Q10 = rate of reaction at (× + 10)°c
Rate of reaction at ×°c
If Q10 equals 2 then this will show that every 10°c rise in temperature does produce a doubling of the rate of reaction.
I think the temperature at which the enzyme will work the fastest (optimum temperature), and therefore make the rate of hydrolysis of starch the fastest is going to be between 37°c and 40°c. I think this is because body temperature is around 37°c and enzymes are designed to work at their maximum output.
I think after 40°c the rate of reaction will slow down very rapidly. This is because the amylase molecules will start to vibrate so energetically that the bonds holding them in their precise shape will not be able to withstand the high temperatures and so will start to break. The hydrogen bonds between the oxygen of the –CO group of one amino acid and the hydrogen of the –NH group of another amino acid are fairly weak and so will break at temperatures above 40°c. The hydrophobic interactions occurring between the non-polar side chains will also break. This means that the active site of the amylase to which the starch molecules bind to will start to lose its shape as more and more bonds break. Therefore the starch molecules will fit less and less well into the amylase’s active site and so cannot be hydrolysed, decreasing the rate of reaction. Eventually which I think will be at 55°c the starch molecules will no longer be able to fit into the active site, so that the amylase enzymes will become denatured. No starch will be hydrolysed making the rate of reaction 0.
Analysis of Variables
Independent Variable
The independent variable is going to be temperature. The range that I am going to use is firstly 0°c, to see whether the reaction takes place at all, then, 10°c, 20°c, 30°c, 37°c, 40°c, 50°c, and 60°c. The dependant variable is going to be the percentage of starch hydrolysed. I am going to use a thermostatically controlled water bath to obtain the different temperatures.
The influential variables are firstly the concentration and volume of the amylase in the solution. This must be kept constant because if different concentrations or volumes of amylase is used at different parts of the experiment, then those tubes with more amylase enzymes will have more active sites for the starch molecules to bind to. This will increase the rate of hydrolysis of the starch, as there will be more of a chance of the enzyme-substrate complex forming. I will keep this constant by measuring exactly the same amount of amylase solution each time, at exactly the same concentration using a syringe. Secondly, the starch concentration and volume must be kept constant. This is because the more starch molecules there are for a given enzyme concentration the more chance there is that they will collide with the amylase molecules, and therefore bind to its active site. Hence, increase the rate of hydrolysis of starch. I will keep this constant by measuring exactly the same amount of starch each time at exactly the same concentration. I will use a syringe to be more accurate than for example using a measuring cylinder. The pH must also be kept constant. This is because changing the pH changes the hydrogen ions in a solution which can ‘alter the ionic charge of the acidic and basic R groups which help maintain a certain shape or conformation of the molecule’. If the shape of the active site of the amylase is disrupted then the starch molecules will not fit as well, and therefore decrease the rate of reaction. I do not think the pH will change very much, but to be certain I will use litmus paper to check that the pH remains constant. The time that we leave the amylase and starch molecules to react has to be kept the same. This is because keeping it longer at different temperatures will allow the amylase and starch molecules to have a longer time in reacting, so that one that has been kept in for longer will have more time to collide and form the enzyme-substrate complex and hydrolyse more of the starch than one that has not. I will keep the time constant by using a stopwatch and taking the colorimeter readings at the same time. It is important that these influential variables are kept constant as they could affect the validity of my results. This is because I will not be sure whether it is the temperature or some other variable that has given me a certain result.
Apparatus
3 × boiling tubes to put the amylase solution into two of the tubes, and distilled water into one of them.
3 × boiling tubes to put the starch solution into and to put the amylase and distilled water when digestion is started.
Thermostatically controlled water bath to accurately maintain the temperature at the eight different ones I am investigating.
Test Tube holder to hold the boiling tubes and colorimeter tubes.
Thermometer, which is going to be used to check whether the solutions are at the right temperatures.
Amylase solution as that is the enzyme that I am using to investigate the effects of temperature.
Starch solution as that is what amylase enzyme is specific to break down.
Iodine in potassium iodide solution as that is what I am using to investigate how much starch is remaining or has been hydrolysed.
Colorimeter to check the intensity of the blue colour.
18 × colorimeter tubes in which iodine will be placed and the solution of amylase and starch.
Syringes to measure out the solutions and iodine out accurately.
Stopwatch to make sure each one has been kept in for the same time.
Safety
As iodine is an irritant to the skin, I will wear gloves to protect my hands in case spillages occur. I will wear a lab coat at all times during conducting the experiment to avoid any iodine falling onto my clothes or skin. However, if accidents do occur I am going to wash the area with soap and water and then seek help. As iodine irritates the eyes I will wear safety goggles, however if any does fall into the eyes I will irrigate them with water and seek medical attention. I will be careful not to get burnt when using the water baths at high temperatures. I will try to avoid any accidents by working in plenty of light and in a well-ventilated area.
Method
- Firstly, place 1cm³ of iodine solution in each of the 18 colorimeter tubes and place them in the test tube holder.
- Prepare the colorimeter by setting it to zero using a colorimeter tube filled with distilled water.
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3 boiling tubes should be labelled A, B and C. To each tube, 30cm³ of 0.1%(g/100cm³) starch solution should be added and then placed in a water bath at 0°c.
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Another 3 more boiling tubes should be taken and labelled 1,2 and 3. 10cm³ of 1% amylase solution should be added to tubes 1 and 2, but 10cm³ of distilled water should be placed in tube 3. This will be my constant. All three tubes should be put in the water bath at 0°c.
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A thermometer should be used to check that the contents of all 6 tubes are at 0°c.
- The digestion should be started by pouring the contents of tube 1 into tube A and the contents of tube 2 into tube B and the contents of tube 3 into tube C. All the tubes should be mixed well with a glass rod and the timer started immediately.
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5cm3 of the solution should be withdrawn at 0min from tubes A, B, and C and each placed into one of the colorimeter tubes with the iodine in. Shake the tubes gently to mix, and then place them in the colorimeter one by one to record the absorbance. The colorimeter should be set to zero before each reading.
- Step 7 should be repeated for the times 2, 4, 6, 8, and 10 minutes.
- The results should be recorded in a table and an average of the absorbance took for each time.
- Numbers 1-9 should be repeated but at temperatures of 10°c, 20°c, 30°c, 37°c, 40°c, 50°c, and 60°c.
I am going to use this method because I think it will be more accurate than the dimple tile method. This is because in the dimple tile method you have to decide the colour intensity of the blue by comparing it to the colour of one where the starch has been completely hydrolysed, whereas with a colorimeter it is a machine, which accurately determines the absorbance. In addition, you cannot tell the colour intensity in between with the dimple tile method, and so will have to wait for when all the starch has been digested which could take a considerable amount of time. Also timing how long it takes for all the starch to be hydrolysed instead of measuring the intensity at set times would be further inaccurate, because you could never be sure if all the starch has been hydrolysed by just comparing the colours. Although it would be easier and more practical, I am not going to mix the starch, iodine and amylase solution in one tube and then take regular readings of the intensity in this one tube in the colorimeter, because the iodine will interfere with the rate of the reaction and slow it down.
Control
In my plan, the control is going to be tube C, which will have the starch and the distilled water in. I need to use the control to show that it is the amylase enzyme that is causing the hydrolysis of starch and not some other factor. The absorbance of the control must stay the same during each reaction. I also need the control to determine whether any starch is hydrolysed without the use of an enzyme, as at the higher temperatures the starch may have enough activation energy to form into the product. The greater the change in absorbance between a sample of starch without enzyme (which is going to be my control) and the mixture containing the enzyme, the greater the amount of starch hydrolysed by the enzyme, therefore the greater the rate of reaction.
I will repeat the experiment twice, and once with the control. I will also compare my results with other investigators to determine any anomalous results or if the entire experiment has not gone to plan.
Results
I will extend this table for the temperatures 10°c, 20°c, 30°c, 37°c, 40°c, 50°c, and 60°c.
Graphs
I will draw my graph percentage starch remaining against time for each of the eight temperatures that I am investigating. I am going to use my calibration curve to look at how much starch has remained at the specific absorbances. I got my calibration curve by taking 12 boiling tubes. In the first one 0.5% starch solution was placed, then along the remaining 11 tubes the concentration of the starch was halved each time using water to dilute them, so that the starch concentration became less and less. Each of the 12 solutions were put into colorimeter tubes with iodine in and the absorbance was recorded. From the results, I drew a calibration curve; absorbance against starch concentration, so I was able to see how much starch remained at a certain absorbance.
I think the trend I am going to get from my experiment is that the temperatures of 0°c and 60°c will produce a straight line on my graph of which the gradient is always zero and therefore the rate of reaction is zero. This is because I think none of the starch will be hydrolysed. I think for the temperatures 10°c, 20°c, 30°c, 37°c, and 40°c the curve of the graph will become steeper and steeper, with 40°c having the steepest curve. I think the graph for 50°c will be less steep than that for 37°c and 40°c.
I am going to calculate the rate of reaction for each temperature by calculating the initial rate of reaction of each temperature, from the above graph. That is calculating the slope of the curve right at the beginning of the reaction by finding the gradient. I think the rate of reaction graph is going to rise steadily from 0°c then it will peak in between 37°c and 40°c and then decline quite rapidly
Bibliography
Jones. M, Fosbery. R, Taylor.D. (2000); Biology 1 pp Cambridge University Press
Ann Fullick (2000); Biology Heinemann Advanced Science
Exam.net textbook; Enzymes
Teacher’s notes