Variables:
There are many factors which will effect my investigation.
Input Variable: Temperature- this will be controlled using a thermostatically controlled water bath.
Enzymes have an optimum temperature at which they work fastest. For mammalian enzymes this is about 40°C, but there are enzymes that work best at very different temperatures, e.g. enzymes from the arctic snow flea work at -10°C, and enzymes from thermophilic bacteria work at 90°C.
Up to the optimum temperature the rate increases geometrically with temperature (i.e. it's a curve, not a straight line). The rate increases because the enzyme and substrate molecules both have more kinetic energy so collide more often and also because more molecules have sufficient energy to overcome the (greatly reduced) activation energy.
The increase in rate with temperature can be quantified as a Q10, which is the relative increase for a 10°C rise in temperature. Q10 is usually 2-3 for enzyme-catalysed reactions (i.e. the rate doubles every 10°C) and usually less than 2 for non-enzyme reactions.
The rate is not zero at 0°C, so enzymes still work in the fridge (and food still goes off), but they work slowly. Enzymes can even work in ice, though the rate is extremely slow due to the very slow diffusion of enzyme and substrate molecules through the ice lattice.
Above the optimum temperature the rate decreases as more and more of the enzyme molecules denature. The thermal energy breaks the hydrogen bonds holding the secondary and tertiary structure of the enzyme together, so the enzyme (and especially the active site) loses its shape to become a random coil. The substrate can no longer bind, and the reaction is no longer catalysed. At very high temperatures this is irreversible. Remember that only the weak hydrogen bonds are broken at these mild temperatures; to break strong covalent bonds you need to boil in concentrated acid for many hours.
Controlled Variables: pH
Enzymes have an optimum pH at which they work fastest. For most enzymes this is about pH 7-8 but a few enzymes can work at extreme pH, such as protease enzymes in animal stomachs, which have an optimum of pH 1. The pH affects the charge of the amino acids at the active site, so the properties of the active site change and the substrate can no longer bind. For example a carboxyl acid R groups will be uncharged a low pH (COOH), but charged at high pH (COO-).
3. Enzyme concentration
As the enzyme concentration increases the rate of the reaction increases linearly, because there are more enzyme molecules available to catalyse the reaction. At very high enzyme concentration the substrate concentration may become rate-limiting, so the rate stops increasing. Normally enzymes are present in cells in rather low concentrations.
4. Substrate concentration
The rate of an enzyme-catalysed reaction shows a curved dependence on substrate concentration. As the substrate concentration increases, the rate increases because more substrate molecules can collide with enzyme molecules, so more reactions will take place. At higher concentrations the enzyme molecules become saturated with substrate, so there are few free enzyme molecules, so adding more substrate doesn't make much difference.
Out Put Variable: - is the oxygen collected at different temperature:
I got the above information from ()
In order to make certain the experiment is a fair test and control all present factors I will:
- I will make sure the temperature is dead accurate before commencing with the experiment. The water bath will to do this since its thermostatically controlled,
- Guarantee the strict amount of substrate is used each time.
- I will also use the same potato; keep the size and surface area of the potato the same. A knife and a cork borer will assist with this.
Health and Safety Risk:
Goggles and Lab coats must be worn at all times to prevent solutions from contacting the eye or the skin. However, if the solutions make contact with eyes rinse thoroughly, if irritating continues alert teacher. If solutions make contact with skin wash off immediately if irritating continues alert teacher. In addition, if there is any spillage clean and dry the area effectively. Hydrogen Peroxide should be kept away from heat since it is an oxidant. Moreover, I will be using a sharp knife to cut potato which requires to be kept away from the experimental area if it is not in use.
My tables of results:
At 0ºC
Replicates: Volume of 0xygen collected in cm³
At 24ºC
Replicates: Volume of 0xygen collected in cm³
At 37ºC
Replicates: Volume of 0xygen collected in cm³
46.5ºC
Replicates: Volume of 0xygen collected in cm³
At 65ºC
Replicates: Volume of 0xygen collected in cm³
Analysis:
From my results I can quite clearly see that as the temperature increases so does the production of oxygen, which means as the temperature increased so does the rate of reaction for their to be more oxygen produced. I also noticed a trend between the results in that the volume of oxygen produced approximately doubles as temperature increases by 10º C. for example at 24ºC the volume of oxygen produced after 30 seconds is 0.42cm³ however, for 30 seconds but at 37.5º C the volume of oxygen obtained is 1.25cm³.
From the results obtained and the graphs drawn they clearly support my prediction. I predicted as I increase the temperature the rate of reaction will also increase. This will be up to the temperature the catalyst works best (optimum). From then on the rate of reaction will decrease until it stops because of the denaturing of the enzymes third structure.
From looking at my graphs you can see that the rate of reaction increased but only at the temperature the reaction worked best at, I found this to be 37.5º C. This is were the reaction was at its optimum temperature because this is the maximum rate the enzyme and substrate can react. Moreover, from here on the volume of oxygen obtained decreased. This was due to the enzymes third structure denaturing. This can also be seen on the graph as the temperature exceeds 37.5º C the volume of oxygen produced decreases.
The denaturing of the enzymes third structure occurs because the energy given into the reaction by the increasing temperature becomes too much some of the enzyme bonds begin to break due to vigorous vibration.
Evaluation:
Overall, the result I collected where in the region of expectation. In a sense, I felt as though my experimental procedure was appropriate even though a small number of problems were encountered while carrying out the experiment. the procedure was suitable as I noticed a clear trend in the graph I had drawn therefore the results were accurate and reliable for example, in the results table for temperature 24celcius which was room temperature I obtained at 30seconds were twice 0.5cm³ and once 2.5cm³ of oxygen was produced.
The problem I come across in the experimental techniques came out as sources of errors which can be analysed in my graphs where I have anomalous results. The anomalous results are circled on the graph and mainly the following factors have contributed for theses anomalous results.
Firstly let’s consider the concentration of the Catalase source and the surface area. This factor could have contributed to anomalous results on my Graph because had the potato disc been larger than they were supposed to be then there would be more Catalase present. Inevitably, there is more active site to bind with the substrate as a result increasing the rate of reaction. However, I controlled the volume and concentration of the Catalase source by making sure they had the same surface area by using a cork borer, they were also the same size by using a knife. These two methods helped to enhance the meticulousness of my result.
Secondly, had the temperature been less than it should have been, then the result obtained would have been void because I am not measuring to the temperature I stated. As a result the rate of reaction would have been slower than it should have been due to the less energy provided and fewer collisions. Even though I controlled the temperature by using a thermostatically controlled water bath I still stumbled across some problems which in more detail I will mention in my sources of error.
Thirdly, had I used more substrate than I should have than the increased number of substrate molecules, means that they produce more of the enzyme substrate complex as more of them bind to the active site. If the volume is increased a lot, than at one point all the enzymes active site would have been bound to a substrate meaning that the remaining substrate would be waiting for an active site to be free. However I controlled the volume of the substrate by using a syringe (cm³) which was problematic. In addition I also ensure that the bung was very tight inside the boiling tube that contained the reactants. This prevented any loss of substrate complex.
Sources of Error:
- As hydrogen peroxide was been delivered to a boiling tube using a syringe then instantaneously closing the tube with a bung, may have decrease the reliability of my results. The reason being is the reaction is fastest at the beginning when the most collisions are occurring, therefore, some enzyme substrate complex are most likely to have escaped.
- An easily avoidable error was not to have used room temperature to record results for 20º. This mediocre judgement reduced the reliability of my results for this specific result because the room temperature is not always 20º.
- The usage of more then 1 potato also hindered the reliability of my result as different potatoes have diverse level of concentration mainly depending on the environment it thrived in and depending how fresh it is or not.
- My biggest source of error occurred because it took too long for the thermostatically controlled water bath to heat up. This issue collided with the fact that I never had enough time to carry out the experiment. As a clear result of this dilemma I started some recordings of different temperature at a 1º or maximum 2º.
Improvements:
In the short or long term future if I was to do this experiment again there would be slight improvements made to overcome obstacles that hindered my results. For example;
- Firstly as an alternative I would use a thermostatically controlled water bath for 20º to represent room temperature instead of assuming room temperature is 20º.
- I will also ensure the same person reads the volume of oxygen produced from the measuring cylinder because different people might slightly read the measurements differently.
- If I also repeated the experiment more often then my results would have been more consistent which would have further increased the reliability of my results.
- Most importantly I would have allocated more time for the experiment so that I could have ensured that experiment was not rushed and that the thermostatically controlled water bath was at the correct temperature instead of being 1degree or 2 off.
