As mentioned above, an enzyme can denature if the surrounding temperature is too high. Surplus heat energy disrupts bonds in the enzyme. This means the structure of the enzyme will be changed, including the shape of the active site. If the active site is changed then the enzyme can no longer interact with the substrate. This is the reason for consideration, and is also the reason that the water bath, regulated at 60 degrees centigrade will be used for these experiments. Both the substrate and the enzyme will be placed in the water bath before the experiment, in separate test tubes, in order that the reaction will take place at the desired temperature. These factors must be kept constant to ensure a fair test.
The concentration of the amylase:
Not only is the concentration of the substrate important, but also the concentration of the enzyme is also important and must be controlled to ensure that it does not affect the rate of reaction. If there is a higher concentration of enzymes, therefore a higher number of active sites, in the solution then the rate of reaction will increase, therefore all experiments must have the same enzyme concentration to ensure fairness. If the concentration of amylase were to change, then the concentration of the substrate would have to remain static, in order that one factor can be measured against the other. All the samples of the amylase must be taken from the same pre-prepared solution.
The volume of the enzyme amylase used:
The same volume of amylase must be used throughout all experiments. A greater volume of amylase will not increase the concentration of the solution but it will increase the availability of enzymes (as there will be more of them) specific to starch within the solution. As a result the rate of reaction will be greatly enhanced, and so the products of this enzyme-catalysed reaction will be in a higher concentration, after a fixed time. This factor must be kept constant to ensure reliable results.
The pH level of the solution, assisting the enzyme:
All enzymes have an optimum pH, at which they will interact with other molecules at a faster rate. pH levels either side of the optimum range of an enzyme will begin to decrease the enzymes ability to react. When an environment is too acidic or too alkaline the enzymes will cease reaction completely (see graph below). This is due to the denaturation of the enzyme, specifically the active site. Care will be taken to ensure nothing is accidentally added to the mixture
Amount of time allowed for reaction to occur:
As this is an experiment testing qualitative results, there is no fixed time, within which each experiment must be completed. All experiments will be continued until there is no change in the colour of the iodine. All experiments begin once the substrate has been added to the enzyme.
Controlling the factors will mean a fair test and relevant results. If a factor was not controlled results produced would be inaccurate and a relation could not be drawn between the substrate concentration and amylase catalysis.
Controlling the independent variable:
The independent variable in this investigation will be the substrate concentration. Different percentages of starch solution will be used. The highest starch solution will be 2%, and from this solution all other percentage solutions will be made. Diluting of the starch will be done using distilled water. There will be constant intervals in between each solution, and the volume of the starch solution, of any concentration, used in each experiment will be 5ml.
Measurement needed to make the solutions:
Using a range of concentrations allows for a range of results that are more accurate. A trend will be easily identifiable and so the connection between the independent and dependent variables will be distinguishable.
The solution will have to be stirred before a sample is extracted to ensure all areas of the solution have the same concentration of starch in them. This will give more reliable results.
Apparatus:
Starch. Concentrations 2%, 1.6%, 1.2%, 0.8%, 0.4%. 5ml needed of each one for every experiment.
A wide range of concentrations allows for trends to occur and also anomalies can be easily spotted. A wide range of results will also give more reliable results.
Amylase. Concentration of 0.1%. 5ml needed for each experiment.
The concentration must be fixed to ensure that the independent variable can be measured against it. 5ml of amylase is a sensible amount as it allows for good reaction, from which a sample can be extracted.
Distilled water. Distilled water is to be used to wash all the equipment before the experiment in order to clear it of any contaminating substances. Also the distilled water is to be used to dilute the starch solutions to their correct concentrations.
Test tubes, test tube racks. The test tubes are to be used as the medium for the reaction. Also, separate test tubes will be used to mix up the different concentrations of the starch solutions. The test tube racks will be needed when the test tubes are not.
Water bath 60oC. The water bath must be used to ensure that the temperature of the enzyme is kept constant. 60 degrees C is the desired temperature for amylase as this is its optimum reactivity temperature. Variations in temperature would result in changes in reactivity and would therefore produce invalid results. The amylase and starch will be placed in the water bath, in separate test tubes, before the experiments begin, to ensure the whole experiment takes place at the required temperature.
Iodine. This will be used to test the presence of glucose. The colour of the iodine will start as a . Iodine will stunt the reaction of the amylase on the starch and so the drops placed in the iodine will not continue to produce glucose, and so the iodine will not continue to change colour.
Pipettes. In order to extract a small volume from the test tubes every thirty seconds a pipette will be needed. Two drops will be added to the iodine in the dropper palette. The pipette could also be used to stir the solution before extraction.
Stopwatch. Every thirty seconds a sample of the solution will be extracted and dropped into the iodine solution. In order to know when thirty seconds is up, a stopwatch must be used. If the timing were estimated then results would be inaccurate and unreliable.
Thermometer. The thermometer is needed to ensure that the water bath is at the correct temperature throughout the experiment. Fluctuations in temperature could cause inaccurate results.
Syringes. Needed when the solutions are being diluted. Small volumes of water and starch need to be added together. Syringes are able to very accurately measure the volumes. Accuracy is important, as changes in volume could determine the reliability and accuracy of the results.
Small beakers. These are not an important part of the experiment. They are needed to hold a small volume of the solution so they can be easily accessed during the experiment.
Dropper palette. Each specialised groove will be filled with two drops of iodine in preparation. The dropper palette is easily accessible, and as it has 12 grooves in it, the colour change will be easily visible.
Method and diagram:
First of all, all the equipment must be thoroughly rinsed out with the distilled water to ensure there is no contamination from previous experiments. Then place roughly 30ml of amylase in a small beaker and 30 ml in another beaker for easy access.
5ml of 0.1% amylase extracted and placed in a test tube, which in turn is placed in the 60 degrees C water bath, to prepare it for the reaction. Also 5ml of the 2% starch is placed in the test tube, then in the water bath to prepare it. Whilst these are getting to the correct temperature, preparation of the other solutions can take place.
4ml of the 2% starch added to the 1ml distilled water to get 5ml 1.6% starch solution in a test tube, and put in the test tube holder/rack.
3ml of the 2% starch added to the 2ml distilled water to get 5ml 1.2% starch solution in a test tube, and put in the test tube holder/rack.
2ml of the 2% starch added to the 3ml distilled water to get 5ml 0.8% starch solution in a test tube, and put in the test tube holder/rack.
1ml of the 2% starch added to the 4ml distilled water to get 5ml 0.4% starch solution in a test tube, and put in the test tube holder/rack.
Add two drops of Iodine to each groove in the dropper palette and place it next to the water bath.
Use the thermometer to test the temperature of the 0.1% amylase and 2% starch that have been put in the water bath. When the temperature reaches 60 degrees centigrade, add the amylase to the starch carefully and stir with a glass rod for five seconds. At the same time starting the stopwatch.
Every thirty seconds extract a very small volume of the solution with the pipette and drop two drops in the dropper palette, starting with the second groove. This is so the colour of the pure iodine is still visible by looking at the first groove:
In between extractions the temperature on the thermometer must be checked to ensure there has been no changes or fluctuations in the water bath, therefore affecting the rate of reaction in the test tubes.
The experiment ends when the colour of the iodine is no longer changing. The process will be repeated for all the other experiments involving the 1.6%, 1.2%, 0.8% and 0.4% starch solutions.
This method is a reliable method that was established by performing pilot experiments. Two changes were made from the pilot experiments:
Before carrying out the pilot experiments, it was not known that the iodine would completely halt the reaction of the amylase on the starch. In the first experiments the iodine was added to the starch at the same time as the amylase, and so there was no colour change. This is due to the fact that the iodine will disrupt bonds in the amylase, therefore rendering it useless for the experiment.
Secondly the temperature of the water bath was changed from 40 degrees C in the first experiment to 60 degrees C in the final experiment. It was assumed that the optimum temperature of the amylase was 37-40 degrees C as it is found in human digestive systems, but in fact the true optimum temperature is near to 60 degrees C.
Consideration and prediction:
Taking in to consideration all the information researched and shown above, my prediction has close correlation to my original hypothesis.
The rate of the enzyme-catalysed reaction will occur at a faster rate with an introduction of a higher concentration of substrate, and the rate of reaction will only be bounded by the availability of active sites.
Graph of prediction:
Safety and ethical considerations:
Lab coats and goggles should be worn at all times.
Care must be taken when handling the iodine, as it is toxic and corrosive. A clean pipette should be used to transport the iodine from the beaker to the test tube.
There are no ethics to take into consideration, as there are no animals being used in this experiment.
Results:
Here are the results of all the experiments:
The first and second trial experiments were wrong and so the results are useless.
Third trial experiment:
First exam experiment:
Repeat exam experiment:
Second repeat exam experiment:
Average results table, for all four experiments:
Graph showing average results:
Analysis:
There is an obvious trend visible from the graph. There is a strong positive correlation. This shows that as starch solution percentage decreases the time taken for the experiment to end increases.
This means that the starch is being broken down much more quickly, when there is a higher percentage of starch molecules in the solution. A higher concentration of starch molecules means that a collision with an amylase enzyme is more likely and so the production of glucose should theoretically, be faster.
There was one anomalous result in the repeat exam experiment. The time taken for the experiment to end at 1.2% starch solution is much quicker than expected.
This could be due to many factors. Firstly the equipment used in the experiment may not have been thoroughly washed and so there may have been contamination from outside sources. Secondly the 1.2% solution may have been diluted badly. The results would suggest that the solution had a starch solution nearer to 1.6% than 1.2%. This would have been down to human error. Not only the concentration of the starch may have been at fault, possibly the wrong amylase solution was added to this particular experiment.
Conclusion:
A higher concentration of starch solution reacting with 0.1% amylase will have a higher rate of reaction and therefore produce the desired products, in this case, glucose more quickly.
It can be assumed that if the 0.1% amylase were to react with a starch solution of increasing concentration, the results would continue the trend shown by the results graph.
Evaluation:
Variability. This could be due to any of the variables altering at any point in the experiment, but this kind of alteration would be down to human inaccuracy and fault. As this experiment relies upon human perception, due to its qualitative nature, variability is bound to occur due to differences in human perception. A perfect graph for this experiment would theoretically look like this:
Reliability and Accuracy. Inevitably this experiment will have a degree of inaccuracy and unreliability, as it involves human perception. But as the graph of results show, there is a strong correlation and a good trend, so we can assume that the experiment was, to a degree, successful and reliable. Also the experiment was repeated four times to gain an average and from these experiments there was only one anomalous result present, which shows consistency and reliability. Therefore due to the reliability of the experiment, the conclusion drawn from it proves to have a reliable source.
Limitations.
- accuracy of human involvement
- time
- ethics and morality
- safety concerns
Human involvement limits the accuracy of the results and time limits the range of results being more expansive.
Further investigations:
As explained above, this kind of experiment relying upon human perception gives results that are the opinion of the experimenter and not discrete results, i.e., one value or another. As a result, further experiments testing the rate of reaction of substrate concentration and amylase will have to involve a way of measuring the glucose concentration produced from the reaction. This experiment would also show that the rate of reaction increases as substrate concentration increases, but it would have quantitative results, which are more reliable, instead of qualitative, based upon opinion.
Further experiments could involve changing the amylase concentration, whilst the substrate concentration remains constant. Results would show that the rate of reaction increases as the enzyme concentration increases and would only be bounded by the availability of substrate to break down.
Varying the temperature of the experiment would discover the optimum temperature of amylase. The rate of reaction would be fastest at 60 degrees C, and would become slower the further away from 60 degrees C the temperature was, and could even denature the enzyme at excessive temperatures.