METHOD
All the equipment above was collected and arranged as follows; Clamps attached to tripod, water basin filled with tap water, test tubes placed in holder, Bung placed in test tubes, tile placed on work surface and hydrogen peroxide kept in safe place until required
Throughout the whole experiment safety was paramount. Gloves, apron, and goggles were worn as hydrogen peroxide is highly poisonous and toxic and extra care was taken whilst using razor.
The potato was placed on the tile and 5 circular rods were bored. Using the razor blade and ruler, 75 discs were cut and each disc weighed was
approximately 0.4g. Each disc was also cut into 4 quarters in order to increase the surface area of the potato catalase.
These discs were placed in groups according to their weight, 5 discs weighting 2g, 10 discs weighting 4g, 15 discs weighting 6g, 20 discs weighting 8g, 25 discs weighting 10g, and 30 discs weighting 12g. These groups were placed in individual test tubes.
Each test tube was labelled with the specific weight to avoid confusion. The test tube containing the group of 5 discs weighting 2g was then attached to the clamp half way down the tripod. The delivery tube and bung was placed in the top of the test tube and the tube placed into the water in the basin. A measuring cylinder was placed in the water basin. The measuring cylinder was filled with water in the basin. A syringe was used to draw 10ml of the 6% hydrogen peroxide, which was inserted into a hole in the bung. Holding the measuring cylinder vertically insert the hydrogen peroxide into the test tube and start the clock immediately. The syringe was left in the bung to prevent oxygen escaping. After 1 minute the amount of oxygen was measured and collected in cm3 and this result was recorded.
The same procedure was repeated for 4,6,8,10 and 12g groups.
This experiment was repeated a second time so to obtain a mean average of the results and also to minimise the extent of any inaccuracies.
RESULTS
The results obtained from each experiment were then transferred onto a table showing the Potato catalase concentration, amount of Hydrogen peroxide and time taken for oxygen to be released in 1 minute.
This table is to show the rate at which hydrogen peroxide is broken down into oxygen, when potato catalase was varied in concentration.
The mean average was calculated by adding each result, then halving it.
The data was transferred onto a graph so the results can be analysed in a clear way.
The graph shows the rate of oxygen released per 1 minute up to 12g potato catalase.
At 0, there was no reaction as no enzyme/ substrate was present. This was included to show that the variable, potato catalase, was being measured.
As the potato catalase concentrations increased, 2, 4, and 6g, the rate of reaction steadily increased on the graph; hence the rate of oxygen produced each minute increased. This steady increase showed that more active sites of the enzymes were being introduced into the reaction and more substrate molecules were able to bind and be broken down each minute. The enzyme concentration is directly proportional to the rate of reaction.
Between 10g and 12g the graph show that the rate of oxygen released per 1 minute, slowed down and the graph began to tail off, a slight increase was observed. The potato catalase is no longer proportional to the rate of reaction. To add more catalase would not increase the rate at which hydrogen peroxide is broken down, as this is now the limited factor.
This shows that the rate of reaction increases with increasing enzyme concentrations, up to a point.
Conclusion
The reaction was observed to be the fastest at the enzyme concentration of 12g potato catalase. At this point the mean average of oxygen produced was 32.0cm. At this point the enzyme concentration was at an optimum concentration, to that of substrate. Substrate molecules occupy the majority of the enzymes active sites and more oxygen was produced per 1 minute. The rate of oxygen released at 12g potato catalase appears to slow down and tail off on the graph. It appears that a further increase in enzyme concentration would have little effect on the rate of oxygen produced, as the substrate concentration is the limited factor.
DISCUSSION
Within the above experiment the enzyme, catalase had to be present in order for the substrate, hydrogen peroxide to be broken down to form products. Enzymes played an important role within this experiment, as they act as biological catalysts that speed up the reaction. For any reaction to occur a certain amount of energy is required, this is known as activation energy. Where enzymes are absent in a reaction, energy can be given in the form of temperature or pressure that provides molecules with kinetic energy. This overcomes the energy barrier and, enables them to move faster thus more collisions occur. An increase in collisions gives a greater rate of reaction. In the presence of an enzyme the activation energy is lowered and the reaction will occur more readily, even at low temperatures. An increase in temperature above ( 40 to 45 ) can break delicate hydrogen and Ionic bonds which give the enzyme this specific structure. The substrate molecule will no longer be able to bind to the active site as the enzyme is denatured. The experiment was performed at a constant room temperature ( 15 ) and no source of heat was introduced. A possible source of error could have been slight variations in the room temperature, which may have led to some inaccuracies between varying enzyme concentrations. An improvement to this experiment would be to measure the temperature of the reaction with a thermometer. Some reactions transfer energy to the environment (exothermic reaction), which increases the temperature of the reaction. Energy can also be absorbed from the environment (endothermic reaction), in this case temperature decreases. In both these cases, a more accurate measurement would be obtained by using a thermometer.
Enzymes are complex globular protein molecules, which have a specific protein tertiary structure. This structure is formed from basic amino acids, which are formed in a particular sequence. This sequence is held together peptide bonds that link each amino acid. The specific structure of the enzyme is given when a polypeptide chain folds back on itself. This folding occurs because there are different areas of negative and positive charge, which attract each other. This specific structure is held in position by hydrogen, ionic and disulphuric bonds. These attractions form a specific surface area, which is known as the active site on the enzyme. It is the shape that determines the position of the active site. This specific area on the enzyme, active site, is were the substrate molecules of hydrogen peroxide binds to and was converted into products (water and oxygen). The substrate molecule is usually made up of different compounds, which cannot be used unless they are broken down into small compounds. The process when larger molecules are broken down into smaller molecules is known as hydrolysis, where the addition of water is required, within the reaction. In this experiment hydrogen peroxide was broken down from a toxic substance to water and oxygen. It is the role of the enzyme to break down these complex compounds into simple compounds, which are useful and harmless.
The substrate used in the experiment, hydrogen peroxide is also formed continuously as a product of metabolism. The enzyme, catalase, immediately breaks down this toxic product into water and oxygen.
All enzymes are very specific in that they will only react with a specific substrate to form a specific product. In this experiment only catalase will break down hydrogen peroxide into products providing the environment is suitable.
Two models of how the enzyme/substrate molecules work is, Lock and Key hypothesis and induced fit. In a reaction the substrate molecules collide with the enzymes active site. Each enzyme is thought to operate on a lock and key mechanism. A substrate molecule of a particular shape to that of the active site of an enzyme will bind precisely, Just like a lock and key. The enzyme and substrate are complimentary to each other in shape and at this point whilst joined they form an enzyme/substrate complex. This E/S complex changes the nature of the substrate into products, and they leave the active site. At this point it is free, and another substrate molecule can bind to it. Each reaction an enzyme catalyses, it is unaffected by the reaction and can be reused. Emil Fischer (1894) proposed the lock and key hypothesis which states that “Enzyme and substrate molecules combine to form an enzyme substrate complex before the products of the reaction are released”. Taken from (Advanced human biology, pg 34; Simpkins and Williams)
The specificity of the enzyme structure and function supports this theory.
A more recent theory of how enzymes function was devised by, Koshland (1958). This was the induced fit theory. He states that “The shape of the active site is changed when a substrate molecule binds to the enzyme,” taken for (advanced human biology, pg 34; Simpkins and Williams). In this theory the substrate molecule is not Complementary to that of the enzymes active site. The enzymes active site moulds itself around the substrate so that it binds. This can be described like a glove moulding itself around the hand.
Before the substrate binds to the enzymes active site is relaxed. When the substrate binds the active site it is pulled into the correct shape and the enzyme/substrate complex forms. As the substrate is broken down the products fit the active site less well and they fall away from it. The enzymes active site is now available for another substrate molecule.
The results of this investigation are as I predicted in my hypothesis:
“Increasing concentration of potato catalase will show an increase in the rate of oxygen produced, because more active sites are available. This will only occur when excess molecules of hydrogen peroxide are available. If molecules of hydrogen peroxide are limited the rate of products formed will be low.”
The data obtained from the experiment showed that as the enzyme, catalase, increased the rate of oxygen produced per 1 minute increased, up to a point.
After this the rate of oxygen produced per 1 minute slowed down. The reasons for this are that there are other variables that can influence the breakdown of hydrogen peroxide in the presence of catalase.
When I increased enzyme concentration it meant that more active sites were available to a greater quantity of substrate and so more substrates would be broken down into products each minute. Enzymes function efficiently in low concentration as the molecules can be used over and over again.
However, at 12g potato catalase, there many free active sites but insufficient substrate molecules to occupy them. I believe that a further increase in enzyme weight without increasing the substrate concentration would have no effect on the rate of reaction, which would eventually remain constant.
The temperature, substrate concentration, and pH all affect the reaction and must be controlled. These factors above are required for an efficient reaction to take place, even when the enzyme concentration increases; it can also be limited by the availability of others.
PH – Each enzyme has an optimum pH at which its active site best fits the substrate. Any changes in pH can results in hydrogen and ionic bonds becoming damaged and this changes the shape of the enzyme and prevents the substrate from binding to its active site. The enzyme is denatured and a slower rate of reaction will be observed.
The pH of the hydrogen peroxide would have been very acidic, and may have affected the enzymes ability to function effectively. The concentration of hydrogen peroxide was kept constant throughout the whole experiment, but the pH of the reaction was not measured. Within this experiment there could have been possible variations either side of the pH that could have possibly affected the rate of oxygen produced. In further experiments the pH could be considered, and made equal to that of the natural environment of the potato catalase enzyme (around pH 7).
Inhibitors- Competitive inhibitors compete with the substrate for the enzymes’ active site. Non-competitive inhibitors attach themselves to the enzyme, but not the active site. This changes the active site so the substrate can no longer bind to it. Inhibitors slow the rate of reaction. No inhibitors were included within the experiment, therefore would not have affected the rate of reaction
Many factors can change the activity of the enzyme. Within the experiment I varied enzyme concentration, by using different weights of potato pieces.
However the exact quantity of catalase present within the potato tissue was unknown. I tried to compensate for this by repeating the experiment twice.
An improvement to this would be use 1 molar solution of catalase that could be diluted to form different concentrations, which could measured more accurately.
In trial experiments it was found that when I increased potato catalase, they were not all being covered by the 10ml hydrogen peroxide. From this I decided to cut the circular discs into 4 quarters, and found that they all could be covered in the hydrogen peroxide. This also increased the surface area of the potato catalase. I used this to see how changing potato catalase in weight, influenced the breakdown of hydrogen peroxide, as the quantity and speed of oxygen produced is dependant on the rate of reaction. Initially, once the hydrogen peroxide was inserted onto the potato catalase, it was clear that there was a reaction-taking place as bubbles of oxygen gas were transferred via the delivery tube into the measuring cylinder faster within the first minute. Trail experiments proved that after 1 minute it took too long to get a certain amount of oxygen, so I decided to collect oxygen released in 1 minute.
The hydrogen peroxide was kept at 10ml throughout each experiment. In trial experiments a greater amount of hydrogen peroxide was used, but this caused a violent reaction as froth travelled into the delivery tube, which prevented oxygen entering the measuring tube, therefore this would have distorted results.
In further investigations of enzyme-controlled reactions the experiment could have been repeated more so to obtain more accurate results and the above suggestions and improvements could be taken into consideration.
