An enzyme, which will catalyse reaction, is written as: -
E is the enzyme, S is the substrate, ES is the enzyme-substrate complex and P is the product.
The (S) substrate will bind to a specific site on the surface of the enzyme known as the active site. The reaction will occur on the enzyme surface, after which (P) product and (E) enzyme are released. The enzyme can then bind another substrate
Health and safety
Hydrogen peroxide can damage your clothes, therefore, I will rinse any spills with water immediately. In addition, I will wear safety glasses and if there is any accident I will report as soon as possible
Apparatus
- Gloves
- Stop watch
- Hydrogen peroxide
- Distilled water
- Potatoes
- Cork borer
- Measuring cylinder
- Conical flasks
- Water bath
- Small container box
- A stopwatch
- A ruler
- Cork borer
- Graduate pipette
- Gas syringe
Method
Assemble apparatus as shown below: -
- Connect delivery tube to the conical flask, then fill water bath with full of water and place delivery tube in water bath.
- Extract potato tissue by using cork borer, and then measure the exact size of potato tissue to 3cm by using a ruler.
- Take 6 conical flasks, and label first conical flask as 100% concentration, second conical flask as 83%, third conical label it as 67%, fourth conical flask as 50%, fifth conical flask as 33% and six and last conical flask label it as 17 % of concentration of hydrogen peroxide and water. Use cork borer, to extract 12 pieces of potato tissue from the potatoes, then cut piece of potatoes on the edge to make all in the same size (3cm long – 1.5 cm diameter)
- Measure hydrogen peroxide by using measuring cylinder to 30ml, dilute with 0ml of water, which this will give us 100% of substrate concentration. Secondly, measure hydrogen peroxide to 25ml, dilute with 5ml of water, which this will give us 67% of substrate concentration. Thirdly, measure hydrogen peroxide to 20ml, dilute with 10ml of water, which this will give us 80 % of substrate concentration. Fourthly, measure hydrogen peroxide to 15ml, dilute with 15ml of water, which this will give us 50% of substrate concentration. Fifthly, measure hydrogen peroxide to 10ml, dilute with 20ml of water, which this will give us 33% of substrate concentration. Sixthly, measure hydrogen peroxide to 5ml, dilute with 25ml of water, which this will give us 17% of substrate concentration.
- After having all different desired concentration of hydrogen peroxide diluted with water in conical flasks, and 3 cm long x 12 pieces of potato tissues in the container box; you will be ready to start the investigation.
- Put a piece of potato tissue in the conical flask, this contains enzyme ‘catalase’ and pours 30ml (100% concentration) of hydrogen peroxide solution and immediately put the gas syringe. Bubbles should start to rise up the tube and gas syringe will move outwards, at the same time start the stop watch and leave it for three minutes and measure how much gas is produced within the fixed time.
- Repeat procedure number 6, but do not use the same piece of potato tissue nor the same hydrogen peroxide. Use different piece of potato tissue (3cm long, 1.5cm diameter) with different concentration for each trial (see procedure number 4 for different concentration).
- The table below illustrate how much water and hydrogen peroxide is needed to make different concentration.
- Repeat the entire test, so that an average can be obtained. Repeating the experiment will help to produce better and more reliable results, as any inaccuracies in one experiment should be compensated for by the other experiments.
- To ensure this is fair test all the variables except for the concentration of hydrogen peroxide must be kept the same through out the experiment. Variable must not be altered include: - Temperature, enzyme concentration – shape and size of the potato (surface area) etc. I have taken the following certain factors and variables into account.
If substrate concentration is low the active sites of the enzyme molecules will not all be occupied, as there are simply not enough substrate molecules to occupy them all. As the substrate concentration is increased, more and more active sites are used. When all the active sites are occupied and the amount of enzyme is the limiting factor, as you increase the amount of the substrate there are more collisions and more enzyme- substrate complexes formed.
If the temperature increases, it can affect the rate of an enzyme-controlled reaction in two ways:
(1) As the temperature increases, the kinetic energy of the substrate and enzyme molecules increases and so they move faster. The faster these molecules move, the more often they collide with one another, and therefore the rate of reaction is faster. In addition, the more often they collide more enzyme- substrate complex is formed and more products are released.
(2) As the temperature increases, the more the atoms, which make up the enzyme molecules, vibrate. If they vibrate, too much it will break the hydrogen bonds and other forces, which hold the molecules in their precise shape. The three-dimensional shape of the enzyme molecules is altered so much that their active sites no longer fit the substrate. The enzyme will be denatured and loses its catalytic properties.
The optimum temperature for an enzyme varies considerably, depending on its surroundings. For many enzymes, the optimum temperature is 400C and denaturation occurs at 600C.
Altering the pH can also break the bonds of the three-dimensional molecular shape. This is because the bonds may be broken by the concentration of hydrogen ions and pH is a measure of hydrogen ion concentration. Any change in the pH can denature enzymes. Each enzyme work best at its own particular pH (Optimum pH).
Changing the size of the surface area of the potato can also alter the rate of reaction. If there is a larger surface area, more enzymes will be released form the cells at the surface of the potato and the reaction will speed up. A larger surface area can be achieved by cutting up the potato into smaller pieces. I will keep the surface area the same by using the same size, shape of the cork borer and potato each time.
By varying the substrate, concentration used each time in the experiment will affect rate of reaction. The more concentration used in the experiment the faster the hydrogen peroxide will react.
I will be varying the concentrations of hydrogen peroxide used in my experiment to find out if that affects the rate at which the enzymes cause the reaction to speed up. I will be controlling the temperature and the pH at which I conduct my experiment. I will also be controlling the surface area of my potatoes by making sure they are cut with the same size cork borer and that they are of equal length. Moreover, I will also use the same volume H O . This is important because if you increase the volume of hydrogen peroxide you will be increasing the number of hydrogen peroxide molecules and therefore, there will be more chance of a hydrogen peroxide molecule colliding with an enzyme molecule. This will increase the number of enzyme –substrate complex molecule formed and the amount of product released.
In order to make my results as precise as possible, I will use the stop clock to help get the exact time and readings and I will use a measuring cylinder that has accurate markings. I will measure all my readings on the same scale – ml. and I will make sure that I only start the stop clock once the bung has sealed off the conical flask. I will get one of my friends to help me in telling me when it is the exact time to read off the measurements and write them down. I must also make sure that I have diluted the substrate precisely in order to get accurate and reliable results.
The temperature has to be constant on room temperature the whole time in order that the results should not be affected. I must also keep the pH of the substrate as neutral as possible so as not to kill off the enzymes or make them denatured. This pH must remain the same throughout the whole experiment so that it should be a fair test. The surface area of the potatoes must also be kept the same throughout so that there are the same amounts of active sites available to the substrate. If there is a larger surface area and more active sites, the reaction will be speeded up and the experiment will not be a fair test in relation to the other times that I will be performing the experiment.
Results table
First results
Second results
Analysis
The Graph shows the amount of oxygen produced over a period of 3 minutes by the reaction between Catalase and Hydrogen Peroxide; in general, as the concentration of Hydrogen peroxide increases, the volume of oxygen produced also increases. Therefore, as you increase the volume of hydrogen peroxide the rate of reaction increases.
My prediction was correct; as the substrate concentration increases the rate of reaction will go up at a directly proportional rate until the solution becomes saturated with substrate hydrogen peroxide. When this saturation point is reached, then adding extra substrate will make no difference. The rate steadily increases when more substrate is added because more of the active site of the enzyme is being used, which results in more reaction, so the required amount of oxygen is made quickly. Once the amount of substrate molecules added, exceeds the number of active sites, then the rate of reaction will no longer go up. This is because the maximum number of active sites is being occupied, so any extra substrate molecules have to wait until some of the active site becomes available.
Evaluation
My experiment worked quite well in the end but if I were to do it again I would change a few things to make my results more accurate. Firstly, I will get more readings by using more potato, different size of potato and I will increase amount of hydrogen peroxide to see if they will have similar trend with my old results.
Before, i started the experiment I should have tested the delivery tube to see if there were any leaks in it by letting water flow through it when it was set up like in the experiment. If there were leaks then I could have either covered these with a thick coating of Vaseline or got a new delivery system. If there were a leak in the tubing that I used it is probable that some oxygen escaped and was not measured in the measuring cylinder. This would directly affect my results and would make them inaccurate. If I had no leaks, however, then my results would not have been affected by this means.
When using the measuring cylinders I used ones, which were too large and amount of water filed in the measuring cylinder, were not the same for each of the trial. I should have adjusted the size of the measuring cylinder to the expected volume of oxygen that I was collecting so that my percentage error would be reduced. In addition, I should have filled the measuring cylinder with the same amount of water for each trial. Whenever, I tried to put water in the measuring cylinder and then turn it up side down, it was very difficult, as some of the water was letting out, I couldn’t maintain the same volume of water inside the measuring cylinder.
Each time, I had to use the same type of potato which was very difficult, as time all the potato was used up and I had to use the same cork borer, it was very difficult to obtain a piece of potato tissue, as each time you use cork bore you had to cut it straight down, not on the side. Moreover, I had to maintain the same surface area of the potato tissue, sometimes, though; you have used the same cork bore, I difficult to have the same diameter of the potato tissue (every time to void some errors I had to cut the straight down).
It was difficult to maintain the same level of temperature, as when the reaction is rapid the substrate concentration is high and energy will be released, which might have increase the temperature of the hydrogen peroxide. In addition, I should have measured first the room temperature, as this might have effect on my results. Moreover, since rates of reaction are so sensitive to temperature I still would have liked to use a water bath during this experiment, as this would have regulated the temperature.
When starting the reaction by adding the hydrogen peroxide and the potato together they reacted immediately so I had to quickly put the bung in very quickly. When I was doing, this some of the oxygen may have escaped before I put the bung in. This would have directly affected my results and therefore my accuracy.
I could not start the timer and put piece of potato into the hydrogen peroxide at the same time therefore the timing was a bit inaccurate. I would have been better if I had had a partner to start the stop clock for me once the potato tissue was introduced to the hydrogen peroxide. This may have improved my results slightly.
