Catalase
Hydrogen Peroxide Water+Oxygen
2H2O2 2H2O+O2
FACTORS EFFECTING ENZYME ACTIVITY.
With help from
There are certain variables that can alter the rate of a reaction. They are:
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Concentration of enzyme. For this to work properly there needs to be an excess of substrate. Essentially the reaction should speed up when more enzymes are added. This is because if there are more enzyme molecules so there are more active sites for the substrate molecules to collide with and also there will be more reactions able to take place at any given time.
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Concentration of substrate. As the quantity of substrate is increased then the probability of a reaction occurring between the enzymes and substrate also increases. This should carry on increasing proportionally.
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Temperature. An increase in temperature should increase the amount of kinetic energy the molecules have therefore result in them reacting more frequently and with more force. However, as I have mentioned before, the active sites of the enzymes can become denatured at higher temperatures (above 40 degrees Celsius) so there is a limit as to how much you can increase the rate with temperature. The rate of reaction will decrease steeply after this point.
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pH. Enzymes have an optimum pH where the work best. I have already roughly explained pH effects. Enzymes will only work in their favourable pH range. This range will vary between different enzymes and if an enzyme is placed in unfavorable conditions it will become denatured.
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Inhibitors. These are substances that alter the reaction therefore slowing it down or stopping it. There are 3 types of inhibitors.
1, Competitive. These have a form similar to the substrate so they can fit into the active site but a reaction cannot take place so the enzyme gets rid of the inhibitor. This uses up time consequently reducing the rate of the enzyme reaction.
2, Non-competitive. This is where the inhibitor affects and alters the shape of the enzyme so that it can no longer bind with a substrate.
3, Substrate inhibition. This sometimes occurs when there is an excess of substrate. It is where there are too many substrate molecules fighting to react with the enzymes so in actual fact they block the active sites preventing a reaction.
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Surface area. This is to do with the state of the substrate. It involves the collision theory. If a substrate is a solid it will have a smaller surface area than if it was a liquid therefore the enzymes will only be able to react with the substrate molecules on the outside of the solid slowing the reaction down.
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Pressure of a reaction. This is slightly more advanced. If a reaction is forced to take place under a higher pressure then the molecules will be more forced together so should react faster. With the equipment that I will be using it will not be possible to investigate this.
AIM.
In this investigation I am going to investigate the effect of substrate concentration on an enzyme-catalysed reaction. I have chosen to investigate substrate concentration as it is the most practical to do with the equipment I have got. I will be using the enzyme catalase in the following reaction:
Catalase
Hydrogen Peroxide Water+Oxygen
2H2O2 2H2O+O2
I will change the concentration of the hydrogen peroxide by adding water.
PREDICTION.
I predict that as I increase the substrate concentration there will be more reactions able to take place between enzyme and substrate molecules, there will be more oxygen gas collected therefore there will be an increase in the rate of the reaction. This is known as the collision theory, the more substrate molecules there are then the higher probability that they will collide with the enzyme molecules and react. This should increase proportionally. However after reading various texts I believe that the rate will start tailing off later on as the enzymes will have limitation as to how many substrate molecules they can convert at one time. Once the enzymes have reached their optimum working ability then the rate of the reaction cannot increase any more and it levels off. This is known as saturation.
If my prediction is true then my graph should have a positive correlation and its gradient should get gradually smaller.
APPARATUS.
- 3x100ml beakers. One for the catalase, one for the hydrogen peroxide and one for the distilled water.
- 3xpippettes. One for the catalase, one for the hydrogen peroxide and one for the distilled water.
- 3x10ml measuring cylinders. One for the catalase, one for the hydrogen peroxide and one for the distilled water.
- Conical flask.
- Bung and delivery tube.
- Washing up bowl ½ filled with water.
- 100ml measuring cylinder.
- Goggles.
- Stopwatch.
The variable that I will change in my investigation will be the concentration of the substrate. I will try to keep every other possible variable the same for each experiment to reduce the risk of affecting my results.
I will measure the amount of oxygen produced in a set amount of time that should give me the changing speed of the reaction.
METHOD.
- I will start by setting up the apparatus as shown above. I will put my goggles on as instructed by the hazchem code for these substances.
- Using the pipettes as they are a more precise way of measuring, I will measure out first 5cm3 of catalase from the beaker to the measuring cylinder and then do the same for my chosen amounts of water and hydrogen peroxide. The amounts will vary according to the concentration that I am doing for that particular experiment. Below is a table containing the concentrations of my experiments.
I will keep the volume of enzymes at 5cm3 for each experiment. I will keep the volume of substrate solution at 10cm3 for each experiment.
- I will then add the distilled water to the hydrogen peroxide to get my concentration of substrate.
- I will fill up the 100ml-measuring cylinder full of water and tip it upside down in the washing up bowl so that the pressure of the water in the bowl holds up the water in the measuring cylinder. There must be no air in the top of the measuring cylinder otherwise my results will be inaccurate. I will then push the delivery tube up through the bottom of the measuring cylinder so they are both vertical.
- I will first pour the catalase in the conical flask and then add the substrate solution. As concurrently as possible I will put in the bung to stop any oxygen from escaping and also start the stopwatch.
- The oxygen produced should rise out of the conical flask and through the delivery tube. It will then come out of the end in the form of a bubble in the water and rise to the top of the measuring cylinder displacing some of the water there and causing an air pocket that will give me the reading of oxygen produced.
- I will time the experiment for two minutes and at the end of those two minutes I will read off the amount of oxygen produced in the measuring cylinder as accurately as possible and record them in a table like the one below.
- I will repeat the experiment three times for each concentration to ensure that I can get a more accurate average at the end and compensate for any anomalies that I may get. I have chosen to do 6 separate substrate concentrations at 20% intervals to give a wide range of results.
- I will then look at my results and if any of them look like anomalies then I shall do that particular experiment again.
- Before I do my main experiments, I will carry out a few preliminary tests to test my plan so that I can improve on anything that needs improving on.
- I will do a test at a high concentration and a low concentration to find out the approximate size of measuring cylinder I will need to collect my gas in and also to see if my experiment runs smoothly therefore what alterations need to be made. Below is a table of the results that I gathered. It tells me that I will need an approximately 100ml measuring cylinder as this is the largest one available for this experiment. I also recognized the need to move swiftly when adding together the two substances, placing on the bung and starting the stopwatch all at the same time. Good co-ordination is needed.
HOW WILL I KEEP MY PLAN FAIR?
- I will label my beakers, cylinders and pipettes as well as washing them out after I every test so that the three substances don’t mix before I start my experiment and start the reaction early.
- I will not inhibit the reaction by moving the conical flask while the reaction is taking place. This agitates the molecules and increases the probability that some of them will collide.
- I will try and keep human error to a minimum, therefore I will try and measure out the same amounts of substances each time and make sure I read off the volume of gas correctly.
- I will use the same batch of catalase and hydrogen peroxide at all times at one batch is never identical to another and it could affect my results.
- I will keep all the variables apart from the concentration of the substrate constant throughout all the experiments.
- I will repeat the experiment three times for each of the temperatures incase I get one of the experiments wrong I can compare it with the others and see what might have gone wrong.
- I will try and be very quick when placing the bung in the conical flask as the reaction starts as if I don’t then I may lose a lot of the oxygen produced at the start of the reaction.
- I will try not to swap measuring cylinders in the middle of an experiment and if I do I must change it swiftly so that I do not lose too much oxygen.
- I will also make sure that the time I choose for the experiment to take place in stays the same for each experiment. This will be 2 minutes.
- I will write down my results to the highest possible degree of accuracy so my results are as accurate as possible.
- I will always use 5cm3 of enzymes for all of my experiments.
These precautions will make sure my final results are as reliable as possible and make my investigation successful.
SAFETY PRECAUTIONS.
I will take note of the precautions on the hazchem cards,
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I will wear my goggles throughout the experiment.
- I will tie my hair back so it will not get in the way of the experiment.
- I will follow any other lab rules such as no running and chairs under the desks.
- I will not look directly over the reaction while it is taking place incase it goes wrong.
- The hydrogen peroxide that I will be using is corrosive so I will have to be cautious when using it.
- The hydrogen peroxide is also a bleaching agent so I will be careful not to spill any of it on my clothes.
OBTAINING EVIDENCE.
This is a table of results collected during my experiment.
Non of these results look very anomalous so it is my decision not to take any of these results again.
ANALYSIS OF RESULTS.
From the results I have collected in my table I have drawn a graph with a line of best fit (page 11). This graph shown the velocity of the reaction as I have plotted concentration of the substrate against the volume of oxygen collected in 2 minutes. In my prediction I said that as increase the concentration of the substrate, the amount of gas that I collect would also increase. Therefore the rate of the reaction would increase. I said that it would increase proportionally for a short while then level off gradually to a plateau where the amount of oxygen being produced will neither increase nor decrease. My graph reflects just this. This means that I have succeeded in proving my prediction is correct. I have drawn 2 dotted lines on the graph. The first one shows the initial rate of the reaction and the second one shows the final rate. If the reaction did not slow down at the end then the two lines would not have a gap between them. They show how the rate differs from start to finish. At the beginning of the graph up to a point around 24% substrate concentration the graph is straight and it goes through the origin. This shows a steady, proportional increase in volume produced. Above that point (25%), the graph begins to curve so it is here that the increase fails to be proportional. The line curves with a gradually decreasing gradient. I can make a good guess that the line would carry on curving to form a plateau of gradient 0 if I were to extend my graph, however my results were not broad enough to show this plateau. Most of my points seemed to follow the line of the curve with the exception of the point at 40% which I noticed after I had drawn my line was an anomaly.
Firstly, the simple increase in volume with concentration can be explained by the collision theory. This tells us that the higher the substrate concentration, the more substrate molecules there are, therefore more collisions can take place over a set amount of time as there is an increased probability that an enzyme and a substrate molecule will collide. The collision theory explains the steady increase in the graph but it doesn’t explain why it starts to level off as the concentration increases.
Up to 25% concentration the increase is proportional. This is where there are not enough substrate molecules for the catalase to work at a maximum velocity so as I added more substrate, the enzymes worked harder proportionally. At the point where the graph levels off, the reaction is starting to become ENZYME SATURATED.
