When looking at the ‘induced fit’ theory, it can also be applied to my experiment. My enzyme Savinase could structurally change shape so that the active site fits exactly around my substrate molecule and therefore hydrolyse the protein substrate.
There are various factors affecting the activity of an enzyme; pH, concentration of the enzyme, concentration of the substrate and temperature. In my experiment, I will be investigating the effect of temperature. A rise in temperature will give the enzyme molecules more kinetic energy that will cause the rate of reaction to increase. However, when the temperature is increased it can affect the stability of an enzyme which will alter the shape of the active site. If this happens, substrate molecules can no longer combine with the enzyme. All enzymes have an optimum temperature at which maximum activity is reached. At temperatures above the optimum temperature the enzyme quickly loses its activity and becomes denatured. As all enzymes are proteins they can be denatured by high temperatures. These high temperatures can cause hydrogen bonds and hydrophobic interactions to break and therefore resulting in the change of the shape of the enzyme molecule. As soon as the shape of the active site is altered the enzyme can no longer act as a catalyst as it can no longer hold its substrate molecules.
Another factor that can affect the activity of an enzyme is an inhibitor. An inhibitor joins to the enzymes active site but is not the true substrate. Therefore, it prevents the enzyme from working properly
A diagram to show
how an inhibitor works
Diagram from
Preliminary Work
In my preliminary work, I investigated the effect of temperature on the mass of casein present after ten minutes. I used Marvel™ powdered milk as a substrate and I made up my enzyme from its powdered state. I made my enzyme solution up to have a concentration of 3%. To obtain this concentration, I weighed out 3g of the enzyme powder and to this I added 97g of distilled water. The amount of substrate that I used was 5ml each time and to this I added 5 ml of the enzyme. The solution was placed in a water bath of 40°C and another solution was placed in a water bath of 50 °C.
List of equipment:
- Powdered enzyme- Savinase
- Marvel powdered milk
- Distilled water
- 2x 10ml syringe
- 7x test tube
- 2x beaker
- Mass balance
- Test tube rack
- Stopwatch
- ‘Albustix’
- Water baths of temperatures 10°C-60°C
Preliminary method:
- Firstly, the enzyme must be made up to the appropriate concentration. I chose to use a 3% concentration as it would be more effective than a 1% as more active sites would be available for the substrate to combine with. How I made my enzyme to this concentration is mentioned above. The solution was placed in a beaker.
- Next, I made my substrate solution from Marvel powdered milk and distilled water. This was placed into a beaker also.
- I then placed five test tubes into a test tube rack. To two of these test tubes I added 5mls of my substrate solution and to the other two, I added 5mls of my enzyme solution. To the other I just added some of my substrate solution.
- I added the four test tubes with the substrate and enzyme solutions in them to water baths of temperatures 40°C and 50°C.I left the four test tubes in the water baths for one minute before adding the test tubes containing the enzyme to the test tubes containing the substrate.
- I started a stopwatch and left the test tubes in the water baths for ten minutes.
- Whilst the test tubes were in the water baths I tested the test tube containing only the substrate solution to see how much protein was present in it. I did this using ‘Albustix’. The results are shown in the table below.
- After ten minutes, I removed the test tubes from the water baths and I tested each of them for the mass of protein present in them. The results are shown in the table below. The mass of protein is measured in grams/litre (g/l). The ‘Albustix’ are coated in an indicator that changes colour depending on the mass of protein present in the solution. There is a colour comparison chart on the side of the container in which the Albustix are contained and this chart acts as a scale, informing you of what mass of protein is present in the solution. The scale reads from negative up to >20g/l.
Results table 1
Analysis of preliminary work: When looking at my results, my hypothesis is proved correct, however, I can not base my experiment on these results entirely as only two temperatures were used and these will not give me a wide enough range of results. However, at 50°C there was a smaller mass of protein present compared to 40°C. This means that more protein was broken down at the higher temperature, therefore proving my hypothesis correct. I used Albustix to determine what mass of protein was present. A dark green colour represented more than 20g/l of protein whilst a lime green colour represented a trace of protein. There is a colour comparison chart on the side of the Albustix container that gives the colour and the mass of protein that the colour represents.These indicator coated strips give the amount of protein present, however they only read the following masses of protein: negative, trace,0.30g/l, 1g/l, 3g/l and >20g/l. Because of this I do not know the precise mass of protein present in my solutions however in order to provide quantitive data, the Albustix had to be used.
Health and safety/Risk assessment: I am going to be using a powdered/granulated enzyme in my experiment. As the enzyme will be in a powder form, there may be some dust present when the enzyme is being prepared for use, for example when the product is being measured out. Eyes and skin should be covered to prevent dust from coming into contact with them. Dust from enzymes can cause asthma or hay fever.
If any of the enzyme is spilt then it should be cleaned up immediately using cold water. When disposing of the enzyme it should be dissolved in water. If I come into contact with the enzyme dust, I must wash the affected area immediately with water. Eye protection must be worn at all times when dealing with enzymes and general safety in the classroom should be carried out such as putting stools underneath work benches etc. There are various water baths being used in my experiment that are all set to a variety of different temperatures. Care should be taken to prevent burns and scalding from the hot water. Also, various glass wear is going to be handled and care should be taken to prevent breakages and therefore injury. If any glass wear is broken it should be cleaned up immediately and a member of staff should be informed. (www.ncbe.reading.ac.uk)
Fair test/ Variables: In my experiment, I am going to be using temperature as a variable. This is the only variable that I am going to be using as the enzyme, the concentration of the enzyme and the substrate are all constants. All of the same equipment will be used throughout the experiment and the time that each test tube is left in the water bath will be kept the same also - fifteen minutes. The temperature of the water baths will be varied, I will be using temperatures of 10°C, 20°C, 30°C, 40°C, 50°C and 60°C as I feel that these temperatures will provide me with a wide range of results and will hopefully provide a trend that coincides with my hypothesis. However, a water bath’s temperature can fluctuate and because of this reason, a thermometer was placed in the bath to monitor the temperature. This thermometer was checked every minute to ensure that the temperature was constant.
Bibliography:
NAS Molecules and Cells- John Adds, Erica Larkcom, Ruth Miller- pages 33-35
page ‘enzymes for schools’.
Diagrams are from-
Information on biological washing powders- http://www.lsbu.ac.uk/biology/enztech/detergent.html
Method for actual investigation:
-
The first thing to do is to make up the chosen enzyme,Savinase, to the desired concentration. I have chosen 3% and this concentration is obtained by measuring out 3g of powdered enzyme and adding this to 97cm3 of distilled water. A mass balance should be used in order to achieve accurate measurements. The solution should be placed into a beaker.
- Next, the substrate must be made up from its powdered form into a liquid. This was done by adding distilled water to it. The substrate that should be used is casein which is present in milk. I am using Marvel powdered milk as it is a source of protein, of which my enzyme hydrolyses. The solution should be placed into a beaker.
- Seven test-tubes should be placed into a test tube rack. These should be marked with what temperature water bath they‘re going to be placed into using a chinograph pencil. To these test tubes add 5mls of the substrate.
- To the substrate add 5mls of the enzyme solution apart from one test tube. The test tube containing both the substrate and the enzyme should be added to a water bath of the desired temperature. The test tube containing only the substrate should be tested to see what mass of protein it contains using ‘Albustix’. This will be used as a comparison to which the solution containing the enzyme will be compared to. It will be a control.
- The test tubes should be placed in water baths of temperatures ranging from 10°C-60°C. As soon as they are placed in a stopwatch should be started. I have chosen to use a water bath as a desired temperature can be maintained.
- The test tubes should be left in the water baths for fifteen minutes. The temperature of the water baths should be monitored throughout to ensure that they are at the correct temperature.
- After fifteen minutes the test tubes should be removed from the water baths and placed back into the test tube rack. They should be tested with Albustix to see what mass of protein the solutions within them contain. The results of which are in ‘Results Table 1’.
- The practical should be repeated in order to make sure that no errors have occurred and also to provide a second set of results. The repeat practical results are shown in ‘Results Table 2’.
- All of the equipment should be cleared away appropriately paying particular attention to the proper removal of the enzyme. It should be dissolved in water before being removed.
My actual method differs slightly from my preliminary method. This is because I have corrected anything in my method that could lead to inaccurate results or it simply improves my experiment which will lead to more accurate data. I have added to my actual method that the test tubes should be marked with a chinograph pencil with initials and the temperature of the water bath that they are going to be placed into. This is so that the test tube is identifiable and therefore nobody else picks it up by mistake. I have also explained in more detail how the Albustix work and I have tested my substrate to use as a control. By doing this I can see how much protein is in the plain substrate and this will act as a figure to which I can compare my results involving the enzyme with. I am expecting the mass of protein to be smaller than that result I obtained from the control test. Also, I have chosen to repeat my practical as I feel that it will provide me with a greater insight to how the enzyme hydrolyses the protein and also it helps to identify any mistakes that may have occurred whilst the practical was being carried out.
Results Table 1
Results Table 2
The end point in my experiment is related to time. All of my solutions will be placed in the water baths for the same time- 15 minutes. After this time they will be removed and the solutions will be tested with Albustix to establish what mass of protein remains in the solutions.
This is the scale that is on the side of the Albustix container. It is measured in grams per litre. The light green colour indicates a negative result. The dark green colour indicates more than 20g/l of protein is present.
This is my control test. It is a reading of the mass of protein present in my substrate. The reading is 3g/l.
This stick shows the mass of protein present after the enzyme and substrate were placed together in a water bath of 20°C. The reading is again 3g/l.
This stick shows the mass of protein present after the enzyme and the substrate were placed together in a water bath of 60°C. The reading is 0.3g/l. This was the temperature at which most of the protein was hydrolysed as the Albustix gave the lowest reading at this temperature.
Test tube contains:
1.Plain substrate
2.Enzyme and substrate heated to 10°C
3.Enzyme and substrate heated to 20°C
4.Enzyme and substrate heated to 30°C
5.Enzyme and substrate heated to 40°C
6.Enzyme and substrate heated to 50°C
7.Enzyme and substrate heated to 60°C
NB. All test-tubes above also contain biuret reagent which consists of sodium hydroxide solution and copper sulphate solution.
This picture shows an obvious colour change in my solutions. After I had finished my experiment, I added biuret reagent to my solutions to show that the amounts of protein present in my solutions had changed. The control is on the left, the dark violet colour. The other test tubes follow this control in the order 10°C- 60°C. The colour seems to be getting weaker as the temperature increases. The test tube that was placed in the water bath measuring 60°C is the lightest colour. This would indicate that there is less protein present in the solution. However, this test does not contribute to my experiment; it was only carried out to present an obvious colour change. The biuret test only determines whether there is a protein present or not, it doesn’t provide you with how much protein there actually is.
Analysis: The results that I obtained whilst carrying out my experiment are presented in Results Tables 1 and 2. The results that I achieved from my experiment are as I had expected; they correspond to my hypothesis, as the temperature of the water baths increased from 10°C to 60°C, the mass of protein lowered considerably from what mass of protein there was present in the control test. At temperatures 10°C- 30°C, the mass of protein present in my solutions was the same as the mass in the plain substrate; 3g/l. However, as the temperature reached 40°C the mass of protein present lowered to only 1g/l and this was also the case at 50°C. The lowest mass of protein was obtained when a test tube was placed in a water bath measuring 60°C. This is because the higher the temperature, the more kinetic energy is transferred to the enzyme( Savinase) and substrate particles( powdered milk), meaning that they will collide more frequently and bind; therefore the rate of reaction will increase, in this case, more protein will be hydrolysed in the time limit- 15 minutes.
My results follow a trend; the higher the temperature of the water bath, the smaller the mass of protein. However, the scale of the Albustix is not sensitive enough, a reading could be 2g/l, however the scale does not show a colour result for this mass and therefore a colour nearest to 2g/l will be used. Therefore my results are not entirely accurate as the precise mass of protein is not established. However, from my results, I do not have any anomalies, my results follow a trend and I even repeated my experiment to ensure that I obtained a set of results that could be as accurate as possible and minimise the risk of error. The results from my second experiment proved to be identical to those from my first experiment, and I put this down to the poor scale of the Albustix as they were not sensitive enough to pick up on masses in between those shown on the scale.
By looking at my graph, the line steeps downwards from 30°C to 40°C and then from 50°C to 60°C. This is because as the temperature increases, the mass of casein decreases. However, I obtained the same reading; 3g/l, for the following temperatures: 10°C, 20°C and 30°C. When looking at the graph these results cause a ‘flat’ line on my graph before the line steeps downwards as the mass decreases when the temperature reaches 40°C. The same reading was obtained for 50°C which caused another ‘flat’ line. Once again as the temperature increases to 60°C the line steeps downwards.The reason that my graph steeps downwards is that as the temperature of the water bath increased, the mass of casein decreased.
Reliability and precision of data: The results that I have obtained from my practical experiment, I feel are as accurate as possible as I ensured that all variables were kept constant. The results that I have obtained however are not precise enough, as the ‘Albustix’ that I used to test for the mass of casein present in the solution were not sensitive enough. In some cases I attained the same reading for varying temperatures which, if a more sensitive analytic stick had of been used, the results would have almost certainly been different as a rise in 10°C would in fact change the mass of casein present in the solution if only by a small margin. Although the above reasons represent that my test was not sensitive enough, I can still draw conclusions from them. For example, when the temperature of the solution was 10°C, I obtained a reading of 3.0 grams per litre. In comparison to the reading that I got from a solution of 50°C,1.0 g per litre, this shows that temperature does have an effect on the mass of casein present in the solution. This is because the kinetic energy that is given to the enzyme and substrate molecules from the heat mean that collisions between the substrate and the enzyme happen more frequently which in turn speeds up the rate of reaction. The increase of the rate of reaction means that the protein present in the solution is therefore being hydrolysed at a faster rate by the enzyme Savinase, at a higher temperature.In the time limit, fifteen minutes, more protein is broken down.
Limitations of the experiment: Whilst completing my experiment I came across numerous limitations, the first being the insensitive ‘Albustix’. They did not pick up a minor change in the mass of protein which means that some of the results that I obtained for different temperatures are identical. If a more sensitive type of analytic stick was used, I’m sure that there would be a change in the mass of protein at different temperatures; even if it was only minor.
The second limitation of my experiment was that only three water baths were set up to varying temperatures. This meant that to achieve the correct temperature to place my test tubes into, I had to fill a beaker with the hot water from the highest temperature water bath and add cold water from the tap until my desired temperature of water had been reached. I then had to observe the beaker of water at all times to ensure that the temperature did not fluctuate and therefore make my experiment an unfair test.
When looking at my results, they are as I had expected them to be. However, I would have expected more protein to have been hydrolysed then the amount that actually was. For example, at 60°C, I would have expected to see a trace of protein remaining however, 0.30 grams still remained. At 60°C, the Savinase and powdered milk molecules would have a lot of kinetic energy and therefore a high mass of protein should be hydrolysed.
Overall, I feel that my experiment was a success; I acquired a set of results that are as accurate as possible and I feel that I now have evidence to suggest that the change in temperature alters the rate of reaction between an enzyme and a substrate, between the enzyme Savinase and powdered milk molecules .An increase in temperature increases the rate, a decrease lowers the rate.
In order to establish more precise results, in a future experiment I could use a more sensitive device to test for the mass of protein present, such as a colorimeter which passes light through the substance to analyse how dense it is. This would detect if there were more or less protein molecules present compared to the control test. I could also use different enzymes belonging to the protease group of enzymes to see whether a specific enzyme is more efficient at hydrolyzing protein at a lower temperature which would reduce the amount of energy needed. This would be useful in cleaning items of clothing that had a protein based stain on them, as the enzyme would possibly function efficiently at a low temperature, therefore preventing excessive energy loss. Another method I could use would be to use different sources of protein or I could extend my experiment further by monitoring the temperature at which the most protein was hydrolysed; therefore finding the optimum temperature of the enzyme.
