As the substrate concentration increases, the initial rate of reaction also increases. Again this is only what we would expect: the more substrate molecules there are around, the more often an enzymes active site can bind with one. If we keep increasing the substrate concentration keeping the enzyme concentration constant, there comes a point where every enzyme’s active site is working continuously. If more substrate is added the enzyme cannot work faster: substrate molecules are ‘queuing up’ for an active site to become free. The active site of an enzyme may be used again and again. Therefore enzymes work efficiently at very low concentrations.
As the enzyme concentration increases, the rate of reaction will also increase. This is because there are more active sites available for the substrates to bind with.
Factors
The factors that I am investigating are:
- Temperature
- pH
- Substrate and Enzyme Concentration
Controls, Variables and Constants
The temperature, pH and Substrate Concentration are the controls that will be changed, as these are the factors that I am testing and the variables are the different temperatures concentrations and pH.
-
The temperatures that will be used are: 0 oC, 25 oC, 37 oC, 60 oC, 70 oC, 40 oC
- The pH’s to be used are: pH3, pH5, pH7 and pH 9.2
- The concentrations I am going to use are: 1%, 0.5%, 0.25%, 0.125%, 0.625 and 0% (H20)
- The time that the experiment is carried out within is 15 minutes and must remain constant.
- The iodine solution must be added at 30 second intervals
Risk Assessment
- Don’t spill the starch and avoid contact with hands as it is very sticky.
- Wear safety goggles at all times to protect yourself from the chemicals.
- Put all glass equipment away from the edge of the table.
- pH buffers are corrosive so avoid contact with hands and other parts.
- Handle water bath carefully as it is hot.
- Tie back hair/ headscarves to avoid any interference with the experiment.
- All bags and stools must be kept well out of the way to avoid accidents.
Apparatus
- 1% Starch Solution
- Water Bath
- pH Buffer 3,5,7 and 9.2
- Test Tubes
- Test Tube Rack
- Well Trays
- Iodine Solution
- Pipettes
- Stop Clock
Method
pH
-
Take 2cm3 of 1% amylase in pH7 buffer
- Add 2cm3 of pH3 buffer
-
Incubate at 37oC for 5 minutes
-
At the same time incubate the starch also at 37oC for 5 minutes
- Prepare a well tray with drops of iodine
- Mix the solutions together
- At 30 second intervals test for colour change
- Repeat for pH5-11
Temperature
- Take 4cm3 1% amylase
-
Incubate for 5 minutes at 37oC
-
At the same time, incubate 4 cm3 1% starch at 37oC
- To a well tray, add drops of iodine
- At 30 second intervals add 1 drop of reaction mixture to the iodine
- Note change
- Repeat for other temperatures
Substrate Concentration
-
Mix 5cm3 of 1% starch suspension in buffer pH 7 with the same volume of 0.1% amylase, also made up in the same buffer.
-
Set up a control containing 5cm3 of buffer only instead of amylase.
-
Set up a second control containing 5cm3 of boiled amylase only.
-
Incubate the tubes at 37oC.
- At 30 second intervals, remove drops of the reaction mixture and place in a well tray.
- Add drops of iodine in iodine solution.
- Note the colour change.
- Note the time taken for the blue-black colouration to fail to appear.
Enzyme Concentration
-
Mix 5cm3 of 1% starch suspension in buffer pH 7 with the same volume of 0.1% amylase, also made up in the same buffer.
-
Set up a control containing 5cm3 of buffer only instead of amylase.
-
Set up a second control containing 5cm3 of boiled amylase only.
-
Incubate the tubes at 37oC.
- At 30 second intervals, remove drops of the reaction mixture and place in a well tray.
- Add drops of iodine in iodine solution.
- Note the colour change.
- Note the time taken for the blue-black colouration to fail to appear.
During the experiment try and measure out the chemicals as carefully as you can and try keeping the time as close to 30 seconds if not exactly.
Results
Temperature
Fig. 1.1
Temperature (oC) Time taken (Minutes)
0 No change
- 21
- 8.5
- 25+
- 30+
pH
Fig. 2.1 pH Level Time taken (Minutes)
- 18
5 6.1
8 15
9.2 25+
Substrate Concentration
Fig. 3.1
Substrate Concentration (%) Time Taken (Minutes)
1% 15
0.5% 7.6
0.25% 6
0.125% 2.3
0.0625% 0.5
0 Water No change
Enzyme Concentration
Fig. 4.1
Enzyme Concentration (%) Time Taken (Minutes)
1% 4
0.5% 7
0.25% 12
0.125% 16
0.0625% 22
0 Water No change
Analysis
Fig. 1.1 shows the temperatures that I used to see how the enzymes work at certain temperatures.
You can see that when the temperature was 0oC there was no change because no reaction took place, as the temperature was too low. It also shows that at 36oC the rate of reaction was at its quickest. This is said to be the optimum temperature for the enzyme. The rate of reaction was higher at the higher temperature of 70oC. This is because as the temperature is raised, the energy levels of the enzymes and substrates are also raised. They have more kinetic energy and so they collide more often and therefore more reactions take place between them. This agrees with my prediction because I said that as the temperature rises so will the rate of reaction until the optimum temperature after which it starts to fall again. You can see this on the graph, Fig. 1.2.
At 0oC nothing happens because the enzymes have lost energy and therefore they can’t move around and bind with the substrates. As the temperature increased the rate of reaction also increased until around 36oC. As the temperature increase the particles gain more energy and move faster and find substrates. As they can moved around more they had a higher chance of finding a substrate and therefore the rate of the reaction increased. From the table and graph I concluded that the rate of reaction was at its fastest at 36oC.
The rate of reaction increased after the optimum temperature of around 36oC. This is because as the temperature rose after the optimum temperature, the enzymes began to destabilise the hydrogen bonds. These bonds begin to break and in doing so alter the shape of the active site, which makes it harder for substrates to fit into them. The enzyme is then denatured after a certain time.
Fig. 2.1 and 2.2 show the time taken for amylase to work with starch at different levels of pH.
Looking at these figures, you can see that at pH 3, 8 and 9.2 the rate of reaction was high. This was due to the fact that those pH’s were too acidic or alkaline and therefore caused the hydrogen bonds to destabilise. This in turn altered the shape of the active site so that it is no longer complementary to the shape of the substrate. The rate of reaction was at its fastest at pH 5. Enzymes generally work faster in neutral conditions, pH 7 and seeing as pH 5 is closer to pH 7 it’s fair enough to say that the optimum pH around 5.
Fig 3.1 and 3.2 show the time taken for the amylase to react with the starch at different substrate concentrations.
The graph shows that as the substrate concentration increased so did the rate of reaction. This was because as the substrate concentration increases there are more substrate molecules available and this increases the chances of a substrate binding with an active site. At a lower concentration the rate of reaction was at its fastest. However as the concentration increased so did the rate of reaction. This was because as there are more substrate molecules the active site is working continuously. The substrate molecules ‘queue up’ for an active site to become free because more substrate molecules appear as the concentration rises. Therefore I conclude that enzymes work more efficiently and effectively at low concentrations.
Fig. 4.1 and 4.2 show the rate of reaction for the amylase to work on starch at different enzyme concentrations.
Looking at fig.3.2 you can see that as the enzyme concentration increases the rate of reaction decreases. This is due to the increase in active sites. Therefore if there are more active sites available then the rate of reaction will be quicker as the substrate molecules can fit into the active site. This pattern can be seen clearly on the graph.
Evaluation
Overall the results show me that:
-
As the temperature rose the rate of reaction also rose until it reached the optimum temperature which was 37oC after which it started to rise again. But there comes a point where the enzyme denatures.
- If the pH was too acidic or too alkaline the rate of reaction increased but if it was in a neutral condition of pH 7 then the rate of reaction was faster at this level. In this experiment it was found that pH5 was when the rate of reaction was at its lowest.
- As the enzyme concentration increased the rate of reaction decreased.
- As the substrate concentration increased the rate of reaction so did the rate of reaction. Therefore it would be fair to say that the enzyme concentration is proportional to the rate of reaction.
The results I obtained stand to reason with my prediction.
There were some anomalous results and they could have occurred in the experiment because of the following reasons:
- The measurements may not have been accurate enough. To improve this it would be sensible to take all measurement readings at eye-level.
- The time at which the iodine drops were dropped at may have been to early or too late. To improve this I would drop the iodine solution exactly at the 30 second intervals.
- Another reason may be that as the test tubes were taken out of the water bath the temperature may have decreased while putting the iodine drops in.
- Also the test tubes were to remain in the water bath for 5 minutes but they may have been taken out too early or too late. To improve this I would take the tubes out exactly at 5 minutes.
If I were to do this investigation further then I would do the following experiment:
Experiment to test the hypothesis that catalase (in yeast) breaks down hydrogen peroxide
- Half fill the beaker with tap water and use water bath with the Bunsen burner.
- Place approximately, half the yeast-glucose mixture into the boiling tube and leave in the water bath for 5 minutes.
- Use the spirit marker to label one test-tube A and the other B.
- Place the test tubes in the test-tube rack.
-
Wearing safety glasses and gloves, pipette 5cm3 hydrogen peroxide into tubes A and B.
- Cool the boiled yeast-glucose solution.
-
Using a clean pipette, place 5cm3 of the cooled yeast solution into tube A.
-
Place 5cm3 of the unboiled yeast mixture into tube B.
- Start the clock immediately
- At 2minute intervals, use the ruler to measure the height of the froth (In mm) which may appear above the liquid in each tube.
- Record the data in the table.
References
Shari gave us the methods (Simons.S.2001)
Glenn Toole And Susan Toole, (1991), Understanding Biology For Advanced Level; Stanley Thornes (Publishers) Ltd., Second Edition, Pages 50 & 51
Ted Lister & Janet Renshaw, (1991), Understanding Chemistry For Advanced Level Stanley Thornes (Publishers) Ltd., Second Edition, Pages
Eileen Ramsden, Jim Breithaupt, David Applin, (1991), Key Science 4 Book 1. Stanley Thornes (Publishers) Ltd.Second Edition, Pages 254 & 225
Acknowledgements
I would like to thank Shari for all her help on how to do the experiment and how to write it up. I would also like to thank the technicians, especially Linda for guiding and helping us get all the right equipment and with the experiment.