Sc1 Peroxide Investigation - James Baxter
1AS
Introduction
This experiment will investigate the effect of an environmental factor on the rate of Hydrogen Peroxide breakdown using Peroxidase enzyme in celery. We will be adding Peroxide (H202), which is a toxic product of several different metabolic reactions, to liquefied celery. The peroxidase enzyme in the liquefied celery has a specific active site like every enzyme and the hydrogen peroxide is a substrate that fits the active site of the peroxidase enzyme. Every enzyme as an active site, like a cleft or depression in its side and this enzyme has a substrate or substrates that fit this cleft exactly. In this experiment the interaction of the substrate (hydrogen peroxide) with the active site on the peroxidase enzyme breaks the substrate apart into two different products - water and oxygen. These two product molecules leave the active site, leaving the enzyme molecules unchanged and ready to bond with another substrate molecule.
Hydrogen Peroxide Water + Oxygen
2H202 2H2O + O2
Variables
There are many variables that we could change in the proceedings of our experiment that would change the rate of hydrogen peroxide breakdown and they are as follows: -
) pH - Most enzymes work best at a pH of around neutral (pH 7), however some enzymes have a different optimum pH. Since pH is simply a measure of hydrogen ions and these ions can't interact with the bonding of certain chemicals (for example, amino acids), an enzyme working in a pH very different from its optimum pH will have its bonding interfered with and it will eventually become denatured. We could perform the experiment under a number of different pHs and, keeping all other variables constant, see the effect of pH on the rate of oxygen production, hydrogen peroxide breakdown.
2) Temperature - When temperature is increased, the molecules in a chemical will move faster, thus increasing collisions between the substrate and enzyme, therefore speeding up the reaction. There comes a point however that the molecules are moving so fast that the enzyme becomes fractured and denatured when it collides with the substrate. Also if the temperature is too low there is virtually no molecular movement so no reaction is present. Therefore we could change the temperature in the experiment and record the oxygen production (hydrogen peroxide breakdown) at different specific temperatures.
3) Peroxide concentration - The more substrate molecules there are present in the solution, the more bonding will take place, assuming there are enough enzyme molecules to bond with them, as each substrate will bond with one of these enzyme molecules and so increase the reaction rate compared to when there were fewer substrate molecules. If the peroxide concentration decreases, there will be a decrease in the reaction rate since, assuming there are enough enzyme molecules to bond with, there will be not so many substrate molecules to bond with the enzyme molecules. We could alter the peroxide concentration and record the rate of reaction in accordance to that concentration.
4) Volume of Peroxidase - If the volume of peroxidase is increased there will be more enzyme molecules to bond to the substrate molecules with and so, assuming that there are substrate molecules available, the peroxide breakdown will be increased. However if all the substrate molecules are currently bonding, an increase in peroxidase will not increase reaction rate since the enzymes will not be able to bond, as there are not enough substrate molecules to 'go round'. The reaction therefore will not speed up. Therefore we could change the volume of celery and because the celery has peroxidase in it, this will increase the volume of peroxidase and so we can measure the rate of reaction in accordance to that volume.
5) Stirring - Because stirring increases the speed of the liquid, thus increasing the molecule speed, it therefore increases collisions between the substrate and the enzyme and so increases reaction speed. We could alter the time that we stir the solution and record the amount of oxygen produced at for those particular experiments.
6) Time - Time is a factor that can affect the rate of reaction immensely. If the enzyme and substrate are only allowed to react for a short period of time obviously the overall rate of reaction will be high since enzyme reactions start off fast then slow down later on, and the overall amount of oxygen will be small. If they are allowed to react for a long time, the overall reaction rate will be small since it almost levels out and slows down later on, and the mean average will be therefore smaller than a shorter time. However the total oxygen collected will be much higher than had the reaction been for a shorter time. We could alter the time that we record results for and compare the different average rates of reaction.
7) Mass - Keeping the other variables and the ratio between enzyme and substrate constant, the mass of the solution, when increased, will cause an increase in total amount of oxygen collected, but not rate of reaction, since the ratio between enzyme and substrate remains the same. If it is decreased, as before the total oxygen collected decreases but the reaction rate remains the same. We could alter the mass and record the different amount of oxygen collected at these different masses, despite the fact that reaction rate would remain the same.
8) Surface Area - Increasing ...
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7) Mass - Keeping the other variables and the ratio between enzyme and substrate constant, the mass of the solution, when increased, will cause an increase in total amount of oxygen collected, but not rate of reaction, since the ratio between enzyme and substrate remains the same. If it is decreased, as before the total oxygen collected decreases but the reaction rate remains the same. We could alter the mass and record the different amount of oxygen collected at these different masses, despite the fact that reaction rate would remain the same.
8) Surface Area - Increasing the chemical containing peroxidase, in this case it is celery, will increase the rate of hydrogen peroxide breakdown. This is because a larger surface area increases the number of particles exposed and available for reaction, thus increasing the rate of enzyme activity and consequently the rate of hydrogen peroxide breakdown.
9) Competitive and Non-competitive Inhibition - Enzymes are not required to work at their optimum rate, i.e. a reaction may stop when sufficient product has been formed, and this is called end product inhibition. Another type of inhibitor is a non-competitive inhibitor, which binds to an enzyme at a place other than its active site, altering the enzymes shape and leaving it unable to convert any more substrate to product, thus it is denatured. Competitive inhibitors have a similar shape to that of the enzymes substrate and literally compete with the substrate for bonding at the active site. Therefore if one of these competitive inhibitors enters the active site, a substrate molecule cannot and so the whole reaction is slowed down. The level of molecules which cause competitive inhibition depends on the concentration of the substrate - a higher concentration of substrate lessens the degree of inhibition. These types of substrate could hinder the rate of enzyme activity and therefore the rate of hydrogen peroxide breakdown. Thus the volume of the competitive inhibitor could be altered and the oxygen production recorded at these different volumes.
I have chosen to investigate the Hydrogen Peroxide concentration in my experiment as the variable. The peroxide, already diluted to 40% concentration, will be diluted to a further 5 concentrations and the same experiment, keeping all other variables constant, will be carried out on each concentration. To make it an utterly fair test, the peroxide concentration will be the only variable altered - all the others will remain constant.
Hypothesis
"As substrate concentration increases, the rate of reaction and therefore oxygen production will increase. As substrate concentration decreases, the reaction rate and therefore oxygen production will also decrease."
I predict this because enzymes, defined as biological catalysts, have a precise 3-dimensional shape with hydrophilic side chains on the outside of the molecule to ensure they are soluble, and a specific cleft or depression to which a molecule that fits the cleft can bind. This is the enzymes' active site and in this experiment the hydrogen peroxide substrate fits the peroxidase's active site and therefore bonding and a reaction can occur. The substrate is held in place by temporary bonds which form between the substrate and some of the side chains of the enzymes amino acids. Because the cleft (active site) in the enzyme is such a specific cleft, usually only one substrate fits it exactly - in this case hydrogen peroxide. The interaction between substrate and enzyme breaks the substrate apart and the products, in this case oxygen and water, leave the active site and thus leave the enzyme unchanged and ready to bond with another substrate. Therefore increase in the concentration of Hydrogen Peroxide will increase the number of collisions between hydrogen peroxide molecules and peroxidase molecules, and this will increase the rate of oxygen production.
a) An enzyme has a cleft in its side called the active site. The substrate molecule has a complementary shape.
b) Random enzyme and substrate movement brings substrate into active site. An enzyme-substrate complex is temporarily formed. The side chains of the amino acids in the active site interact with the substrate.
c) Interaction breaks substrate apart. The products leave the active site and leave the enzyme unchanged and ready to bind with another substrate molecule.
I would expect the graph for the rate of oxygen release against peroxide concentration to be a positive curve that gradually levels out since peroxide and oxygen release are not directly proportional. I expect it to level out because the rate of oxygen production will slow down since there will be less and less peroxide molecules to break up as the experiment proceeds.
Method
A preliminary experiment should be carried out to ensure that the concentrations and volumes of enzyme etc are suitable for the time allocated in which to perform the experiment. A net volume of hydrogen peroxide and water solution should be decided and also a net amount of celery extract. Both of these should remain at a constant volume - the only change should be the ratio between water and peroxide.
) Collect your apparatus.
This must include: -
Timer/Stopwatch
60ml of Hydrogen Peroxide
A 250 ml beaker
A delivery tube
A conical flask
Gas Burette
Bung
10 ml Syringe
30ml Liquefied Celery
Water
Set the apparatus up as follows: -
2) Take the 50ml of peroxide solution (at 40% concentration) and divide it into 5 beakers. Then dilute all of these concentrations down to a set of 5 different peroxide concentrations. Since our starting peroxide solution was already diluted to 40% concentration (2 parts peroxide to 3 parts water), therefore all the other concentration had to be lower than 40%. We then decided to split this into five concentrations so the first would be a fifth of 40%, the second would be two fifths, etc. In my case the 5 peroxide concentrations were 8%, 16%, 24%, 32% and 40%.
Test Number
Hydrogen Peroxide
Water
Celery Extract
Concentration
Volume
Concentration
Volume
40%
20 ml
60%
30 ml
3 ml
2
32%
6 ml
68%
34 ml
3 ml
3
24%
2 ml
76%
38 ml
3 ml
4
6%
8 ml
84%
42 ml
3 ml
5
8%
4 ml
92%
46 ml
3 ml
Total Peroxide used - 60ml
Total Water used - 190ml
To keep this experiment fair, the celery extract must remain a constant volume and the net volume of peroxide and water must also remain constant. The only thing that should change in the experiment is the ratio between water and hydrogen peroxide.
3) Divide the celery into five beakers, each containing a constant amount of celery. I divided the 30ml celery solution into five beakers, each containing 3ml of the liquefied celery. I decided this after using 5 divisions of 5ml celery, which produced a reaction slightly too vigorous to produce reliable results. Therefore I decided to use less celery (only 3ml for each of the 5 experiments instead of 5ml) to produce a reaction more under control to give a more reliable set of results. Even though this means that 15ml of the celery is not used, it gives a more controlled experiment than had we divided the celery into 5 beakers of 5ml or 6ml each and used up all or most of the celery solution. Therefore the amount of celery in each beaker should be small rather than large, if a more controlled experiment and a more reliable set of results are desired.
4) Use a syringe to remove one of the celery solutions from one of the beakers and place it inside the conical flask, or alternatively carefully pour it in so as not to lose any celery solution and thus alter the results.
5) Wash the syringe or take a fresh one, then use it to take one of the peroxide solutions and add it to the celery in the conical flask by squirting it down the side of the conical flask. This is necessary to ensure there is no backsplash of the corrosive peroxide. Place the bung into the conical flask quickly so as not to loose any oxygen. The celery should be of 100% concentration, obtained by liquefying the celery into a pureed form.
6) Start the timer and record the amount of oxygen collected in the gas burette at a constant period of time. I decided to time each experiment for five minutes, and measure the amount of oxygen collected every 30 seconds during those five minutes. I came to this decision based on the rate of oxygen production seen in the pilot practical, where a recoding every 30 seconds for 5 minutes seemed to give reliable results and was not too difficult to record.
7) Repeat steps 4 to 6 for the other four peroxide concentrations chosen.
Fair Test
To make it a fair test the total amount of water and peroxide must remain constant for every experiment - only the ratio of water to peroxide must change. Again to keep it a fair test the temperature and pH should also remain constant in the reactions. To do this no foreign chemicals that might change the pH of the experiment must be added, and no foreign heat sources, for example drafts from open windows or heat from radiators, must be brought near the experiment. This will interfere with the reaction and thus with the results and will alter the conclusion and make the investigation invalid. The experiment should not be moved while the reaction is occurring since that this moves the liquid and therefore alters the reaction results since this increases collisions between substrate and enzyme, thus speeding up the reaction. The time chosen for the reaction should be also kept constant (in my case, five minutes) and so should the surface area of the liquid as this will again increase collisions between substrate and enzyme and will obviously alter the results. To keep the surface area the same, the total solution (consisting of a constant total amount of water and peroxide, and a constant amount of pureed celery) should be added to the same size container. The most important thing in the proceeding of the experiment is to make sure the conical flask is sealed so that no oxygen escapes and drastically affects the results. The reasons behind the importance of keeping these variables consistent can be found in the Variables section.
Safety
The issue of safety was taken into account during my pilot practical and so played a large part in forming the results. Firstly safety goggles must be worn at all times since peroxide is a corrosive material and will do damage to the eyes. If any peroxide, despite concentration, comes into contact with any part of the body during the experiment, measures must be taken to ensure that it is removed properly, preferably by putting the exposed area under running water. Care must be take in the handling of the peroxide due to its corrosive nature, and so if any is spilt, it must be cleaned up immediately, and also care must be taken not to splash the peroxide and causing backsplash when adding it to the celery. Therefore the advised method of adding the peroxide to the celery is to us the syringe to squirt it down the side of the conical flask so that it does not splash. Since the equipment is glass, special care must be taken to make sure no breakages occur and, if they do, they must be cleaned up and disposed of immediately before others injure themselves on the breakage. Sensibility overall is the issue that needs most care - be sensible and take care of the delicate glass object in the surroundings and the corrosive peroxide that is in use.
Amendments
The main change in the proceeding of the experiment was that the 5 concentrations of hydrogen peroxide were pre-prepared to concentrations of 40%, 30%, 20%, 10% and 5%. I also changed the net volume of these concentrations used in each experiment from 50ml to 5ml and the net celery extract volume from 3ml to 1ml since both of these give a more controlled experiment. I also timed each experiment for 6 minutes instead of 5, but still recorded levels of oxygen every 30 seconds. I used a bung with only one hole instead of two since this eliminates the possible released of oxygen from the experiment and so produces a better and more reliable set of results.
Results
Time (minutes)
Volume of Oxygen Collected (cm3) at Peroxide concentration of
40%
30%
20%
0%
5%
40% (repeat)
0
0
0
0
0
0
0
0.5
0
0
0
0
0
0
.5
.3
.6
.2
.5
.5
2.5
.8
2.2
.9
.7
2
3.1
2.1
3
2.5
2.2
2.5
3.8
3.1
3.7
3.1
2.5
3
4.1
3.5
4.4
4.1
2.8
3.5
4.8
4
5
4.7
3.2
4
5.1
5
6
5.7
3.8
4.5
5.1
5.7
6.8
6.6
5
5.4
6.7
7.5
7.9
5.5
5.4
7.3
8.5
8.9
6
5.4
8
9.5
9.8
After the experiment, a more controlled experiment was carried out with different peroxide concentrations.
Peroxide Concentration (%)
Oxygen (cm3) collected after
0 minutes
20 minutes
20
7
33.4
5
5.9
30.8
0
5
29.8
5
4
27.8
2.5
9
8.8
Analysis
The graph from the first experiment showed that with a concentration of 40%, the general shape of the graph was as expected since it was reacting fast at the beginning and then the rate of reaction leveled out towards the end of the experiment. However the repeat of 40% did the opposite - it started off rather more slow than it finished since at the end there were greater increments for every 30 seconds of oxygen productions at the end of the experiment than at the beginning. The 30%, 20% and 10% concentrations followed a fairly steady line but as the peroxide concentration decreased, the total oxygen collected and thus the steepness of the graph increased. I did not expect the 30%, 20% and 10% concentration to also follow such a straight line since as the reaction rate should have decreased as more and more substrate was broken down, and thus there were less and less substrate molecules to bond with the enzyme molecules as time elapsed. There were no anomalies in the experiment graphs, however there were slight fluctuations in the results which were expected since the rate of oxygen production, although steady , comes in short bursts and so if the oxygen production is slightly delayed and the usual 30 secon measurement is taken, the amount of oxygen would be slightly lowered than it could have been. There for this causes slight variations in the results but no larger fluctuations or anomalies were experienced. These results lead us to believe that there was a foreign factor affecting the experiment, possibly one of the factors as discussed in the variables section. I expected the rate of oxygen production to increase as the peroxide concentration increased since the more substrate molecules there are present in the solution, the more bonding will take place, assuming there are enough enzyme molecules to bond with them, as each substrate will bond with one of these enzyme molecules and so increase the reaction rate compared to when there were fewer substrate molecules. If the peroxide concentration decreases, there will be a decrease in the reaction rate since, assuming there are enough enzyme molecules to bond with, there will be not so many substrate molecules to bond with the enzyme molecules. Therefore these results were the inverse of what I had expected and so a factor was looked into as to why these results occurred. A pH test on the solutions for the first experiment was done and these results were collected:
Hydrogen Peroxide Concentration (%)
pH
40
3
30
5
20
6
0
7
5
7.5
Therefore from these results we can observe that at the higher concentrations, the acidity of the solution was having an adverse affect on the enzyme and so, as the concentration decreased, so did the acidity and therefore the oxygen rate increased. This is because the enzyme obviously works better at pH of around neutral, and thus not at an acid pH. This would explain the adverse results from what I had expected. The enzyme obviously works best in a solution of neutral pH and not of one of acid/ alkali pH since if the pH is very different from a chemicals optimum pH, it has trouble bonding with other chemicals and eventually becomes denatured. This would explain the results since as the acidity increased, the oxygen production decreased, thus meaning that the enzyme is working worse in those conditions. The enzyme works by creating temporary bonds, which form between the substrate and some of the side chains of the enzymes amino acids. Because the cleft (active site) in the enzyme is such a specific one, usually only one substrate fits it exactly - in this case hydrogen peroxide. The interaction between substrate and enzyme breaks the substrate apart and the products, in this case oxygen and water, leave the active site and thus leave the enzyme unchanged and ready to bond with another substrate. However if, in this case, the pH is far from the optimum pH of the enzyme, the acidity/alkalinity interferes with the hydrogen ions ability to bond with certain chemicals and, if the pH reaches a level where the enzyme becomes too badly affected, the active site can change its shape and so not react to the substrate - it has become denatured.
In the controlled experiment, the pH of the hydrogen peroxide solutions remained constant and here the results show as the peroxide concentration increases, the rate of oxygen production increases. This is because as the concentration increases there are more substrate molecules for the enzyme molecules to bond with and so there are more reactions performing at any given time. When the peroxide concentration decreases, there are less substrate molecules for the enzyme molecules to bond with and so there are less bonds performing at any given time - reaction rate decreases.
To get reliable results the experiment should be repeated, making sure that there are no foreign factors, especially pH, affecting the experiment.
Evaluation
The main error of my experiment was the fact that the pH of the hydrogen peroxide solutions did not remain constant and so there were other variables affecting the experiment. This means although while my first experiment gave inconclusive results as to the relationship between peroxide concentration and oxygen production, it concluded that the pH adversely affects the reaction rate as it becomes more acid. This foreign variable (pH level) could be kept constant by neutralizing it and keeping the pH at a constant level, as performed in the second, controlled experiment, and this would give conclusive results about the relationship between peroxide concentration and rate of oxygen production. The air temperature of the room seemed to increase rapidly throughout the experiment, and this would have affected the results by varying the temperature at reaction and thus adversely affecting the results. This could be kept constant by making sure that no external heat sources are altered throughout the investigation, and that the room temperature is also kept constant. This is important since if more than one variable is affecting the reaction, in this case peroxide concentration, pH and possibly temperature, the results become inconclusive to any one of those variables and so the whole experimental results become invalid. These factors should be investigated separately to avoid this, for instance the peroxide concentration and room temperature could be kept constant while the pH of the solution increased/decreased, to see what effect this has on the rate of oxygen production. Overall the main flaw in my experiment was the fluctuation of pH level which increased as peroxide concentration decreased, thus drawing no conclusive results to my aim. This should be prevented my making sure the pH level remains constant and so results drawn will be conclusive to the relationship between rate of substrate-enzyme reaction and hydrogen peroxide concentration.
