSO4 means that the average chance of meeting copper ions will increase by a scale factor 2. Thus I quantitatively predict that as the concentration of CuSO4 doubles then the time for my reaction to take place will also double. Thus the concentration of CuSO4 will be proportional to the time of the reaction (see graph A) hence the rate of reaction will be inversely proportional the CuSO4 concentration (see graph B).
Predicted Graph A
CuSO4 Concentration (mol dm-3)
Predicted Graph B
Scientific Background
Amylase is a digestive enzyme which speeds up the rate of a reaction by reducing the activation energy required for the enzyme to hydrolyse starch into maltose, however at the end of the reaction the enzyme remains chemically unchanged. The enzyme hydrolyses the polysaccharide starch by breaking the glycosidic bonds converting it into the disaccharide maltose. This enzyme may be derived from animal, fungal and plant resources. (1)
Enzymes are biological catalysts which speed up chemical reactions that would normally happen very slowly. Enzyme molecules have a complicated three-dimensional shape due to the particular way the amino acid chain that makes up the protein is folded. This tertiary protein structure gives the enzyme its catalytic ability. A few of the amino acids on the surface of the molecule fold inwards to make a specific indentation, called the active site, into which a particular substrate can fit. Once the enzyme and the substrate are joined they form an enzyme-substrate complex. The formation of an enzyme-substrate complex makes it possible for substrate molecules to be brought together to form a product. The product is released and the enzyme is free again to take part in another reaction.
Enzymes function by the 'induced fit' method. The substrate does not simply bind with the active site. It has to bring about changes to the shape of the active site to activate the enzyme and make the reaction possible. So small molecules may enter the active site, but they cannot induce the changes in shape to make the enzyme behave like a catalyst. The method suggests that when the enzyme's active site comes into contact with the right substrate, the active site slightly changes or moulds itself around the substrate for an effective fit. This shape adjustment triggers catalysis and helps to explain why enzymes only catalyse specific reactions.
The functioning of an enzyme is influenced by a number of factors such as temperature, pH, enzyme concentration, substrate concentration and the addition of inhibitors and activators.
Inhibitors are any substances which interfere with the formation of enzyme substrate complexes. They can act directly on the active site (competively) hence competing with the substrate molecule for the active site, or they can act indirectly on another area of the enzyme other than the active site called the allosteric site (non competitively) thus disrupting the enzyme's tertiary structure hence preventing any substrate molecules binding with the enzyme. Competitive inhibitors are reversible however, non competitive inhibitors are irreversible.
Figure 1.1: Diagram to show non competitive inhibition (2)
Figure 1.2: Diagram to show Competitive Inhibition (3)
CuSO4 is likely to be non competitive inhibitor. This is since the Cu2+ ions present in CuSO4 is a heavy metal and heavy metals tend to be non-competitive inhibitors. This means that they inhibit the action of the enzyme-controlled reactions by attaching themselves to the enzyme molecule to an area other than the active site preventing the formation of enzyme substrate complexes. This therefore means that with fewer successful collisions fewer enzymes substrate complexes are produced thus the time period for the hydrolysis of starch into maltose increases. In this case, the extent of the inhibition depends entirely on the concentration of the inhibitor i.e. the copper sulphate and cannot be varied by changing the amount of substrate present i.e. the amount of starch. In this investigation the effect of varying concentration of CuSO4 is indicated by measuring the effect on a solution of amylase and starch using iodine.
Preliminary Experiment
Apparatus
* Bacterial amylase solution 0.2M, 100cm3
* Starch solution 0.1M, 100cm3
* Copper sulphate solution 0.1M, 25cm3
* Iodine solution 1.0M, 25cm3
* 3 10cm3 syringes
* 9 test tubes (3 for amylase, 3 for starch and 3 for the CuSO4 concentrations)
* 1 test tube rack
* Water bath set at 60oC since this is the optimum temperature for amylase. A water bath will be used to provide the solutions with a constant temperature.
* Spotting tiles to monitor the changes in the reaction mixture with iodine.
* Stop clock
* Thermometer
* Buffer solution pH 7.0
Spotting tiles will be used in this experiment since this will enable me to time my endpoint at which amylase is no longer hydrolysing the substrate starch. Comparisons in any colour change between each spotting well can also be observed when using a spotting tile thus my end point decision making can be carried out thoughtfully providing me with more accurate results.
Risk Assessment
. CuSO4 is harmful if swallowed and may cause vomiting. Therefore hands must be washed after the experiment preventing the salt coming in contact with any food. If vomiting ensues then wash out the mouth and have a glass or two of water. If further vomiting takes place then seek medical attention. (4)
2. CuSO4 may also be irritating to the eyes and the skin thus must be handled with care. If any solution comes into contact with the eye then flood the eye with gently running tap water for 10 minutes. Seek medical attention. Similarly, if any solution comes in contact with the skin then wash off the skin with plenty of water. (4)
3. The enzyme amylase is a potential allergen, and should be handled so as to minimise contact with skin or inhalation. However, fortunately no one in the class including myself was allergic to enzymes.
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2. CuSO4 may also be irritating to the eyes and the skin thus must be handled with care. If any solution comes into contact with the eye then flood the eye with gently running tap water for 10 minutes. Seek medical attention. Similarly, if any solution comes in contact with the skin then wash off the skin with plenty of water. (4)
3. The enzyme amylase is a potential allergen, and should be handled so as to minimise contact with skin or inhalation. However, fortunately no one in the class including myself was allergic to enzymes.
4. Amylase can cause asthma and or irritate the membranes of the eyes and the nose. If swallowed wash out mouth and give a glass or two of water, and then seek medical advice as soon as possible. If the solution gets into the eyes then flood the eye with gently running tap water for 10 minutes and seek medical attention. (5)
5. Iodine is harmful therefore contact with the eyes and the skin must be avoided. Wash hands after use. If the solution is spilt onto the skin flood the area with water immediately or bathe with sodium thiosulphate solution if available. (6)
6. The glassware must be handled with care and normal safe laboratory practice should be carried out i.e. wearing overalls and remembering to wash hands in case of a spillage as well as after the experiment has been completed. If glassware is broken then it should be cleared using appropriate equipment such as a dust pan and brush and disposed in a designated glassware bin.
Copper sulphate, amylase, starch and iodine could all be disposed down the sink due to very dilute concentrations of the solutions were being used.
Bacterial amylase was used therefore there were no possible sources of objections.
Variables
It is important to ensure that the variable being tested (dependant variable) in this experiment is not affected by other independent variables such as the following. These variables must therefore be kept constant throughout the entire experiment.
. Temperature- variations in temperature changes the energy supplied to the reaction. An increase in temperature would result in an increase in kinetic energy thus a greater number of molecules in this case the substrate starch having energies greater than or equal to the activation energy. This results in more collisions of the substrate starch molecules with the enzyme amylase molecules per second resulting in more maltose product being formed thus the rate of reaction increasing. The temperature will be controlled using a water bath which will be set at 60°C and monitored using a thermometer.
2. pH- This must be kept constant at pH 7 since this is the optimum pH at which amylase works at its best. A change in pH will affect the hydrogen, ionic and disulphide bonds holding the enzyme's tertiary structure. This will result in the enzyme's active site changing thus the substrate molecule, starch, not being able to bind with the enzyme's active site. Hence resulting in the rate of reaction decreasing. Thus a buffer solution of pH 7 will be used in order to control this variable.
3. Starch concentration- 0.1M of starch must be used throughout the whole of the experiment. As the substrate concentration increases, there are a greater number of substrate molecules which can collide with the amylase molecules. This results in more successful collisions per second thus the rate of reaction increases. At higher concentrations the enzyme molecules become saturated with substrate, so there are few free enzyme molecules, therefore increasing the substrate concentration does not make any difference.
4. Amylase concentration- 0.2M of amylase must be used throughout the experiment. If the concentration of amylase is increased then the rate of reaction will also increase. This is since there will be a greater number of active sites available for the starch molecules. Therefore there will a greater number of successful collisions per second and the rate of reaction will increase.
5. Iodine concentration and volume- 2 drops of 0.1M of iodine will placed in each spotting well since varying iodine concentrations and volumes could result in different colour observation.
6. Time- Every 10 seconds a sample of the solution will be removed from the test tube and placed in the spotting well. The time will be kept constant.
7. Volume of sample placed in spotting well- 3 drops of the sample will be placed in each the spotting tile well until no further colour change is observed. This is since if a greater or less volume is placed then this could affect the colour observation.
8. Volume of inhibitor, enzyme and amylase- 2cm3 of CuSO4, 4cm3 of 0.2% bacterial amylase and 4cm3 of 0.1M starch solution will be placed in each test tube resulting in a total volume of 10cm3 in each test tube.
Preliminary Method
. A test tube was taken into which 2cm3 of 0.0M of CuSO4 i.e. distilled water was placed. No CuSO4 was placed into the first test tube to ensure that the enzyme amylase was actually functioning. A second test tube was taken into which 4cm3 of 0.2% bacterial amylase was placed and finally a third test tube was taken into which 4cm3 of 1% starch solution was placed.
2. Different syringes were used for each of the three different solutions to avoid any contamination between the solutions.
3. The three test tubes were labelled to avoid any confusion between the solutions.
4. The optimum temperature for the bacterial amylase was researched to be 60°C (source 4). Thus the temperature of the water was set to 60°C.
5. Whilst the water bath was equilibrating to 60°C, 8 spotting tiles were taken into which two drops of iodine solution was placed into each well.
6. Once the water bath had reached 60°C the three test tubes were placed into the water bath.
7. Thermometers were placed into each of the test tubes to monitor the temperatures of the 3 solutions. When the solutions had reached 60°C they were mixed and at the same time a stop clock was started.
8. At 10 second intervals a sample of the mixed solution was removed using a pipette and placed into a well of the spotting tile.
9. The time taken for the iodine to change from a blue black colour to the colour of the iodine solution at the start of the experiment i.e. from blue black to an orange colour was recorded.
0. The experiment was repeated another 2 times however, different concentrations of CuSO4 solution were used. The concentrations and formation of these concentrations are shown in the table below.
Concentrations of CuSO4 Used in Preliminary Experiment 1
CuSO4 Concentration (mol dm-3)
Volume of CuSO4 / 0.1M
(cm3)
Volume of Distilled water
(cm3)
0.00
0.00
2.00
0.05
.00
.00
0.10
2.00
0.00
Results of Preliminary Experiment 1
CuSO4 Concentration (mol dm-3)
Time taken for amylase to hydrolyse starch (s)
0.00
40
0.05
2020
0.10
3600
These concentrations of CuSO4 were used at the start. However, the time taken for the amylase to hydrolyse the starch took far too long i.e. for 0.1M of CuSO4 the time taken for amylase to hydrolyse starch took 60 minutes. Thus in my actual experiment this would be inappropriate since repeats of 3 would have to be carried out meaning that for that specific concentration carrying out 3 repeats would take around 3 hours. This would thus result in my experiment taking far too long hence not being able to finish the experiment in the time allocated.
Therefore a second preliminary experiment was carried where the method was the exact same to the first preliminary however, this time lower concentrations of CuSO4 was used. The concentrations used and their formation can be observed in the table below.
Concentrations of CuSO4 Used in Preliminary Experiment 2
Concentration of CuSO4
(mol dm-3)
Volume of CuSO4 / 0.1M
(cm3)
Volume of Distilled water
(cm3)
0.000
0.00
.00
0.015
0.15
0.85
0.030
0.30
0.70
Results of Preliminary Experiment 2
CuSO4 Concentration (mol dm-3)
Time taken for amylase to hydrolyse starch (s)
0.000
20
0.015
50
0.030
320
This time the time taken for amylase to hydrolyse starch for the highest CuSO4 concentration i.e. 0.30M took no longer than 5 minutes 30 seconds. This reaction length is suitable for my actual experiment since sufficient repeats will be able to be carried out hence resulting in my results being reliable. Thus for my actual experiment the highest CuSO4 concentration used will be 0.03M.
The volumes of distilled water and of CuSO4 (0.1M) used to make up the different concentrations of copper sulphate are very small as shown in the table on the previous page. Thus in order to increase the accuracy of the copper sulphate concentrations, a 1cm3 syringe will be used.
My actual experiment method will be improved in many ways.
* A water bath will be set up at 55°C since fluctuations in the water bath occur. The optimum temperature for amylase is 60°C and temperatures above this cause the hydrogen and ionic bonds holding the enzyme amylase's tertiary structure to break. If the molecular structure is disrupted, amylase will cease to function since the substrate molecule i.e. starch will no longer be able to bind with the enzyme.
* A buffer solution of pH 7.0, 1cm3 will be used in order to control pH.
* A greater number of CuSO4 concentrations will be used 0.000M, 0.050M, 0.010M, 0.015M, 0.020M, 0.025M and 0.030M.
* The whole experiment will be carried out three times to provide reliable results.
Actual Experiment
Apparatus
* Amylase solution, 100 cm3, 0.2M
* Starch solution, 100 cm3, 0.1M
* Copper sulphate solution, 25 cm3, 0.1M
* Distilled water, 30 cm3
* Iodine solution 1.0M
* 2 10cm3 syringes (each for amylase and starch) and 7 1cm3 (each for the different CuSO4 concentrations)
* 9 beakers ( 1 for amylase, 1 for starch and 7 for the different CuSO4 concentrations)
* 7 pipettes
* 21 test tubes (7 for amylase, 7 for starch and 7 for the CuSO4 concentrations)
* 1 test tube rack
* Water bath set at 55oC to provide the solutions with a constant temperature
* Spotting tiles to monitor the changes in the reaction mixture with iodine.
* Buffer solution pH 7, 1cm3- to prevent any fluctuations in pH
* Stop clock
Actual Method
. A test tube will be taken into which 1cm3 of 0.0M of CuSO4 i.e. distilled water will be placed. No CuSO4 would be placed into the first test tube to ensure that the enzyme amylase is actually functioning. A second test tube will be taken into which 4cm3 of 0.2% bacterial amylase will be placed and finally a third test tube was taken into which 4cm3 of 1% starch solution will be placed.
2. Different syringes would be used for each of the three different solutions to avoid any contamination between the solutions.
3. The three test tubes will be labelled to avoid any confusion between the solutions.
4. The optimum temperature for the bacterial amylase was researched to be 60°C. However, the water bath will be set to 55°C due to possible fluctuations in the temperature of a water bath resulting in the temperature being able to exceed the optimum (60°C). This could thus result in the enzyme being denatured hence producing unreliable results. The temperature of the water bath will be monitored using a thermometer.
5. Whilst the water bath would be equilibrating to 55°C, 8 spotting tiles will be taken into which two drops of iodine solution will be placed into each well.
6. Once the water bath had reached 55°C the three test tubes would be placed into the water bath.
7. Thermometers will be placed into each of the test tubes to monitor the temperatures of the 3 solutions. When the solutions have reached 55°C they will be mixed and at the same time a stop clock will be started.
8. At 10 second intervals a sample of the mixed solution will be removed using a pipette and 2 drops of the sample will be placed into a well of the spotting tile. If any excess solution is left inside the pipette then this will be disposed into a separate beaker which would be disposed at the end of the experiment.
9. The time taken for the iodine to change from a blue black colour to the colour of the iodine solution at the start of the experiment i.e. from blue black to an orange colour will be recorded.
0. The experiment will be carried out another 2 times however; a greater number of CuSO4 solutions will be used. The concentrations used will be 0.005, 0.010, 0.015, 0.020, 0.025 and 0.030. For each of these concentrations a new pipette will be used to avoid any contamination. The formation of these concentrations can be viewed in the table on the following page. Each concentration of copper sulphate will be made in a bulk since this would prevent me from making each concentration three times in each experiment. This would therefore result in the exact same concentrations being used each time the experiment is carried out hence enabling my results being more reliable.
Concentration of CuSO4 (mol dm-3)
Volume of 0.1M CuSO4
(cm3)
Volume of Distilled water
(cm3)
Total Volume
(cm3)
0.000
0.00
9.00
9
0.005
0.05
8.95
9
0.010
0.10
8.90
9
0.015
0.15
8.85
9
0.020
0.20
8.80
9
0.025
0.25
8.75
9
0.030
0.30
8.80
9
IMPLEMENTING
Results
Concentration of 0.1 M CuSO4
(mol dm-3)
Time taken for amylase to hydrolyse starch (s)
2
3
Average (1.d.p)
0.000
20
20
20
20.0
0.005
50
50
50
50.0
0.010
*250
40
30
73.3
0.015
90
90
200
93.3
0.020
260
270
270
266.6
0.025
330
340
350
340.0
0.030
400
420
410
410.0
Concentration of 0.1M CuSO4
(mol dm-3)
Rate for amylase to hydrolyse starch (1000 ÷ time) (1.d.p)
2
3
Average
0.000
50.0
50.0
50.0
50.0
0.005
20.0
20.0
20.0
20.0
0.010
*4.0
7.1
7.7
6.3
0.015
5.3
5.3
5.0
5.2
0.020
3.8
3.7
3.7
3.7
0.025
3.0
2.9
2.9
2.9
0.030
2.5
2.4
2.4
2.4
* = Anomaly
ANALYSIS
I have drawn four graphs:-
Graph 1-Graph to show the effect of CuSO4 concentrations on the time taken for amylase to
hydrolyse starch into maltose (s).
Graph 2- Graph to show the effect of CuSO4 concentrations on the rate of reaction.
Graph 3a- Graph to show the reliability of data using error bars
Graph 3b- Graph to show the reliability of data using error bars on a larger scale.
Graph 1
The results of the experiment shown in the implement section have been plotted. From graph A it is evident that as the concentration of copper sulphate increases the time taken for amylase to hydrolyse starch into maltose increases. This can be seen where at 0.00M of CuSO4 the time taken for the hydrolysis of starch to take place was 20 seconds, at 0.010M was 120 seconds, at 0.02M was 266.6 seconds and at 0.030M was 410 seconds.
It is also evident that as the concentration of CuSO4 was doubled then the time taken for amylase to hydrolyse starch also doubled. This can be seen clearly as the concentration of CuSO4 is proportional to the time taken for the reaction to occur excluding my control where no CuSO4 was added. In my graph my control i.e. 0.00M of CuSO4 was also plotted to show the effect the inhibitor had on the time taken. It can be seen that as soon as the inhibitor was added i.e. at 0.005M then the gradient increased.
Graph 2
From graph 2 a general trend can be observed. It is evident that as the concentration of CuSO4 is increased then the rate of reaction decreases. This can be seen at 0.00M where the rate of reaction was 50, at 0.010M was 6.3, at 0.020M was 3.7 and at 0.030M was 2.4. At the start the initial rate of reaction was very steep showing that at the start the rate of reaction was very high since the gradient was very steep. As the CuSO4 concentrations increased the initial rate of reaction decreased more and more resulting in the graph beginning to level off hence the graph resulting in the graph being inversely proportional.
The decrease in the rate of reaction shows that CuSO4 had an effect on the activity of amylase i.e. the copper ions were inhibiting the reaction taking place. Copper sulphate does not affect starch since these substrates are chemically stable thus the copper ions could not have displaced any other element present in the starch molecules. Hence the copper ions must have affected the enzyme amylase's tertiary structure.
As shown by the graph on page 5, an increase in the mass of copper sulphate will be more effective, preventing more enzyme-substrate complexes to form, and subsequently making the reaction take longer. The solution with 0.02 grams of copper sulphate dissolved into it could have taken longer than the control solution due to the inhibitory effect, or because the time difference is so little, this may have been coincidental. However, the solution with 0.04 grams of copper sulphate dissolved in it had a more significant effect on the time taken, and also the reaction rate. The increased concentration of copper sulphate in this solution has a greater effect on the amylase activity, preventing a higher proportion of enzyme-substrate complexes being formed than in the solution with 0.02 grams. Between 0.04 and 0.1 grams, the difference in the time taken increases steadily, but less significantly than between 0.02 and 0.04 grams. The difference in rate of reaction is also very small between these concentrations. It is likely that the presence of a mass in excess of 0.04 grams does not have such as significant effect on the rate of the reaction and the time that it takes. This is because at this stage a large proportion of enzyme molecules are being inhibited already. Therefore increasing the concentration of the copper sulphate past a certain point will have less effect, as there are less enzyme molecules available to be inhibited.
Graph 3a and 3b
In graph 3a I have marked my error bars on the average rate of reaction at which amylase hydrolyses starch into maltose. However, my scale was not large enough to plot the accurate error bar points thus my error bars were not very clear. In order to overcome this problem I have drawn another graph (graph 3b) in which I have enlarged my scale enabling my error bars to be seen more clearly.
EVALUATION
My results supported my hypothesis reasonably well. I will now evaluate the reliability and precision of my data obtained. Firstly, I will weigh up the reliability of the data and I will undertake this by making deductions from the error bars that I drew onto the graph.
Number of repeats carried out in the experiment
Firstly, the whole experiment was carried out three times i.e. three replicates of each CuSO4 concentration was carried out in the same day. The mean of these replicates were calculated providing me with graph 1 showing the effect of CuSO4 concentration on the time taken for amylase to hydrolyse starch. Then using my individual raw data the rate of reaction was calculated for each concentration for each repeat and later the mean rate of reaction was calculated. This provided me with graph 2, 3a and 3b. The mean of these replicates were calculated to provide me with reliable results from which further analysis could be carried out. However, it must be taken into consideration that this experiment was carried out only 3 times which is not sufficient to state that my results 'prove' my hypothesis.
Graph 3a and 3b- error bars
The reliability of results was also shown by using error bars in graph 3a and graph 3b. These showed the maximum and minimum values of the reading taken during the course of the reaction. The higher the difference between the maximum and minimum value, the higher the spread and therefore lower the reliability.
If we look at graph 3a showing the rate of reaction against the concentration of CuSO4, there are no error bars present at the CuSO4 concentration 0.000M and 0.005M. This shows that there are no fluctuations in the data hence showing that these two points are extremely reliable. On graph 3b, for the remaining concentrations i.e. 0.015M, 0.020M, 0.025M and 0.030M, there are error bars present, however the difference between the maximum and minimum values are very small i.e. the readings vary smoothly. This thus shows that even for these concentrations of CuSO4 my average graph plotted for the rate of reaction for amylase hydrolysing starch is very reliable.
However, at 0.010M of CuSO4 an anomaly was present where an error bar was also marked. At this specific concentration the error bar was large in comparison to the other points thus showing that my raw data collected for this specific point was less reliable. The large fluctuations in the data attained at this concentration could have possibly caused my anomalous result which is clearly indicated on all of my graphs.
Anomaly
In my whole of my experiment I had come across one anomaly which is clearly indicated in all of my graphs. My anomaly could have been due to a variety of factors.
The anomaly could have been due the temperature in the water bath changing. This could have happened, as the water bath I used relied on a negative feedback mechanism, so once the temperature got to a certain level (55oC), the heating would stop. After this, the water would cool, and then the heater switched back on. It is possible that this particular concentration was done at this break, where the temperature of the water bath was not 55oC, but lower than this.
This would decrease the rate of reaction and distort the results. If this did occur then this might explain the anomaly attained at 0.010M of CuSO4 since the rate of reaction was lower than expected.
At 0.010M of CuSO4, the error bar is quite large. This could be an explanation to the anomaly attained, because the error bars indicate that there are fluctuations in the data hence the result for this specific concentration could have been higher. If the rate of reaction was higher, then this would increase the rate of reaction enabling this concentration to be closer to my line of best fit. It is clearly visible from graph 3b that my anomaly is far from the line of best fit.
From my raw data results table showing the rate of reaction the anomaly achieved was at 0.010M of CuSO4. The repeats for this specific concentration deviate from each other by quite a lot in comparison to the rest of the concentrations. For the first repeat at 0.010M of CuSO4 the rate of reaction was 4.0, for the second was 7.1 a difference of 3.1 and for the third 7.7. However, at 0.015M of CuSO4 the rate of reaction attained for the three repeats were 5.3, 5.3 and 5.0; similarly, at 0.020M of CuSO4 the rates of reactions were 3.8, 3.7 and 3.7.
Precision
The method carried out was subjective thus not very accurate. This was since the time taken for amylase to stop hydrolysing starch had to be observed i.e. the time taken for iodine to change from a blue black to an orange colour. Different people have different opinions thus have a difference in opinion to when no more colour change is taking place. It was difficult for the human eye to be precise whilst distinguishing between the colour change since the eye can only make a subjective analysis not a quantitative one. A specific apparatus called the colorimeter could have been used to provide me with more accurate data. This method could have been carried out in the exact same way however, the iodine along with the solution would have been placed into cuvettes at regular intervals which would have then been placed into the colorimeter. The reading would have then been noted and repeats would have been carried out.
A water bath was used in order to maintain the temperature throughout the experiment. However, the water bath used
1. The time taken for the iodine to change from a blue black colour to the colour of the iodine solution at the start of the experiment i.e. from blue black to an orange colour will be recorded.
In order to make my results more reliable I would do the following thing:
- I would have more replicates of results for each concentration of the substrate in order to make the results more reliable
- I would have a larger variety of concentrations in order to have a larger variety of results.
- I would take more readings by extending the time slightly.
- Larger test tubes can be used so more substrate can be added lowering the percentage error of the apparatus
- Find a method that does not require the bung to be removed and replaced each time as a result stopping the gas loss completely
Overall I can say again that the precision and reliability of my results were good. I can prove this as my results proved my hypothesis very well. The error bars on the graph and standard deviation table also proved this was the case.
Bibliography
. http://www.greatvistachemicals.com/biochemicals/amylase.html
2. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html
3. http://en.wikipedia.org/wiki/Image:Comp_inhib.png
4. Hazard card 27: copper salts.
5. Hazard card 33: enzymes.
6. Hazard card 54: iodine.
Sabeen Siddiqui Page 1