In my experiment I will keep my enzyme concentration constant and so will be controlled in all my experiments. It will stay 10cm3 at all times.
Temperature
Increasing the temperature will increase the rate of reaction because the molecules are moving faster with greater kinetic energy. As the molecules have more energy they are able to overcome the activation energy barrier forming products. However in an enzyme controlled reaction, the rate of reaction can only increase up to a certain point called the optimum temperature. Enzymes are globular proteins and so increasing the temperature above optimum level will cause the enzymes in the reaction to denature due to bonds in the tertiary structure breaking and changing the shape and active site of the enzyme.
I will control this factor by doing all my experiments under room temperature so that the temperature will not change. My test tubes will not be heated at any point in the experiment because this will cause the temperature to rise.
PH
PH has a key impact on enzyme activity. A sudden change in PH can affect the molecular structure of the enzyme and therefore reduce the chances for it to bind with substrate molecules. An explanation for this is that enzymes are proteins which have ionic bonds formed during the making of its structure. Change in PH affects these ionic bonds causing them to break and change the enzymes shape. We now say that the enzyme has been denatured and so the rate of reaction stops. Substrate molecules cannot longer fit into enzymes active sites therefore no enzyme/substrate complexes are formed. It all depends on the concentration of hydrogen ions (H+) in the solution. The more hydrogen ions there are, then the lower the PH will be.
In my experiment I will control the PH by using the same solutions that I take out of the bottles for each experiment. This will keep my PH the same for all my tests.
Number of Iodine Drops
Adding iodine drops to my solution will help measure the rate of reaction. It will give the solution a blue/black colour which therefore means I can measure the time it takes for the blue/black colour to turn colourless. However increasing the number of iodine drops will decrease the rate of reaction. This is because more iodine molecules are present therefore will collide more often with the enzyme molecules leading to less successful collisions between the substrate molecules and enzyme molecules. Because of this fewer enzyme/substrate complexes will be formed and rate of reaction will decrease.
I will control the amount of iodine drops added to each experiment by keeping the same number of drops added to each test I carry out. Only 2 drops will be added making the number of iodine drops constant.
RELIABILITY:
To make my results reliable I will use 3 repeats for each chosen range of substrate concentration. Having 3 similar results will help me work out an average value and so will be helpful when I draw up my graph. I have decided to use a range of 5 in order to gain results and a range spread which follows a pattern of even numbers to make sure I get reliable results. My range spread will be 0.2%, 0.4%, 0.6%, 0.8% and 1.0% of substrate concentration. Having these ranges will reduce any possible anomalies from occurring. My equipment I use will be accurate and appropriate when I carry out my experiment so it’s more likely for me to obtain reliable data.
Firstly I will use a graduating pipette which will give me a more accurate reading of what I am measuring and putting into my test tubes. And secondly I will use a stopwatch to measure the time taken in milliseconds correct to 2 decimal places. The temperature will be the same for all my experiments because all of them will be under room temperature. This make the conditions appropriate to carry out a fair test and provide reliable data.
SAFETY:
- Make sure all bags and coats are kept far away from working area. Can cause accidents such as other pupils tripping over.
- Goggles should be worn at all times during the experiment.
- Iodine can be poisonous so should be kept away from contact with skin and can be absorbed easily by the skin leaving a stain.
- Stools should be kept tucked underneath the table and out of the way.
- Handle amylase with care because can be irritating if gets contact with eyes.
EQUIPMENT/APPARATUS:
- Test Tube rack
- Test Tubes x15
- White Tile
- Black Marker
- Goggles
- White Labels x15
- Dropping Pipette
- Graduating Pipette
- Beaker x3
- Measuring Cylinder
- Stopwatch
- Iodine
- Distilled Water
- 1% Starch (substrate)
- 1% Amylase (enzyme)
METHOD:
1] To avoid contamination, clean all glassware with distilled water before carrying out the experiment.
2] Set out all equipment, put goggles on and make sure you are working in a safe environment.
4] Use 1 test tube to mix 10cm3 of amylase and 10cm3 of starch. You will see a clear yellow colour solution and this will be used as a control.
3] Put 5 test tubes into a test tube and label each one according to the range of substrate concentration chosen.
Test tube 1 will be labeled 0.2%
Test tube 2 will be labeled 0.4%
Test tube 3 will be labeled 0.6%
Test tube 4 will be labeled 0.8%
Test tube 5 will be labeled 1.0%
4] Use a graduated pipette to measure 2cm3 of starch and place into a test tube. Do the same for 4cm3, 6cm3, 8cm3 and 10cm3 of starch.
5] Now again using a clean graduated pipette, measure the correct amount of distilled water to put into each test tube and make sure that the volume of each test tube adds up to 10cm3.
Test tube 1 will have 8cm3 of distilled water
Test tube 2 will have 6cm3 of distilled water
Test tube 3 will have 4cm3 of distilled water
Test tube 4 will have 2cm3 of distilled water
Test tube 5 will have 0cm3 of distilled water
6] Add two drops of iodine to each test tube using a dropping pipette. A blue/black colour should appear once added.
7] Draw a black cross on a white tile and hold it up against the test tube that you will be testing first. You should not be able to see the cross anymore.
8] Add 10cm3 of amylase into the test tube. As soon as the amylase concentration is in contact with the starch and distilled water concentration, start the stopwatch. A reaction should now start to take place.
9] When the reaction is over, you should see a colorless solution and the black cross should be visible. Compare your result to the control and if both solutions look the same or similar than stop the watch and record your results in table.
10] Do the same for all the other labeled test tubes. (10cm3 of amylase added to 0.4%, 0.6%, 0.8% and 1.0% of substrate concentration)
11] Now repeat each chosen range another 2 more times to get reliable results using the exact same method.
ANALYSIS
I drew my rate of reaction graph by putting substrate concentration/% along the x axis and rate of reaction x10-3/s-1 along the y axis. I got my x axis values by taking the substrate concentrations from my table and y axis values by using a formula (1/t), where t equals average time taken for the blue/black colour solution to go clear.
Looking at my table I can see that as substrate concentration increases, the average time taken decreases. This means that the rate of reaction will increase as more substrate concentration is added to the solution because a colourless solution starts to appear more quickly. When 0.2% substrate concentration is added the average time taken is 261.46 seconds and the rate of reaction is 3.82 x10-3/s-1. When 1.0% substrate concentration is added the average time taken is 19.46 seconds and the rate of reaction is 51.39 x10-3/s-1. The figures clearly show that the reaction gets faster and time taken for a colourless solution to appear is quicker as substrate concentration increases.
The trends and patterns my graph shows is that as substrate concentration increases, the rate of reaction will also increase. Between 0.2% and 0.4% substrate concentration the rate of reaction line shows us that there is a general rise but, between 0.4% and 0.6% there is a rise but the line tends to get less steep. Between 0.6% and 0.8% the line gets steeper and stays similar to the line between 0.2% and 0.4%. However between 0.8% and 1.0% there is a very sharp rise and very steep line showing that the rate of reaction increased rapidly. The overall trend and pattern that the graph shows if that the line is always going up indicating that the rate of reaction increases as more substrate concentration is added.
I can back my statements I made about rate of reaction increasing as substrate concentration increases by using scientific biological information. As substrate concentration increases, the number of substrate molecules increase. This means there is a higher chance for the substrate molecules to collide with the enzyme molecules and therefore more successful collisions between the two as they collide with each other more often.
I can use the lock and theory to explain that the formation of enzyme/substrate complexes will be quicker because there are more substrate molecules available for the enzymes.
The enzyme molecules have a specific active site and the substrate molecules have a specific shape which causes them to bind with each other when they collide to form enzyme/substrate complexes. At the moment where they both have been locked on to each other, the substrate molecule will slightly change shape and repel from the enzymes active site because the specific shape the changed. Products will now be formed and the enzyme is free to take on other substrate molecules. Linking this to my work I can say that because there are more substrate molecules present for the enzymes to work with, more enzyme/complexes will be forms and therefore more products. When enzyme/complexes are forming, at the same time other enzyme/substrate complexes are forming as well when the substrate molecules bind with the enzymes specific active site. This suggests that the total concentration of enzyme/substrate complexes increase and so the time taken decreases as more substrate concentration is added and so rate of reaction increases.
Going back to my graph I stated that my line gets steeper at some points and less steep at other points. I can now explain that by using my scientific biological information. Between 0.2% and 0.8% there is an overall steady rise in rate of reaction of 3.82 x10-3/s-1 to
12.34 x10-3/s-1 as the substrate concentration increases. This is because there are more substrate molecules to bind with the enzyme molecules forming more enzyme/substrate complexes. There are more collisions between the two because they tend to be more crowded together. Between 0.8% and 1.0% there is sharp increase in rate of reaction meaning that as the number of substrate molecules continue to increase, the collisions are greater due to the two molecules being even more packed within each other. The substrate molecules are binding with the enzymes specific active site much faster producing enzyme/substrate complexes more rapidly at a quicker pace.
As concluded that the rate of reaction will increase when more substrate concentration is added, however it will only increase up to a certain point. When the concentration of substrate molecules is much higher than the concentration of enzyme molecules, the enzymes will get filled up and the rate of reaction will not increase anymore. On a graph, the line will level off because increasing the amount of substrate concentration will not increase productivity. The enzymes will work at the same rate but because there are so many more substrate molecules compared to the enzyme molecules, fewer collisions will take place which will decrease the rate at which enzyme/complexes will be formed. In other words the enzymes are working at their maximum rate and substrate molecules have to wait a long time in order for them to catch a free enzyme which is available.
EVALUATION
In overall I think my investigation on finding the relationship between increasing substrate concentration and keeping enzyme concentration constant was successful. The results I gained from all my experiments showed me that the theory I knew was true and trends were clear to see from both my table and graphs.
There was one anomaly which took place as the second reading for 0.6% substrate concentration. The reaction took place slower for this reading compared to the other two. The average reading was 106.99 seconds and the second reading was 100.21 seconds. The first reading was 111.50 seconds and the third reading as 104.28 seconds. I can see that the difference between the second reading and average reading is 6.78 seconds which is the largest difference out of the three. (Difference between first reading and average reading is 4.51 seconds and difference between third reading and average reading is 2.71 seconds)
I know this must be an anomaly because if I calculate every difference between each reading and its average reading, the difference is will not be higher than 5.00 seconds. The second reading for 0.6% substrate concentration however does give a higher difference than 5.00 seconds and is the only reading which does.
There were many possible errors that may have occurred as I was carrying out my experiment. The room temperature may have affected my results because there might have been a slight increase or decrease in temperature inside the room at times. If it had decreased then it may have caused the substrate and enzyme molecules in my solution to have less kinetic energy therefore fewer collisions will have been successful decreasing the rate of reaction. This therefore meant that the time taken for my solution to go colourless would have been longer than expected and so I will be taking down unreliable data.
I can control this error in the future by using a water bath to keep the temperature of the solution constant so that it will not keep changing. Also doing this will make sure the enzymes are working at optimum temperature therefore work with the substrate molecules at a faster rate forming more enzyme/substrate complexes. The enzymes will not worker slower or denature because the advantage of using this water bath is that I will be able to control the temperature myself.
When I was measuring the time taken for the reaction to go colourless, I used a black cross on a white tile which used placed against the solution. This technique was not very accurate and reliable because it was hard to exactly tell when the cross can be seen and therefore caused error in my experiment because I may have stopped the stopwatch too early or too late. The results may have been affected by this because when calculating the rate of reaction unreliable figures would have been used.
This technique can be improved by using a colorimeter usually meaning “measuring the absorbance of particular wavelengths of light by a specific solution” (wikipedia definition). It helps determine the end point when a colourless solution has appeared more accurately.
As I added iodine to my solution, the drops were not all even in size. This made the concentration of the solutions in each test tube different leading to error. This had an influence on my results because if slightly larger drops of iodine were added, than the blue/black solution would have been thicker and darker. This meant that the time taken for the solution to clear had increased and the rate of reaction had decreased due to more molecules being present for collision. If slightly smaller drops of iodine were added, than blue/black solution would have been thinner and lighter. This meant that the time taken for the solution to clear had decreased and the rate of reaction had increased due to less molecules being present for collision.
I can control this by using a micropipette instead of a dropping pipette to keep the number of iodine drops same in size.
As I poured my amylase, substrate and distilled water concentrations into my test tubes, some of the amounts may have stayed inside the graduating pipette where it could not be seen. Small amounts remaining will cause there to be less substrate molecules or less enzyme molecules. This could have affected my results in terms of rate of reaction. If there was slightly more amylase molecules compared to the starch molecules in the solution due to some starch remaining in the pipette, than the time taken for the solution to clear would be longer and the rate of reaction would have decreased. This is because there will be less substrate molecules to collide with the enzyme molecules forming fewer enzyme/substrate complexes. However, if there was slightly more starch molecules compared to the enzyme molecules in the solution due to some amylase remaining in the pipette, than the time taken for the solution to clear would be quicker and the rate of reaction would have increased. This is because there will be more substrate molecules to collide with the enzyme molecules forming more enzyme/substrate complexes.
I could improve this course of action by taking more time on transferring chemicals with the graduating pipette to make sure all the liquid has been removed and the right measurements have been taken in.
In overall there were no major errors made which affected my conclusions but the results I got from my table didn’t really follow a fine pattern due to errors mentioned above. Trends and patterns I got totally backed up my biological knowledge stating that the rate of reaction will increase and substrate concentration increases. If I had to carry out this investigation again in the future I will use a higher range in order to get more accurate reliable results.
