N.B: Preliminary Experiment:
After preliminary experiments, it was decided to change the Copper Sulphate from a crystalline solution to a liquid form as it combined better with the enzymes and substrate- both liquids. The time that all test tubes were left inside the water bath was also increased from 5 minutes to ten. The amount of both the substrate and the enzyme were exactly halved as the amounts were not practical and did not fit inside the boiling tubes.
Method
Begin by collecting the required equipment described further below and set the experiment up. A variety of different increments of copper sulphate should be gathered of 0.2 mol dm-3 / 0.4/0.6/0.8 and 1.0.These lots of copper sulphate should each be kept in separate containers.
Once the amounts of copper sulphate have been collated, set up six boiling tubes (one for each of the amounts) with 20 millimetres of amylase in each, place the tubes into a water bath at a controlled temperature of 37◦C
Do the same with six boiling tubes containing 20 ml of starch and place them into the water bath for around 10 minutes to get them all at an even temperature.
When the optimum temperature has been reached, combine the tubes of starch with the amylase adding one of the amounts of the copper sulphate at the same time to each tube, labeling them as you go along. Replace the finished tubes back into the water baths for 10 minutes to allow the enzyme and inhibitors to work.
After the ten minutes have passed firstly take a sample of each and pipette it into a spotter tray pre-filled with iodine. At the side take 6 millimetres of each of the final products and place into six separate test tubes adding 10 cm cubed of Benedict’s reagent. Heat the tubes in boiling water baths for around 8 minutes, then leave it to cool.
Filter off the precipitate- if any, and dry. Weigh the precipitate and record the mass. Record the different shades of blue in the iodine dimples and record. Repeat the entire experiment once or twice more to allow for anomalies.
Independent Variable
The varying mol dm-3 of copper sulphate used.
Dependant Variable
The weight of the precipitates and the colour of the iodine at the end of the experiments.
Contol Variables
The following are the variables that need to be controlled in order to allow for a fair test and to give accurate results.
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The temperature of all the test tubes – the temperature can effect enzyme behaviour by denaturing it at too high a temperature. At certain levels of heat enzymes wok best but once the temperature increase past that point the bonds within the enzyme begin to break down and the enzyme is rendered useless.
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The amount of substrate (starch) – the levels of starch are important as they can effect the rate of the reaction. If there is far too few substrates around the experiment would not show an accurate reading as the enzymes aren’t being used. However if there is too much substrate the experiment once again slows down as substrate inhibition occurs ( too much substrate fighting for the same enzyme and blocking the path allowing nothing to get through)
- The amount of the enzyme (amylase)
- The amount of iodine in each spotter dimple.
- The amount of Benedict’s Solution
Apparatus and Materials
- Amylase – solution used as the enzyme
- Starch – 1% starch suspension used as the substrate
- Copper Sulphate (CuSO4) of 0.2/0.4/0.6/0.8/1.0mol dm-3 to be used as the inhibitor
- Benedicts Reagent as the main apparatus in the reducing Sugar experiment.
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Water Bath – this will be at a temperature of 37 degrees C at first for the first part to provide the optimum temperature of the enzyme Amylase. From there it will be a regulated boiling water bath to allow the Reducing Sugar experiment to react.
- Spotter Tray – to place the iodine in and amylase the results.
- Iodine- to show the levels of starch present.
- 18 boiling tubes for each of the different processes. 6 for the Starch.. A further 6 for the Amylase and the final 6 for the Benedicts reaction to test for reducing sugars.
- Test tube holders/racks.
- Scales to weigh the amount of Copper Sulphate and the mass of the precipitate.
- Measuring Syringe to provide accurate measurements of samples for benedict’s test.
- Stirrers to allow for even heat throughout the samples.
- Tissue to dry the precipitate.
Risk Assessment
Copper Sulpahte CuSO4 is harmful by inhalation or ingestion as the dust may ulcerate membranes. Therefore as a safety issue the experiment should take place in a well ventilated area. For an extra precaution safety goggles should be worn.
Ethical Considerations
Copper sulphate is very toxic to aquatic organisms and may cause long term damage in the environment. To dispose of it properly, small amounts of dilute copper sulphate solution can be flushed down a with a large quantity of water, unless local rules prohibit this. Larger quantities should be collected as solid waste and disposed of via professional waste disposal.
Analysis:
Below is the main raw table of results compiled from the qualitative iodine test and the quantitative data prescribed from the benedicts test.
Trends:
By looking at the graphs, one can see that the general trend is a decrease in the weight of the precipitate as the concentration in mol dm-3 of the Copper Sulphate increases. This is due to the inhibitor decreasing the effect of the enzyme amylase on starch. Amylase breaks down starch into maltose which is a reducing sugar- meaning it can be picked up with the Benedicts Test by forming a precipitate. The weight of the precipitate determines the amount of maltose present
If the inhibitor is lowering the enzymes’ effectiveness via non-competitive inhibition then that means less maltose will be produced and therefore less precipitate. This theoretical result is reflected in my experiment as shown by the graphs and tables.
In regards to the iodine test, the same reflections are made within the colour change- as the level of starch increases due to breakdown into maltose the colour turns from a dark navy indicating that starch is present to a lighter and lighter orange.
The next trend is that there is an average decrease in weight of around 0.03 grams for every 0.2 mol dm-3 increase of the inhibitor Copper Sulphate (CuSO4)- apart from at 1mol dm-3 solution where there is a slight increase (see anomalies).
Anomalies:
Straight off, one can immediately spot two anomalies within the results table and on the graphs –(both the scatter graphs and the bar).
On the raw data scatter graph on the second experiment for the third 0.4 % mol solution there is a slight increase in the weight of the precipitate instead of a decrease which reads on the graph as 0.13 (g) and should perhaps be more around 0.11/0.12 (g). A similar anomaly can be seen at 1% molar solution. There, the second reading for that solution reads extraordinarily high at 0.1 grams as apposed to something in the region of 0.04/0.05 grams.
