I think that the starch in the reaction will be catalysed by the amylase using the lock and key method, because the amylase is a very specific enzyme and can only catalyse a reaction with the substrate starch. Therefore it can not be using the induced fit hypothesis, because if it did, then the active site of the amylase should be able to change it’s shape and catalyse other reactions with a different substrate.
PREDICTION
I predict that at very low temperatures such as 10 c, the reaction will take place very slowly. This is because the starch, water and enzyme molecules have very little kinetic energy and so the frequency and energy of the collisions will be very low, therefore slowing down the reaction. At this moment the enzyme is known to be deactivated.
I also predict that as the temperature increases the rate of reaction will also increase, but only to a certain point, the optimum temperature. After the optimum temperature, I predict that the reaction will slow down because the high temperatures will start to denature the enzyme molecules. This temperature is known as the optimum temperature.
I predict this because the collision theory states that there must be two things for a reaction to take place.
1) There must be particles colliding frequently.
2) The collisions that take place must have sufficient energy for the product to be produced.
So therefore, the increase in temperature will increase the kinetic energy making collisions more frequent and the particles will be moving with greater energy.
The collision theory also states that as the temperature increases by 10 c, the rate of reaction will approximately double so we can say that the increase in temperature is proportional to the increase in rate of reaction, but only to a certain point, the optimum temperature. The collision theory states this because the effect of temperature on the rate of reaction can be expressed as the coefficient Q10. The formula to work out Q10 is:-
Q10 = Rate of reaction at (x+10)c
Rate of reaction at x
This means that between 0-40c, the Q10 is 2, meaning that every 10c raise in temperature, the rate of reaction should approximately double. Outside this range of 0-40c, in an enzyme catalysed reaction, the rate of reaction falls and at approximately 60c, it stops completely.
As I said before, once the rate of reaction reaches the optimum, the reaction will slow down due to the denaturing of the enzyme molecules. The reason enzymes denature is because an increase in temperature causes the kinetic energy to increase, and the bonds holding the globular structure(tertiary structure) of the enzyme molecule are to weak to withhold too much pressure and energy. The bonds are so weak that too much energy can break the bonds, and if these bonds break, then the enzyme molecule will lose it’s three dimensional structure and distort the active site. Once the active site loses it’s shape, it can no longer bind with the substrate molecule. When an enzyme molecule is denatured, it can no longer work as a catalyst and so the more enzymes that denature, the less catalyst molecules in the solution, therefore slowing down the reaction.
I predict that the optimum temperature should lie in the range between 37-45 c. I know that 37 c is not the optimum temperature because these type of reactions can take place in a test tube under a lot of heat and pressure. The only reason why this heat and pressure is not applied in the body is because the living cells in our bodies can not survive it. Another reason why the optimum temperature may be higher than 37 c is because when a person has a fever and the internal temperature has increased by a few degrees, some enzymes in the body may denature, but the amylase still works because our food is still being digested by the use of this enzyme.
I have also predicted what my graph may look like at the end of my experiments. I can use this graph to compare the actual graph at the end to see how close my prediction was.
PRELIMINARY WORK
Before I carry out my main experiment, I had to do some preliminary work to help me decide which starch concentration and what volumes of starch and amylase would be suitable for the experiment and give accurate, reliable results. I also had to choose the right technique in which to carry out my experiments.
Starch concentration
My first preliminary experiment was to find the suitable starch concentration. The apparatus and substances used for this experiment are listed below:-
I made different starch concentrations by diluting the starch with water. The highest starch concentration I could use was the stock solution which was 4%. The volumes of starch and water that were diluted are shown in a table below. The total amount of solution made for each sample was 100cm. As this was a lot of solution for each sample, it would be a waste and so I divided the volumes by 10 so that I would only be making 10cm of solution. The final volumes I used to make my solutions are shown in the table.
To make my solutions, I used the burette to measure and fill a test tube with the starch, and used a pipette filler to measure the water. To this I added a drop of iodine and the solution went blue/black, this indicates that there is starch present in the solution. I did this for all nine samples. Once I had my samples, I took each one and added 10cm of amylase using a pipette and at the same time, I started the stop watch. Once the solution went clear, I stopped the watch as it meant that the starch was completely digested. Just to make sure, I added a drop of benedicts solution to see whether there was glucose present in the solution.
The results are shown below:-
From my results, I have decided that the 0.5% starch concentration is the best to use for my main experiment, as it will take the least time to digest enabling me to obtain my results faster. There will be one disadvantage that could possibly make my final readings inaccurate, and that is that the reaction may occur too quickly at high temperatures and will prevent me from getting an accurate measure of the time taken. In this case, I would have to use a stronger starch solution for the higher temperatures, if necessary.
Volumes of starch and amylase
Next, I carried out another experiment to help me decide what volumes of starch and amylase are suitable for the experiment. The apparatus and substances used for the experiment are listed below.
I took 10cm of 0.5% starch solution from the burette, added the drop of iodine and using the pipette, I mixed it with different amounts of amylase each time. I noted down the time taken for the starch to be completely digested and after each experiment, I washed out the test tube, so that it is a fair test. The results are shown below.
From my results, I decided that I would use 10cm of starch and 2cm of amylase, because it was the most appropriate one. If I used 10cm of starch with 1cm of amylase, it would take a long time to obtain results, especially when experimenting with low temperatures. If I used 10cm of starch with 3cm of amylase, it may be difficult to get accurate results when experimenting with high temperatures because the reaction might take place too quickly.
The technique
There are two possible techniques that I could use for the main experiment. they are the spotting tile technique and the test tube technique.
SPOTTING TILE TECHNIQUE
In this technique, the idea was to put some iodine in each section of the spotting tile before the experiment actually started. I then took 10cm of the starch solution and to that I added 2cm of amylase. At the point when they were mixed, I started the stop watch. I took some sample from the test tube and put it in each section of the spotting tile (which already had the iodine in it) every 30 seconds. Each time I took a sample, the iodine indicated and showed me whether the starch had been digested. At first, the sample should turn blue/black as there would be starch present. If or when the starch is completely digested, the solution should remain the same colour as the iodine, which is brown.
TEST TUBE TECHNIQUE
In this technique, I measured 10cm of starch in to the test tube. To this I added two drops of iodine. Due to the starch present in the solution, the solution went blue/black. To this I added the 2cm of amylase and started the stopwatch. Once the solution went transparent, it meant that the starch had been completely broken down and so I stopped the watch.
The best technique
After analysing both techniques, I chose to go along with the test tube technique as it will give me a more accurate set of results which are more reliable than the results I would get if I went along with the spotting tile technique.
The major problem the spotting tile created was the fact that I could not decide when the sample taken from the test tube remained the same colour as the iodine when reacted together. The reason for this problem may be due to the amount of sample taken each time. Some samples, when taken, had a larger volume than others and so this could have effected the colour, thus making my results unreliable.
The results I get from this technique are also inaccurate because I only knew what was happening in test tube every 30 seconds and didn’t know what was happening in between e.g. If I took a sample that roughly, but not exactly the same colour as the iodine and then after 30 seconds, took another sample, and this time it was identical as the colour of the iodine, I wouldn’t know when in the period of 30 seconds, the reaction was actually over, thus giving me inaccurate results which are unreliable.
Another problem with this technique was that each time I took a sample, I didn’t know how much starch and amylase I was taking out of the test tube e.g. I might have taken more starch out than amylase, changing both of their concentrations in the test tube. This can effect the rate of reaction as I am introducing a new variable, and their for it would not be a fair test.
Unlike the spotting tile technique, the test tube technique is much more quicker. This technique is more accurate and reliable because the iodine in the solution made it easier to clarify whether the starch was digested. This technique also pinpointed the exact time for when the reaction was over, thus giving me a more accurate result.
The test tube technique does have a disadvantage, and that is the fact that the iodine is being mixed in to the solution at the time of the reaction.
The purpose of the iodine is to indicate whether there is starch present in a solution. If there is starch present in the solution, and iodine is added to it, the solution shoulc go blue/black, to indicate that there is starch present. The iodine, in this reaction, acts as a competitive inhibitor. The iodine molecule has an identical three dimensional structure as the starch molecule, causing it to bind with the active site of the amylase molecule. If the iodine is joined to the amylase, it prevents the starch from binding to the amylase molecule, and so the starch can not be broken down as the iodine has taken it’s place, therefore effecting the rate of reaction by slowing it down.
This can effect the rate of reaction, but the problem can be resolved. In my main experiment, I will only use 1 drop of iodine to mix into the solution. This way, the pattern of the rate of reaction will not be disturbed, as all the volumes in each experiment will be identical.
From these preliminary experiments, I have decided to use 10cm of 0.5% starch solution, 2cm of 1% amylase, 1 drop of iodine and use the test tube method. I’m also going to use burettes and pipette fillers as measuring instruments as they are more accurate than measuring cylinders and normal pipettes when measuring the starch and amylase. I also took readings from the burettes and pipette fillers at eye level to prevent parallax errors. When measuring a volume, I always took the reading that was at the meniscus.
THE EXPERIMENT
After doing the preliminary work, I started my main experiment. The apparatus and substances are listed below.
I first collected my equipment and set up the experiment. I put some water to boil in a glass beaker on the Bunsen burner. In the second beaker, I had ice. These beakers were there to help me keep the temperature of the solutions constant.
To start my experiment, I measured 10cm of starch which was in the burette, and poured it into a test tube. To this, I added a drop of iodine. I then used a pipette filler and measured 2cm of amylase and poured into the second test tube. I started with the lowest temperature and worked my way up. I put a thermometer in each test tube and until the starch and amylase were the same temperature, I didn’t mix them. To make them the same temperature (10 c as it was my lowest temperature), I kept the test tubes in ice and if the temperature fell too low, I moved them into the beaker of hot water. Once they were the same temperature, I poured the amylase into the starch solution, and at the same time, start the stopwatch.
Once the solution had gone transparent, it meant that there was no starch present, meaning the reaction was over. I stopped the watch and noted down the time it took for the starch to be completely digested. To check whether the solution was transparent, it was best done by placing white paper behind the test tube, and this way, any colour that was present in the solution would be visible. Just to make sure at the end of the experiment, I added a drop of benedicts solution, to show me if glucose was present. I then washed out the test tube and repeated the experiment until I got all my results.
To maintain a fair test, I made sure that I used the same volumes of starch, amylase and iodine. I used only one drop of iodine because otherwise it would take too long for me to clarify whether all the starch was digested.
I also had to maintain a constant temperature for each temperature I was working with, because if the temperature decreased or increased, it could effect the resulting outcome and the results wouldn’t be very reliable.
To keep the temperature constant, I had to keep a thermometer in the test tube with the reaction taking place, and keep moving it to and from the hot and cold beakers. Although this was the only option, this method didn’t work very well because the temperature did rise or fall by a couple of degrees and so it makes it difficult to believe whether the results are reliable or not. Due to this, I repeated the experiment three times for each temperature and I will use this to find an average enabling me to achieve a more reliable set of results.
MY RESULTS
Table one shows the results I obtained from the experiment. I recorded the time in minutes correct to one decimal place. I then found the average time taken for the starch to be digested at each temperature. The formula to work out the rate of reaction is:-
RATE = 1 / time
TABLE 1
*Anomalous results, therefore repeated. The new result is shown under the old.
From looking at my results table, I can see that my results look very reliable, because after repeating the experiments three times, my reading for each tempreture are quite similar when comparing.
My results show me that the rate of reaction increases, as I increase the temperature, which is what I predicted. At 10 c, the rate of reaction was very low and the reaction took very slowly. On the other hand, at 60 c, a more higher temperature, the rate of reaction was very high, infact the reaction took place almost immediantly. At 70 c, I did not get a reading because the amylase was not able to survive the high temperature and so it denatured. This meant that it can no longer work as an enzyme. At the end of each experiment, I did add a few drops of Benedict’s solution into the to indicate whether there was any glucose present. Up to 40 c, the Benedict’s solution indicated that there was a very high concentration of glucose in the solution. At 50 c, Benedict’s solution indicated that there was a medium concentration of glucose and at 60 c there was a very low concentration of glucose present in the solution. This shows that although the rate of reaction was increasing, the amylase was not actually breaking down the glucose at high temperatures, such as 60 c. The reason this might be is because as the temperature increased after 40 c, more and more amylase molecules were becoming denatured.
To see whether my results are accurate, I am going to compare my results to another group’s results. The group used the same volumes of starch, amylase and iodine and carried out an identical experiment. Their results are shown below, with the average time and the rate of reaction.
TABLE 2
Table two used slightly different temperatures, but they still obtained similar readings. At 60 c, there is a huge difference between the rate of reaction in table one and the rate of reaction in table two. This shows that for this temperature, there has been an error. This group had the same outcome for 80c as I had for 70 c.
I will now plot both sets of the average times on a graph to compare them visually.
From the analysis of the graph, there was a big difference with the time taken for the digestion of starch at low temperatures. At about 30 c onwards, there was a huge ressemblence between the results.They even shared the same line of best fit perfectly. This shows that although there was a big difference at low temperatures, the results were very similar. This also shows that our results look quite reliable at this early stage.
Now I am going to compare our rates of reaction along with the Q10 to see if they have a relationship between them. The table below shows the Q10 for my rates and the Q10 for the other group’s rate. The formula to work out Q10 is:-
Q10 = Rate of reaction at (x+10)c
rate of reaction x
The Q10 theory stated that the relationship between the rate of reaction and temperature was that they were prortional, but thats only for the temperatures between the range of 0-40 c. It stated that for every 10 c rise in temperature, the rate of reaction will approximatley double. It will double because if the Q10 is worked out for any temperature within that range, the Q10 will always be 2.
After working out the Q10 for both sets of results, it looks like my resuls look more accurate and reliable than the other results because my results have clear evidence that the temperature was almost proportional to the rate of reaction, as the Q10 was frequently 2. After 40 c, the Q10 for my rates were no longer 2 and so the temperature and the rate of reaction was no longer proportional. I had predicted that after 40 c, the reaction would slow down, but my rate of reaction still did increase all the way to 60 c, and then stopped compltely. However, the other groupes results also showed the rate of reaction increasing untill 60 c, but their Q10 shows that it was not increasing proportionally and that after 40c, the rate of reaction increased slowly. From this I conclude that the Q10 theory is the truth, and my results partially show this, but not completely. This could because my experiments were not 100% reliable and accurate.
The graph of my rate of reaction against temperature has a similar pattern to the Q10 graph. This shows that my results are fairly reliable. The other group’s graph starts of being almost identical to my graph. This also shows that my results are fairly reliable. Both the graphs have a similar pattern to the Q10 graph at the begining. The only difference is the optimum temperature. The Q10 graph shows it’s optimum temperature to be about 40 c, whereas the other two graphs show their temperature to be between 50-70 c. What looks odd though is that at 60 c, my graph shows the rate of reaction to be very high compared to the other group’s rate of reaction. Between 50-60 c, the rate of reaction for the other group hardly increased, whereas my rate of reaction at 60c went up rapidly from 50 c. This shows that at this point, the results are not reliable and that there must have been an error in one of the experiments, or there was a lack of accuracy in the experiments.
When I look back on my prediction, I had predicted that at low temperatures, the reaction would take place slowly as there wouldn’t be suffecient kinetic energy. My graph does agree with this prediction. The lowest rates of reaction did take place at the low temperatures. The low temperatures didn’t seem to be giving particles enough kinetic energy and force for frequent collisions to take place. This is what made the reaction take place very slowly.
I had also predicted that when the temperature is increased, the rate of reaction will also increase, but only to the optimum temperature. A fter the optimum temperature, I predicted that the rate of reaction will start to decrease. However, I predicted that the optimum temperature would lie between 37 -45 c. From looking at the graph, the rate of reaction was still increasing after 45 c. The high temperatures were giving particles a lot of kinetic energy and force, and so collisions were more frequent, enabling the reaction to take place faster.
My graph shows me that the opyimum temperature lies between 60-70 c because at 60 c, the reaction took place almost immediatly and at 70 c, the amylase started to denature due to the high temperature. The amylase could not withold the huge amount of energy given at +70 c, and so the three dimensional globular structure of the enzyme was destroyed, leading to the distortion of the active site. The amylase had precipitated out of the solution, forming a cloudy foam. The blue/black solution of the iodine and starch went clear at 70 c. This may be because the high temperatures may have inturrupted the reaction between the starch and iodine.
Another prediction I had made was that, due to the coeffecient Q10, every raise in 10 c on the temperature would double the rate of reaction, but only to the optimum temperature. After working out the Q10 for my results, I would say that the rate of reaction did approximately double as I raised the temperature by 10 c each time. The rate of reaction may not have exactly doubled due to the lack of accuracy in the experiments.
I had predicted that the optimum temperature must be higher than the internal body temperature and my graph proved that this was true. However, I had also predicted that the optimum temperature would be lower than 45 c. This didn’t agree with my results. The reaction was still taking place at 60 c and it took place much more faster than it took place at 40 c and 50 c.
EVALUATION
After doing this investigation, I think my results are not 100% reliable because there were many factors in the experiments which may not have been completely accurate.
One factor was the iodine. Although the iodine was an inhibitor in the reaction, the pattern of the rate of reaction should not be effected because I used identical volumes of starch, amylase and iodine each time. However, the dropping pipette for the iodine may have had too much iodine at one time than it did at another. If this is true then it could have effected the pattern, making my results slightly unreliable. This could be changed and the volume of the iodine could be made more accurate by using a pipette that measures to two decimal places or by using a burette, instead of a dropping pipette.
Another factor was the starch solution. It may be possible that the starch suspension in the burette may not have been a true solution, because the starch is not fully dissolved in to the water and therefore the starch would settle at the bottom of the burette, whilst all the water settled at the top as it is more denser. If this is true, then some of the starch solution at the beginning of the experiment would have a higher concentration than the solution at the end. This would effect the rate of reaction as I would be introducing a new variable, thus making my results unreliable as it wouldn’t be a fair test. This could be changed by stirring the solution occasionally, mixing the starch and water into an equal distribution in the solution.
It was also confusing whether the reaction as over or not. At some points, it was difficult to see whether the solution was transparent or still pale blue. This may have made my results inaccurate. To solve this problem, I could use a device called a colourimiter to help clarify whether the solution is transparent.
The temperature of the solutions during the experiments were not always constant, and always increased or decreased by a few degrees. This also made my results inaccurate which didn’t make them very reliable. To improve this, I could use a number of water baths, each set up to a certain different temperature and to place the test tubes in. This way the solution would stay constant through the process of the reaction. In my experiments, I had used an alcohol thermometer. These thermometers are not very accurate and so in my experiments, when I took a reading from the thermometer, it may not have been a true temperature, meaning it wasn’t accurate and therefore it effected the results. This could be changed by using a mercury thermometer instead as they are the most accurate type of thermometers.
After obtaining my results, I realised that I had two set of results that were completely anomalous. I am not sure what cause them to be strange, but it may have been the temperature keeping. It was difficult to keep a constant temperature and I may have mistaken the temperature on the thermometer for another temperature.
For the second anomalous result, I realised I had put two drops of iodine instead of one, but I carried on with the experiment to see if it effected the rate of reaction, as iodine is an inhibitor. As the result was anomalous, the iodine had effected the outcome. I did repeat the anomalous results and obtained better readings. I didn’t use the anomalous results to find an average as they were untrue, therefore unreliable.
From looking at my results plotted on the graph, I could investigate further by carrying out experiments in between the range of temperatures, where the optimum temperature lies, to pinpoint exactly where the optimum temperature is. To see whether my conclusions are correct, I could set up another different experiment, for the same variable to see whether I get a similar pattern. I could do this in many ways. I could use the same volumes of starch, amylase and iodine, but apply the changes I made earlier. I could use a more accurate measuring pipette for the iodine, use set temperature water baths, use a starch suspension which is true and making it true where all the starch and amylase is equally distributed in the solution. I could also use the colourimiter to pinpoint exactly when the reaction is over.
From the analysis of my results and method, I can’t say that my results are 100% accurate and reliable, but I could investigate further in many ways by creating different experiments, which will give me a more accurate reliable set of results and a conclusion which is 100% reliable.
