Amylase is found in pancreatic juices released by the pancreas in the small intestine and in the saliva released by the salivary glands in the mouth. The enzymes that make up diastase hydrolyse the different glycosidic bonds that hold the glucose residues together in starch. Amyloglucosidase hydrolyses the α-1,4 and α-1,6 glycosidic bonds, as a result terminal glucose units are removed at the end of the chain. Pullulanase hydrolyses the α-1,6 glycosidic bond at the branching points, giving dextrins. α-amylase hydrolyses the α-1,4 glycosidic bonds, generating dextrins, maltose and glucose. β-amylase hydrolyses alternate α-1,4 glycosidic bonds and accordingly produces maltose.
In this experiment I will be using α-amylase glucoamylase (amyloglucosidase), this functions best between pH4 and pH5.
Apparatus
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150cm3 0.5% starch solution – to be used as the substrate, it is use as a solution to increase the surface area on which the enzyme can act and to be able to mix with the enzyme.
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250cm3 2% diastase solution – to be used as the enzyme that breaks down starch, this is also in a solution to increase the surface area and so it can mix with the starch.
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Iodine in potassium iodide solution – the iodine reacts with the starch to turn blue/black in colour, so it can used as an indicator for starch. The iodine is in the potassium solution as iodine is able to dissolve in water when in the presence of potassium. The colour is formed as a result of a complex formed between the iodine molecules and the amylose helix. This is also in solution so it can mix with the other solutions.
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10cm3 measuring cylinder – this is used to measure out the bigger volumes of liquid accurately.
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2 x dropping pipettes – these are used to transfer the liquids from one container to another, more safely and accurately than pouring.
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pH buffers 4,5,6,7,8 and 9 – these are used to control the pH of the solutions so we can assess the effect of pH on enzyme activity. There is no need to go all the way from pH 1 to pH 14 as we already know that enzymes are denatured at extreme pHs.
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6 x beakers – these are needed to hold the pH solutions so we can extract them easier.
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Stopwatch – this is needed to time how long the starch and diastase solutions have been mixed.
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Colorimeter with 700nm (red) filter – the colorimeter is used to accurately establish the intensity of the colour of the solution, as when the solution is a blue/black less light is able to pass through and so the reading is lower. The red filter is used as it is on the opposite side of the spectrum to the blue.
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6 x cuvettes – these are used to hold the solutions, so they can be put in the colorimeter as regular sized test tubes will not fit in it.
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2cm3 syringe – this is used to accurately measure small volumes of liquid.
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1cm3 syringe – this is used to accurately measure very small volumes of liquid.
Method
- Set up the colorimeter with 700nm (red) filter and set it accurately with water in a cuvette.
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Prepare 2cm3 of 0.5% starch solution in a 2cm3 syringe, making sure that it is from the bottom of the meniscus that you are taking the measurements.
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Prepare 4cm3 of 2% diastase solution in a 10cm3 measuring cylinder, using a pipette, making sure that it is from the bottom of the meniscus that you are taking the measurements.
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Prepare 1cm3 of pH 4 buffer in a 1cm3 syringe, making sure that it is from the bottom of the meniscus that you are taking the measurements.
- Empty the starch solution in a cuvette and add the pH buffer to it.
- Then add the diastase solution and begin timing for 4 minutes.
- At the end of 4 minutes add 2 drops of the iodine in the potassium iodide solution, using a pipette, to the starch/diastase solution and give a quick swirl to mix it.
- Place the cuvette carefully in the colorimeter and place cap.
- Take the reading from the colorimeter and record it.
- Before every experiment reset the colorimeter.
- Repeat the experiment until the results are concordant, and then repeat with the other pHs.
Risk assessment - enzymes
- Irritant – all enzymes are potential allergens and should be handle so as to minimise contact or inhalation, they can cause asthma and/or irritate the membranes of the nose and eyes.
- If it is swallowed – wash mouth out and give a glass or two of water. Seek medical attention.
- If it gets in the eyes – flood eye with gently running tap water for ten minutes. Seek medical attention.
- If spit on clothes or skin – remove clothing, flood area with water and wash thoroughly with soap and cold water
- Wear goggles, a lab coat and gloves for protection.
Variables
To make sure that the experiment is not affected by pH, we need to control any other variables. As discussed previously temperature, substrate and enzyme concentrations have an affect on the rate of reaction. Also the amount of time given for the experiment, the apparatus used and the way in which the solution is handled. To make sure none of these variables affect the experiment
- Temperature – we will take the temperature at the beginning of the experiment and we will not handle the solution too much so we don’t transfer any of our body heat to the solution.
- Substrate concentration – we will make sure that we use the same amount of starch each time and use the same measuring cylinder
- Enzyme concentration – we will make sure we use the same amount of diastase for each experiment and we will use the same syringe
- Time – we will begin timing as soon as all the diastase has gone into the cuvette and as soon as the 4 minutes is up we will add the iodine and put it straight into the colorimeter.
- Apparatus – we will use the same apparatus for each thing, e.g. we will use the same syringe for pH and a different one for diastase. Also when changing pH, the syringe will be washed out.
- Once every thing is in the cuvette we will give a quick shake and then leave it in a test tube holder.
Preliminary
We conducted a preliminary following the method above, but not using any pH buffers so it was a control. We also conducted another where we added the iodine from the beginning and put it straight into the colorimeter, and at the end of each minute for 4 minutes we recorded the results.
Preliminary results
We can establish from these results that the control worked well. However when we took the test tube out of the colorimeter on the second experiment we noticed that the iodine hadn’t mixed well with the other solutions so we decided to keep the method the same.
Results
Temperature = 20°C
Red = anomalies
Analysing evidence and drawing conclusions
The results that were obtained from the experiments have been presented in the table and graph on the previous pages. From looking at the graph it can be established that the optimum pH seems to be pH 5.4, this is quite close to the prediction. Furthermore, it can be seen that from pH 4 the transmission gradually increases and then peaks at pH 5.4, subsequently the transmission gradually decreases until pH 8 where it suddenly drops to pH 9. A low transmission means that the colour of the solution is very dark and the light can not be detected on the other side, so if the solution is dark it denotes that the iodine has reacted with starch and has turned a blue/black colour. Accordingly, when more starch has been hydrolysed by the diastase enzyme the iodine doesn’t react as there isn’t any starch and the transmission is higher. This indicates that after pH 8 the diastase enzyme becomes denatured and can no longer break the starch down. However the error bars on the graph show that the results obtained from pH 8 are not accurate as the error bar it very big, showing a lot of variation. For pH 8 the highest result obtained that wasn’t an anomalous result was 52% transmission and the lowest was 40% transmission, giving a difference of 12 units, which is considerably higher than the errors of other pHs. Also the anomalous result for pH 8 is very far off from the average, the anomalous is 78% transmission which is 32 units away from the average.
Looking at the other results, their error bars are not as substantial. Nevertheless there are still a few anomalous results. The anomalous result at pH 5 is only 10.3 units away from the average; this is not that considerable, however it isn’t in line with the other results. The error bar for pH 5 is also quite small, with only a difference of 4 units. pH 7 has two anomalous results, one at 79% transmission and the other at 78% transmission. These are very close together but they quite far from the average, the lowest distance being 11.25 units. The error bar for pH 7 is once again quite small with a difference of 5 units. pH 8 is the only other pH with anomalous results, this one is at 75% transmission, which is 12 units way from the mean, this is once again quite a considerable difference. The error bar for this pH is slightly larger than the other pHs (excluding pH 9); there is a difference of 6 units for this error bar.
pH 4 does not have any anomalies; also the error bar is the smallest, with only a difference of 3 units. pH 6 is another devoid of any anomalous results, however this error bar is not quite as small with a difference of 5 units.
When looking at the table it is apparent that both pH 4 and pH 6 have large numbers of readings, and a lot of them are very close together. pH 7 is another pH with many readings however the readings are quite scattered and it is hard to tell which should be the anomalies as the 78% and 79% transmission readings are very close together, and the 90% and the two 92% readings are also very close together.
The results obtained from pH 8 are very scattered, the biggest difference being between the 90% and 75% transmission readings, additionally pH 8 only has three readings. This shows great inconstancy deeming these results as inaccurate and unreliable. The same goes for the pH 9 results, even though there are four, they are very erratic and so they too are considered unreliable and inaccurate.
The line of best fit curves very suddenly but only the side with the higher pHs, on the other side of the graph the line curves but only slightly. However the line should be symmetrical if the theory that as the pH get further from the optimum the enzyme activity is reduced. Although, when you look closer at the graph it can be seen that the optimum pH (pH 5.4) is closer to pH 4 than pH 9 where it seems to have denatured. If I started the experiments at pH 2 or 3 then I am sure the graph would look more symmetrical.
Generally it can be seen that the results I have obtained are not very accurate and are not reliable. So any conclusions that have been drawn from them are not necessarily correct.
Evaluating evidence and procedures
From doing the experiment and after analysing my results it has become clear that my results are not accurate. This is due to a number of problems with the apparatus, and method. The results I have obtained are quite inconsistent and therefore unreliable. However they do follow a general rule, that as you get further away from the optimum pH the rate of reaction is lowered. The results also show that at pHs very far from the optimum have dire effects of the rate of reaction, however due to the fact that we only started at pH 4 we can not be sure of this for the lower pHs, but it is expected.
Also this is only one experiment, some of the errors may come down to human error, so the conclusions gained from this can not be widely used. To ensure the reliability of these results they would need to be compared to another experiment that was looking at the same factors as this experiment.
Looking back at our method and the equipment used, I can see many areas in which problems and inconsistencies may have arisen from. When measuring out the solutions in either the syringes or the measuring cylinder, the measurement must be taken from the bottom of the meniscus to be accurate, this may not have happened every time, resulting in more or less substrate or enzyme or pH buffer entering the solution. This would result in the rate of reaction increasing or decreasing. Also when changing pH solution, some of the previous solution may still have been in the syringe, even though we washed them out with water it is not certain that all of it was washed out.
The colorimeter also presented a lot of problems as it is a very sensitive and temperamental piece of equipment and so it would be easy to get unreliable results. When setting the colorimeter with water in the cuvette, it is extremely hard to set it exactly to 100%. Even if you did, when you take the cuvette out of the colorimeter and replace it with the solution in another cuvette the likelihood of something disturbing the setting is very high as just a knock on the table can alter the setting.
When setting the timer it is hard to know when to start timing as you could either do it when all the diastase has mixed with the other solution or when the first drop touches the surface of the other solution. We decided to start timing as soon as the first drop went into the other solution, however by the time you have said start, your partner has pressed the start button and the timer actually starting, the reaction has already started. As a result the timing was not very consistent.
When the four minutes had finished, the reaction didn’t just stop, it carried on going. Even when we were adding the iodine the reaction was still happening, and when we were putting the cuvette into the colorimeter the reaction kept on going, it was still going when we were reading the transmission. As a result the reading we got wasn’t for the 4 minute reaction but more like the 4 – 5 minute reaction, so the readings were possibly higher than they should be. Also when we first added the iodine it went black but it soon began to clear and by the time we had taken the reading it was very clear. However we couldn’t keep the solution in the colorimeter and then add the iodine straight after the four minutes, as the light that needed to pass through the solution was quite near the bottom and you needed to swirl the solution after adding the iodine so it would mix.
To get better results we would need more time to get more results so they become concordant and we would be able to get a better understanding of what was happening when you varied the pH. If it were possible, some sort of solution that could stop the reaction would be useful so that when we added the iodine the reaction wouldn’t keep on going. Also I would imagine that by using smaller amounts of each solution would mean that we would be able to add the iodine to the cuvette when it was in the colorimeter as the solution would need to be mixed, so we would get more accurate results. In addition, if we added more iodine we would be able to ensure more of it was mixing and quicker. Additionally we could use new syringes every time we changed pH buffer solution, this would ensure accuracy. Furthermore we could begin timing as soon as the measuring cylinder is overturned, so that by the time the solution has mixed, the timer would have started. Generally we would have to be extremely careful when using the colorimeter, to ensure the table wasn’t knocked. We would also have to place the cuvette in the colorimeter very carefully.
Despite the fact that there were many opportunities for the results to become unreliable and inaccurate, the results that the experiment did yield followed a general trend and followed what was expected when the pH of the solution was altered.
References
Adds J, et al (2000), ‘Molecules and Cells’, Nelson, ISBN 0-17-448293-0
Clegg C J, Mackean D G (1998), ‘Advanced Biology – principles & applications’, John Murray, ISBN 0-7195-5078-5
Taylor D, Jones M (1994), ‘Foundation Biology’, Press Syndicate of the University of Cambridge, ISBN 0-521-421993
Roberts M, et al (2000), ‘Advanced Biology’, Nelson, ISBN 0-17-438732-6