In plants hydrogen peroxide is produced during an intermediate stage of nitrogen fixation.
Regardless of whether it is produced in plants or animals, it needs to be detoxified rapidly before it causes any damage to cells.
A single catalase (enzyme) molecule can break down 5.6 million molecules of hydrogen peroxide in one minute. It is therefore not very surprising that catalase is found in high concentrations in cells.
Enzymes are also a principal mechanism in many industrial processes. They are key in the production of vinegar, yoghurt and cheese, in brewing, washing powders, processing of leather, etc.
This is not only because they speed up the process, saving time, but because they are efficient, with a very high turnover rate and can be used again and again, saving money.
There are several factors that determine the rate of enzyme activity. These factors include the pressure of enzymes/substrate present, the temperature and pH of the environment they are in and whether or not an inhibitor is present.
Concentration: Obviously the higher the concentration of substrate, the more molecules are present and so the faster the rate of reaction will be, because there will be more molecules to bind with enzymes at any one time.
This, however, is only true for the initial rate of reaction, and only to a certain extent. If you only have a limited amount of enzyme molecules, the increase in the rate of activity will not continue forever. At very high substrate concentrations each enzyme molecule will constantly have a substrate molecule bound to its active site. This leaves substrate molecules, in effect, ‘queuing up’ waiting for available active sites. When this happens, the enzymes have reached their Vmax, which means they are working at their maximum possible rate.
Naturally, the more enzyme molecules present the faster the rate of reaction will be, because there is a higher chance of substrate colliding with a free active site. Nevertheless, this is only possible provided that ample substrate is present. If there is only a small amount of substrate, no matter how much enzyme is added the reaction will not quicken, as there will be no free substrate units to be broken down. When this happens, the substrate concentration has become the limiting factor in the reaction.
Temperature: Raising the temperature will normally increase the rate of enzyme activity because the molecules have more kinetic energy, and are therefore moving more rapidly and with more force. This increases the chance of successful collisions between particles as they are bumping into each other more often and have a higher chance of overcoming the activation energy.
Raising the temperature, by 10°C, will in the majority of reactions, double the rate of enzyme activity. This only applies until the optimum temperature is reached, which is when the enzyme molecules will work at their fastest rate.
Once the optimum temperature has been exceeded the heat will cause the molecules to vibrate too violently causing hydrogen bonds to break, hydrophobic interactions to cease, and reform in other places, altering the shape of the enzyme molecule. On the distortion of the active site, the substrate molecule will no longer be able to bind to the enzyme, as it will not fit. The enzyme has become denatured and can therefore no longer function as a catalyst.
The optimum temperature for catalase is between 40-50°C, beyond this and it will begin to denature.
pH: The measure of the concentration of hydrogen ions in a solution is referred to as the pH. The higher the pH is the lower the concentration. An acidic solution has many free hydrogen ions, whereas an alkali solution has many hydroxyl ions.
These ions can affect the hydrogen and ionic bonds that hold the active site in its precise shape, and can also affect the R groups that line the active site disabling its ability to form temporary bonds with the substrate.
Most enzymes only work within a restricted range of pH, and although they do vary, most (including catalase) have an optimum pH of around 7.
Inhibitors: A substance that prevents an enzyme from catalysing a reaction is called an inhibitor. There are two ways in which they work.
Competitive inhibitors resemble the shape of substrate molecules and are accepted by the enzymes active site. They compete with substrate for free active sites, stopping the substrate molecules themselves from binding and being broken down, which as expected, slows down the rate of reaction.
Cyanide is a competitive inhibitor of hydrogen peroxide as it is accepted by the active site in catalase molecules, stopping the H202 molecules from binding with it and being broken down.
Non-competitive inhibitors do not bind to the active site, but to another part of the enzyme, distorting its shape. This alteration causes a change in shape of the active site so that substrate will no longer fit.
Copper sulphate binds to catalase distorting its shape so that H2O2 will no longer fit into its active site.
Non-competitive inhibitors are more successful, in that the degree to which they have an affect does not depend on the concentration of substrate, as they can still successfully bind to enzymes even if a large quantity of substrate is present.
Competitive inhibitors on the other hand will only have a noticeable affect on the rate of reaction if there is small substrate:inhibitor ratio.
aim:
By carrying out this experiment I hope to investigate the effect of different catalase concentrations on the rate at which hydrogen peroxide breaks down into oxygen and water.
prediction:
I believe my results will show that the rate of reaction will be proportional to the concentration of catalase added to the hydrogen peroxide solution, i.e. the higher the concentration of catalase, the faster speed of the reaction.
As stated in my ‘scientific knowledge and understanding’ a higher concentration implies a larger number of catalase molecules will be present to bind with H2O2, leaving less to ‘que up’, and more to be broken down into water and oxygen at any one time. A larger number of catalase molecules also means there is higher probability of collisions occurring between enzyme and substrate.
Based on this I predict that my 25% concentration catalase solution will have the slowest rate of reaction, as there will be fewer molecules present to react with the H2O2 than in any other concentration, and the 100% solution will have the fastest rate of enzyme activity, as it will have most enzyme molecules present out of all of the solutions.
As I have increased the concentration of each catalase solution constantly, by the same amount each time, I assume that the initial rate of reaction for each catalase concentration will increase by the same amount each time.
I believe that the 55% concentration solution will occur just under half the speed of the 25% concentration and at just over a third of the speed during the 70% concentration.
apparatus (and justifications):
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Distilled water; will be used to clean some of the apparatus and to compose the catalase solutions instead of normal tap water, because it has no impurities, which may affect the break down of H2O2, resulting in inaccurate results.
- Celery extracts; will be combined with the distilled water to make the different catalase solutions.
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Hydrogen peroxide; the difference in the rate of break down of H2O2, into water and oxygen, will be
observed of different concentrations of catalase solutions.
- 3 10ml and 2 5ml syringes; 1 of the 10ml syringes will be used for the hydrogen peroxide, whilst one of each size is used for both the distilled water and the celery extracts. I will be using syringes rather than measuring cylinder because they are much more accurate in measuring liquids, and are still practical to use, as I will only be using relatively small amounts of each liquid.
- 3 50ml glass beakers; I will pour each of the different liquids into a different beaker, so that they can be ‘sucked up’ by the syringes.
- 6 25ml glass beakers; to hold each of the catalase solutions.
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Conical flask; the shape of the conical flask allows for the ‘swirling’ of the liquids which are in it. This is a very important feature, as the H2O2 will need to be stirred with the catalase solution in one way or another to ensure that the enzyme and substrate molecules are evenly dispersed, without any being lost in the process.
- S-shaped delivery tube with thistle funnel; this allows entry of a liquid into the conical flask without any gas escaping through the top. Any gas produced will only be able to escape through the delivery tube.
- Petroleum jelly; this will be used to seal the conical flask off, completely, to ensure that absolutely no gas escapes, in the case of the thistle funnel/delivery tube fitting being loose. It is very important that no gas is able to escape because this will dramatically alter my results and make them unreliable.
- 1 burette; the s-shaped delivery tube will lead to the burette which will be filled with water. As the oxygen gas is produced it will travel through the delivery tube, into the burette and float to the top where it can be measured. I have chosen to use a burette because it will be fairly accurate for the measure of gas produced.
- Clamp stand; to hold the burette upright.
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Large plastic container; this will be filled with water and provide an environment for the transfer of O2 gas from the delivery tube to the burette.
- Stopwatch; to measure the production of oxygen during regular time intervals.
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China graph pencil; to label everything, including: which liquid each syringe and beaker is for and to draw a mark on the burette on the level of O2 produced every minute.
method and diagram:
- Label each syringe and beaker according to which liquid/concentration it will be handling.
- Using the syringes make up the following concentrations of catalase using the distilled water and celery extract.
- Fill up the large plastic container with water, making sure the water level is high enough to cover the delivery tube leading to the burette.
- Making sure the burette tap is closed, fill it up with water and turn it upside down, with your finger placed over the top to stop any water coming out. If you were not able to stop the entry of air bubble, with the china graph pencil make a mark to indicate the water level before the experiment has started. Place the top into the plastic container filled with water, and hold it in position with the clamp stand. Adjust the position of the burette so that the delivery tube will fit underneath it.
- Using the labelled syringe, measure out 30ml of the hydrogen peroxide, transferring it from the syringe straight into the conical flask, so as to reduce the risk of any loss of the substance through transferring it from vessel to vessel.
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Cover the delivery tube stopper in petroleum jelly and seal off the conical flask, making sure that the end of the thistle funnel is in the H2O2, i.e. below the liquid level, and that the delivery tube is positions directly underneath the burette in the plastic container with water.
- Ready with the stop watch and china graph pencil, swirl and then pour the first concentration of catalase into the conical flask through the thistle funnel, start the stop watch and then quickly swirl the contents within the flask.
- Every 15 seconds, for the first 3 ½ minutes, draw a mark on the burette to indicate the amount of gas produced in that amount of time.
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Once the experiment has finished, write down any observations you have made, and note down the volumes of gas produced for each time interval. Remove the conical flask and wash it out using distilled water. Remove the burette and fill it up with water again, to set it up as before. Measure out another 30ml of H2O2 into the conical flask.
- Redo this experiment 3 times for each concentration solution.
- Repeat steps 6-10 of the experiment for each of the different concentration catalase solutions.
analysing:
Before I commence the experiment I will need to draw up several tables to accumulate the data collected.
One table, with the concentration of catalase and the amount of oxygen produced every 15 seconds, will need to be created for each different concentration, with three rows, as the experiment will be carried out three times for each concentration.
Once I have gathered all my results I will be able to find averages for the amount of O2 gas produced every 15 seconds for each concentration of catalase, by adding the three times for one time interval and then dividing by three. Finding the average will eliminate any anomalous results, making the data more reliable.
Using these time averages, I will be able to plot an amount of O2 gas produced (y-axis) against time (x-axis) graph. By drawing a line of best fit on the graph (which will be curved, because as the H2O2 is being broken down, it will be decreasing in concentration which slows down the rate of enzyme activity, as explained in my ‘scientific knowledge and understanding’) I will be able to find the initial rate of reaction by finding the gradient of the line, before it begins to curve. The initial rate f reaction will be the amount of O2 produced in the first 15 seconds divided by 15 seconds.
I will need to find this figure for each of the different concentration catalase solutions so that I can plot my second graph: initial rate of reaction (x-axis) against catalase concentration (y-axis), which should have a positive correlation, with a straight line of best fit. This graph will show the relationship between the concentration of catalase present and the rate of reaction, so it is from this second graph that I will be able to conclude whether or not my original predictions were correct.
I will be able to see the difference in times between the different concentrations, to see if my beliefs that the 55% concentration solution would occur at just under half the speed of the 25% concentration, etc.
Using this graph I will also be able to estimate the rate of reaction for any concentration of catalase (between 0.0M and 1.0M) breaking down 30ml of hydrogen peroxide.
method justification:
Before the experiment began the celery extract was filtered to remove any larger parts of celery that would settle at the bottom of the beaker, meaning the catalase would be unevenly dispersed throughout the solution, so any small amounts ‘sucked up’ by the syringe, at the surface, would in fact have a different concentration to begin off with.
Catalase solutions were in fact, swirled before they were transferred from any vessel to ensure the catalase was always evenly disbursed.
The solutions were also swirled together once the H2O2 had been added to better scatter the enzyme and substrate molecules.
During step 6 of the method the mixture in the conical flask will have to be swirled with the delivery tube remaining underneath the burette so that none of the gas produced is lost. This can be achieved by rocking the conical flask from side to side, rather than in full circles.
This is necessary because the loss of any oxygen produced by the experiment would alter my results, making them inaccurate and unreliable.
Even so it must be taken into account that the air already in the delivery tube should not be counted as part of the oxygen produced. I decided to add the hydrogen peroxide to the flask with the delivery tube already under the burette because if I was to place it underneath after the H2O2 had been added, there would be no way of being certain I was letting the same amount of air escape before directing the delivery tube under the burette, or even if I was beginning to let oxygen to escape. So that the experiment was a fair test I decided to include the air that was already in the delivery tube because the difference in volume of gas produced would be the same for every experiment carried out.
Another article used to ensure no oxygen was lost is the petroleum jelly, which sealed the stopper and conical flask together.
I resolved to carry out the experiment three times for each concentration because if I ended up with an anomalous result it would not have as much of an impact on my final average finding, as it would have if I only carried the experiment out once or twice. Although carrying out the experiment as many times as possible would have given me the most accurate result, I only have a limited amount of time to carry out the entire experiment.
I decided on which catalase concentrations to use from my preliminary work, in which I found the amount of gas produced for 25, 50, 75 and 100% concentration catalase solutions. I concluded that there was no need to carry out the experiment for less than 25% because it would happen too slowly to be bothered with. I decided to choose concentrations that had less of a difference, so that when it comes to plotting my graph I will be able to plot points closer together and get a more accurate line of best fit.
During my preliminary investigations I only noted the volume of gas produced every 30 seconds, but found that it was difficult to see the progression, so I decided to shorten the time intervals to 15 seconds for the real experiment so that the difference between the amount of oxygen produced would be more evident.
I decided to carry on each experiment for 3 ½ minutes to end up with 14 results for each concentration. The more results are obtained the easier it is to plot a graph and draw in the line of beat fit. I shortened the time to 3 ½ minutes from 5, which I did in my preliminary because the volume did not alter very much after 4 minutes and this way I would be able to carry out the same experiment more times, to give me a more accurate average result.
safety:
Of the apparatus being used some is made of glass, and therefore very fragile. Care must be taken to ensure that none of the apparatus is knocked over/falls and breaks, as the glass would shatter.
In the event of any breakages, DO NOT TOUCH ANY BROKEN GLASS, but call the supervising teacher immediately.
Hydrogen peroxide must also be handled with caution. It is a corrosive chemical; so a lab coat and goggles must be worn. If contact is made with the skin, wash immediately with soap and water. To reduce the risk of any spillages, the transfer of H2O2 to different vessels should be limited, such as in step 5 of the method where it is transferred from the beaker to the syringe straight to the conical flask.