pH
Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Not all enzymes work best in the same pH, each enzyme has an optimum pH at which its active site best fits the substrate and at which it is working at its fastest rate. For example amylase works best or has and optimum pH of about 8, but stomach protease (pepsin) has an optimum pH of about 4/5. Variation at either side of the enzyme, of pH results in denaturation of the enzyme and a slower rate of reaction.
Proteins can be damaged by very alkaline or acidic conditions and therefore work best at a pH of 7, i.e. a neutralised state.
Substrate concentration
When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate. So therefore enzyme concentration also affects the rate at which enzymes work.
Inhibition
Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors or negative catalysts, therefore slow the rate of reaction. There will be none in this experiment and therefore will not effect the rate of reaction.
Enzyme cofactors
Cofactors are none protein substances which influence the functioning of enzymes. They include activators that are essential for the activation of some enzymes. Co enzymes also influence the functioning of enzymes although are not bonded to the enzyme. Unless enzyme cofactors were present in the potato tissue containing the Catalase, they will not be included in this investigation and therefore will not have an affect on the rate of reaction and the results of this experiment.
Enzyme Concentration
Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect, therefore the greater the number of pieces, the greater the enzyme concentration.
Planning
Aim: to see if surface area effects the rate of catalase action in potato chips
Prediction:
As the surface area increases the more oxygen bubbles will be produced because there will be more enzyme available i.e. more catalase. Therefore more activity with the hydrogen peroxide, i.e. the rate of enzyme reaction will increase. If we double the surface area, then the rate of reaction may double too. Hydrogen peroxide will breakdown to oxygen and water in the presence of Catalase. The reaction will increase with increasing enzyme concentration when molecules of hydrogen peroxide are freely available. Increasing enzyme concentration will mean that there are more active sites for the hydrogen peroxide to lock on to, to release more oxygen. However, when molecules of the substrate are in short supply, the increase in rate of reaction is limited and will have little effect.
The graph expected.
Rate of enzyme
Reaction / ------------
Number of O2
Bubbles
Enzyme concentration = Surface area cm2
The line may curve or plateau, showing that the hydrogen peroxide has run out or that the potato chips are covering one another.
Apparatus list:
-
Hydrogen peroxide - H2O2
- Syringe
- Stopwatch
- Knife
- Tile
- Wet kitchen towel
- 6 potato chips
- Water
- Side arm test tube
- Test tube rack
- Test tube
- Rubber bung
- Ruler
Safety
- An overall should be warn, because the hydrogen peroxide is a dangerous chemical and so gloves and goggles will have to be warn too, at all times, so that it does not react with skin or get into our eyes. If it does by any changes, eyes must be washed immediately with warm water and the teacher should be informed.
- Hair should be tied back so that it does not come in the way as a distraction.
- The apparatus should not be kept too close to the edge of the bench, in case anything spills.
- The side arm test tube should be supported; maybe with a retort stand or test tube rack, the other test tube should be placed in the same test tube rack and kept away from the edge of the bench.
- Hydrogen peroxide is a very dangerous solution; it can burn through clothes, bench surfaces and even skin, and therefore all safety issues should be taken into account and not ignored.
The constants
- The volume of the potato chips should all be kept the same, throughout the whole experiment – I have decided to keep them all 5cm
- The type of potato chips should be the same – Tescos own brand potatoes are going to be used through out the whole experiment
- The room temperature should remain the same – although the temperatures may increase due to the number of people in the lab, so windows may need to be opened when needed as there is no way of keeping this constant, the temperature should be monitored to explain any anomalous results.
- The temperature of the solution should remain the same – there is no way of keep this constant either and so will also b monitored.
- The pH should be kept constant – there is no way of keeping this constant, so we can keep a record the pH through out the experiment to see if there are any changes which could be accounted for anomalous results.
- The volume of hydrogen peroxide should be the same for all the different surface areas - To control the substrate concentration; identical quantities of the substrate should be used for each reading. To ensure that this is measured precisely, 20ml syringes will be used to accurately gauge to exact quantities.
If any of these are not kept constant then it will not be a fair test, as they will affect the rate of catalase action.
The independent variable
- This is the surface area of the chips, which is the enzyme concentration i.e. the amount of enzyme available. I will vary the enzyme concentration by altering the number of pieces of potato that contain the Catalase, in the reaction.
The dependent variable
- This is the number of oxygen bubbles produced per minute from the reaction, which is the rate of catalase action.
Diagram of apparatus
I produces a table in advance, so that I could record my results easily and beable to read them afterwards.
Preliminary experiment
This is an experiment that is done before hand in order for me to know much hydrogen peroxide to use throughout the experiment and how long I should kept the length of the chips. I firstly got 6 long chips and put them in a row, the chips had to be much smaller than the test tube so I decided to cut all the chips to 5cm. I found that this would be a suitable length as I could still manage to cut the chip into 32 pieces.
I then placed one of the cut chips into the test tube. I then got a syringe and sucked up 30mls of water and then syringed a bit of water into the test tube at a time until a level of water covered the potato chip, I then rounded the volume up to the closest number, which volume of hydrogen peroxide used would be 20ml.
Method
Set up the apparatus as shown in the diagram above. Place a side arm test tube in the first hole of the test tube rack, with another test tube two holes down the rack so there is enough space for the side arm in between the two test tubes. Get 6 potato chips from the trough, place them on your tile and make sure that all your chips are the same length. Wrap them all in a wet kitchen towel in order to keep the moisture within them. Make sure that there is a beaker or hydrogen peroxide ready on another table. Then put some water into the second test tube. Make sure at least half the test tube if full of water and that the side arm test tube sits in this test tube properly with the water covering it. Then making sure you have your goggles, overall and gloves on pull out 20mls of hydrogen peroxide from a beaker into the syringe. Then syringe out the hydrogen peroxide into the test tube. Care should be taken to view the syringes from the side to ensure the bottom of the meniscus is lined up properly with the gradations and there are no air bubbles in the syringe.
Now take a piece of pH paper and drop a small piece into the hydrogen peroxide to see what the pH is. It should remain the same through out the experiment. So make sure a reading of the pH is taken before every different surface area of chips is out into the test tube. Then using a thermometer measure the temperature of the room and also of the hydrogen peroxide. Remember to do this before each experiment too.
Then put the first chip into the test tube and then quickly place the rubber bung into the test tube making sure it is secure so that any oxygen produced cannot escape. The oxygen produced will then escape through the side arm test tube and into the second test tube with water. This will enable me to see the oxygen bubbles. The hydrogen peroxide and the enzymes in the potato chip will react creating oxygen bubbles.
Leave an equilibrium time of about 2 to 5 minutes for the reaction to start and settle. Then start the stopwatch and count the bubbles produced for five minutes. Remember to keep an eye on the stopwatch, then repeat the experiment for another five minutes, using the same chip.
After one surface area had been repeated, re do the experiment, placing a chip on the tile and cutting it into 2 pieces, then 4 pieces and 8 and so on, increasing the surface area till there are 32 small pieces. Remember to repeat each surface area for another 5 minutes for reliability.
Average out the two sets of results for each surface area by adding them up and dividing by ten. This will give you the average number of oxygen bubbles released per minute, which is more reliable. As the same number of bubbles will not be released in five minutes.
Calculation
Surface area – show diagram of cut chips.
Obtaining
The results to my investigation have been tabulated in order for clarification so that they can be easily analyzed and read clearly with ease.
I carried out the experiment as planned in the planning. I think I obtained enough results to analyse and draw up a firm conclusion. The data obtained from this investigation has been recorded in a table showing all data that was taken through out the experiment. This means that the results of the experiment are presented in a clear and orderly fashion that allows patterns in the results to become more obvious.
I noticed that in the test tubes it was clear that a reaction was taking place by the observation of bubbles of oxygen gas being released creating a ‘fizzing´ in the test tube.
Analysis
The aim of this investigation was to study the effect of changing enzyme concentration on the rate of catalase action.
The results from my scientific experiment indicate that as the enzyme concentration increases so does the rate of enzyme action.
For example: At the surface area of: 22, average number of bubbles produced: 11.9
At the surface area of: 52, average number of bubbles produced: 50.8
Therefore I can say that as the surface area increased so did the rate of enzyme action so I can say that my prediction was correct. As the surface area increases the more oxygen bubbles were produced because there was more enzyme available i.e. more catalase. Therefore more activity with the hydrogen peroxide, i.e. the rate of enzyme reaction increases. My graph also indicates this to be correct as it can be seen that the line graph is going up. As the surface area increases, there are more sides of the potato chip exposed to the hydrogen peroxide and therefore more collisions, as more catalase is exposed and so the rate or reaction increases.
So I can conclude my results, that as the surface area increases, increasing the enzyme concentration, so does the number of oxygen bubbles produced, i.e. the rate of enzyme action.
At first my graph is steep then curves and flattens out. My graph produced a curve, rather than a straight line, which would have been ideal if the rate of reaction was directly proportional to the surface area. However this did not happen, as my graph plateaued at the end. We can see this by the numerical evidence:
At the surface area of: 52, average number of bubbles produced: 65.1
At the surface area of: 84, average number of bubbles produced: 68
This is a difference of 2.9, which is not much. I could further my investigation to see if the increase in surface area would create a bigger plateau. This plateau may have happened for many reasons, such as; the hydrogen peroxide became limiting because, towards the end, as the surface area had increased, there was not enough substrate. Another reason could be that as there was a larger surface area and the capacity of the test tube was not sufficient enough so the distribution of catalase may not have been constant. Some of the pieces of potato may have been over lapping each other and therefore the hydrogen peroxide was not able to attack all the sides of each chip.
The plateau may also have occurred because as the chips were cut into more pieces and the surface area increased the pieces became smaller and lighter and so sticked to the sides of the test tube rather than mixing with the hydrogen peroxide.
Evaluation
I feel that this experiment worked reasonably well and to my expectation, because I got a reasonable set of results, which proved my prediction to be correct and enabled me to establish a firm conclusion.
I think that my results were accurate enough, for the experiment as they produced a graph and conclusion, although I do feel that if I had a larger range of surface areas, say from it would make my results more reliable. Nevertheless they are still more or less accurate.
The method used was the best way of carrying out the experiment, because it was the most simple and easiest way of doing so and as it worked well. It also gave me encouraging results, although I could have carried out the experiment out in a much more reliable and accurate way, which would improve the reliability of my results.
I could improve the method of the experiment in many ways.
In this experiment, the pH was kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (potato tissue). This was achieved by using a test tube rack and tongs to handle the apparatus so that the heat from my hands did not affect the catalase.
They should not have affected this investigation, however, as none were added.